Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.
Insulin-like growth factors (IGFs) can stimulate skeletal muscle differentiation. One of the molecular mechanisms underlying IGF-stimulated myogenesis is transcriptional induction of myogenin. The current work is aimed to elucidate the signaling pathways mediating the IGF effect on myogenin promoter in mouse C2C12 myogenic cells. We show that phosphatidylinositol 3-kinase (PI3K)/Akt and p70 S6K are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. We have identified three cis-elements, namely the E box, MEF2, and MEF3 sites, within the 133-base pair mouse proximal myogenin promoter that are under the control of the IGF/PI3K/Akt pathway. Simultaneous mutation of all three elements completely abolishes activation of the myogenin promoter by PI3K/Akt. We demonstrate that PI3K/Akt can increase both the MyoD and the MEF2-dependent reporter activity by enhancing the transcriptional activity of MyoD and MEF2. Interestingly, IGF1 does not enhance myogenin expression in Rhabdomyosarcoma-derived RD cells. Consistently, the constitutively active PI3K/Akt fail to activate the myogenic reporters, suggesting the IGF/PI3K/Akt pathway is defective in RD cells and the defect(s) is downstream to PI3K/Akt. This is the first time that a defect in the IGF/PI3K/Akt pathway has been revealed in RD cells which provides another clue to future therapeutic treatment of Rhabdomyosarcoma. Insulin-like growth factors (IGFs) can stimulate skeletal muscle differentiation. One of the molecular mechanisms underlying IGF-stimulated myogenesis is transcriptional induction of myogenin. The current work is aimed to elucidate the signaling pathways mediating the IGF effect on myogenin promoter in mouse C2C12 myogenic cells. We show that phosphatidylinositol 3-kinase (PI3K)/Akt and p70 S6K are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. We have identified three cis-elements, namely the E box, MEF2, and MEF3 sites, within the 133-base pair mouse proximal myogenin promoter that are under the control of the IGF/PI3K/Akt pathway. Simultaneous mutation of all three elements completely abolishes activation of the myogenin promoter by PI3K/Akt. We demonstrate that PI3K/Akt can increase both the MyoD and the MEF2-dependent reporter activity by enhancing the transcriptional activity of MyoD and MEF2. Interestingly, IGF1 does not enhance myogenin expression in Rhabdomyosarcoma-derived RD cells. Consistently, the constitutively active PI3K/Akt fail to activate the myogenic reporters, suggesting the IGF/PI3K/Akt pathway is defective in RD cells and the defect(s) is downstream to PI3K/Akt. This is the first time that a defect in the IGF/PI3K/Akt pathway has been revealed in RD cells which provides another clue to future therapeutic treatment of Rhabdomyosarcoma. myogenic regulatory factor(s) base pair(s) insulin-like growth factor phosphatidylinositol 3-kinase Rhabdomyosarcoma differentiation medium growth medium Mammalian skeletal muscle differentiation has been a model system in the past decade for studying the molecular mechanisms that switch the cellular program from proliferation to differentiation. Intensive studies have led to the discovery and characterization of two families of transcription factors that play pivotal roles during differentiation (1Lassar A.B. Skapek S.X. Novitch B. Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (312) Google Scholar, 2Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (390) Google Scholar, 3Yun K. Wold B. Curr. Opin. Cell. Biol. 1996; 8: 877-889Crossref PubMed Scopus (326) Google Scholar, 4Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (247) Google Scholar). One of them consists of MyoD family proteins (also called myogenic regulatory factors or MRFs)1 which include four members: Myf5, MyoD, myogenin, and MRF4, all members of the basic helix-loop-helix superfamily and exclusively expressed in skeletal muscles. Knock-out and knock-in data reveal the existence of a genetic hierarchy in which MyoD and Myf5 act upstream to determine the myogenic fate of muscle precursor cells, while myogenin and MRF4 act downstream of Myf5 and MyoD to control the differentiation process (3Yun K. Wold B. Curr. Opin. Cell. Biol. 1996; 8: 877-889Crossref PubMed Scopus (326) Google Scholar,4Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (247) Google Scholar). The other group of transcription factors important for muscle differentiation consists of MEF2 proteins which also include four members: MEF2A, -2B, -2C, and -2D (5Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (856) Google Scholar). MRF and MEF2 members can physically interact with each other to synergistically activate many muscle-specific genes (2Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (390) Google Scholar, 6Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (708) Google Scholar). Among four MRFs, myogenin is critically involved in executing the differentiation program. Although myoblasts from myogenin null mice are present, they do not differentiate in vivo leading to severe muscle deficiency and perinatal death of the homozygous mice (7Hasty P. Bradley A. Morris J.H. Edmondson D.G. Venuti J.M. Olson E.N. Klein W.H. Nature. 1993; 364: 501-506Crossref PubMed Scopus (1039) Google Scholar, 8Nabeshima Y. Hanaoka K. Hayasaka M. Esumi E. Li S. Nonaka I. Nature. 1993; 364: 532-535Crossref PubMed Scopus (732) Google Scholar). Expression of myogenin is considered one of the earliest molecular markers for cells committed to differentiation in vitro. The up-regulation of myogenin, in concomitant with induction of the cell cycle inhibitor p21 cip1 , indicates that cells have irreversibly withdrawn from the cell cycle and entered the differentiation program (1Lassar A.B. Skapek S.X. Novitch B. Curr. Opin. Cell Biol. 1994; 6: 788-794Crossref PubMed Scopus (312) Google Scholar). Regulation of myogenin has been extensively studied by both transfection analyses in cell culture systems and transgenic studies (9Cheng T.C. Hanley T.A. Mudd J. Merlie J.P. Olson E.N. J. Cell Biol. 1992; 119: 1649-1656Crossref PubMed Scopus (67) Google Scholar, 10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar, 11Edmondson D.G. Cheng T.C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (257) Google Scholar, 12Buchberger A. Ragge K. Arnold H.H. J. Biol. Chem. 1994; 269: 17289-17296Abstract Full Text PDF PubMed Google Scholar, 13Malik S. Huang C.F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar, 14Johanson M. Meents H. Ragge K. Buchberger A. Arnold H.H. Sandmoller A. Biochem. Cell Biol. 1999; 265: 222-232Google Scholar). A 133-bp myogenin proximal promoter has been found to contain sufficient cis-elements to correctly target a lacZ transgene to specific muscle-forming regions (10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar). Furthermore, an E box, a MEF2 site, and a MEF3 site within this 133-bp proximal myogenin promoter have been identified as critical cis-elements regulating myogenin expression (9Cheng T.C. Hanley T.A. Mudd J. Merlie J.P. Olson E.N. J. Cell Biol. 1992; 119: 1649-1656Crossref PubMed Scopus (67) Google Scholar, 10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar, 11Edmondson D.G. Cheng T.C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (257) Google Scholar, 12Buchberger A. Ragge K. Arnold H.H. J. Biol. Chem. 1994; 269: 17289-17296Abstract Full Text PDF PubMed Google Scholar, 13Malik S. Huang C.F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar, 14Johanson M. Meents H. Ragge K. Buchberger A. Arnold H.H. Sandmoller A. Biochem. Cell Biol. 1999; 265: 222-232Google Scholar, 15Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (184) Google Scholar). MyoD, MEF2, and homeodomain-containing Six (see "Discussion") proteins have been implicated as the nuclear factors binding to these cis-elements. Despite the above progress, how exactly these nuclear factors are activated by various intracellular signaling pathways is less well understood at present. Nor is known about the relationships between these cis-elements/transacting factors and the IGF signaling pathway. Insulin-like growth factors (IGFs) have been shown to potently stimulate myogenesis in cultured myogenic cells and are required for normal skeletal muscle development during mouse embryogenesis (16Powell-Braxton L. Hollingshead P. Warburton C. Dowd M. Pitts-Meek S. Dalton D. Gillett N. Stewart T.A. Genes Dev. 1993; 7: 2609-2617Crossref PubMed Scopus (682) Google Scholar, 17Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar). In rat L6 myogenic cells, IGFs display biphasic action profiles: initially they stimulate proliferation, then function as strong inducers of differentiation (17Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar, 18Rosenthal S.M. Cheng Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10307-10311Crossref PubMed Scopus (145) Google Scholar, 19Engert J.C. Berglund E.B. Rosenthal N. J. Cell Biol. 1996; 135: 431-440Crossref PubMed Scopus (252) Google Scholar). One of the molecular mechanisms underlying the stimulatory myogenic effect of IGFs lies in their abilities to transcriptionally induce myogenin mRNA (20Florini J.R. Ewton D.Z. Roof S.L. Mol. Endocrinol. 1991; 5: 718-724Crossref PubMed Scopus (237) Google Scholar). It remains obscure how exactly this is achieved. Several intracellular signaling pathways have been identified that are activated in response to IGF stimulation. One of them is mitogen-activated protein kinase (MAPK)-mediated signaling pathway. It has been shown that the ERK subgroup of MAPKs can be activated by IGF treatment via the classical receptor tyrosine kinase/Grb2-Sos/Ras/Raf mediated pathway (21Petley T. Graff K. Jiang W. Yang H. Florini J. Horm. Metab. Res. 1999; 31: 70-76Crossref PubMed Scopus (104) Google Scholar). Activation of ERK may be partially responsible for the initial mitogenic effect of IGF (18Rosenthal S.M. Cheng Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10307-10311Crossref PubMed Scopus (145) Google Scholar, 22Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Another IGF-activated intracellular signaling pathway that has attracted much attention is mediated by phosphatidylinositol 3-kinase (PI3K). In response to IGF stimulation, activated PI3K converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate which results in subsequent activation of the pleckstrin homology domain-containing serine/threonine kinases PDK1 and Akt/PKB (23Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 24Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar, 25Leevers S.J. Vanhaesebroeck B. Waterfield M.D. Curr. Opin. Cell Biol. 1999; 11: 219-225Crossref PubMed Scopus (574) Google Scholar). Activated PI3K also leads to activation of p70 S6K , which is mediated mainly by mTOR, PDK1, and atypical PKCs (26Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (608) Google Scholar). It has been demonstrated that PI3K mediates the stimulatory effect of IGFs on muscle differentiation (22Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 27Kaliman P. Vinals F. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1996; 271: 19146-19151Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 28Jiang B.H. Zheng J.Z. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14179-14183Crossref PubMed Scopus (117) Google Scholar). Specific interference of endogenous PI3K activity abolishes myogenic differentiation. Akt is shown to mediate the PI3K effect during muscle differentiation (29Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar). Deliberate activation of either PI3K or Akt greatly enhances muscle differentiation (28Jiang B.H. Zheng J.Z. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14179-14183Crossref PubMed Scopus (117) Google Scholar, 29Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar). However, the downstream targets that couple PI3K/Akt stimulatory signals to myogenic signaling pathways remain to be identified. The present study sought to identify the signaling pathways mediating the effect of IGF on myogenin expression in C2C12 cells, and to define the IGF-responsive cis-elements on myogenin promoter and the nuclear signal receivers/transcription factors that bind these cis-elements. We showed PI3K, Akt, and p70 S6K are critical signaling molecules mediating the IGF effect. Using IGF-mediated myogenin induction as a model, we demonstrated that the stimulatory effect of PI3K/Akt on myogenin promoter activation was mediated by the unique E box, MEF2, and MEF3 sites in the 133-bp proximal myogenin promoter. MyoD and MEF2 family proteins are implicated as downstream targets of PI3K/Akt. p70 S6K was also shown to mediate part of the PI3K signal along with Akt. We also carried out similar experiments in RD cells, a cell line derived from Rhabdomyosarcoma (RMS), a childhood malignant tumor expressing myogenic regulatory factors yet failing to undergo myogenic differentiation (30Anderson J. Gordon A. Pritchard-Jones K. Shipley J. Genes Chromosomes Cancer. 1999; 26: 275-285Crossref PubMed Scopus (143) Google Scholar, 31Merlino G. Helman L.J. Oncogene. 1999; 18: 5340-5348Crossref PubMed Scopus (226) Google Scholar). We found that IGF1 failed to induce myogenin expression in RD cells. While IGF1 could activate PI3K leading to Akt phosphorylation and activation in RD, the constitutively active PI3K/Akt failed to activate the myogenic reporter genes suggesting the defect in the IGF/PI3K/Akt pathway in RD cells lies downstream to PI3K/Akt. The GBBS-Luc was generated by inserting a SacI-BglII fragment from the GBBS-CAT into a HindIII-BglII-digested pXP2 luciferase vector. G133-Luc, G133E-Luc, and G133MEF2-Luc were generated by inserting the XbaI-BglII fragments from the corresponding chloramphenicol acetyltransferase constructs into theHindIII-BglII-digested pXP2, respectively. Various binding site mutants in G133-Luc were generated by a polymerase chain reaction-mediated mutagenesis method (32Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst .). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). The mutations at the E box and the MEF2 site are the same as described in Ref. 10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar. The primers used to make mutations at the MEF3 site are: forward: 5′-TAGAGGGGGGCTGAGCTCTCTGTGGCGTTG-3′, reverse: 5′-CAACGCCACAGAGAGCTCAGCCCCCCTCTA-3′. C2C12 cells and L6 cells were purchased from ATCC and grown in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum (growth medium, or GM) in a 37 °C incubator with 5% CO2. To induce differentiation, growth medium is substituted by differentiation medium (Dulbecco's modified Eagle's medium containing 2% horse serum, or DM) when cells are near confluent. 10T1/2 fibroblasts, HeLa, and RD cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. IGF1 was purchased from R&D systems. The constitutively active Akt contains an in-frame myristoylation sequence. The dominant negative Akt contains a Lys179 to Met mutation. The constitutively active PI3K is Myc-tagged with a CAAX motif (p110α-CAAX). The constitutively active MKK6 contains Thr to Glu mutations at the two conserved Thr residues in the kinase subdomain VIII. 4RE-luc contains four copies of the E box fused to the luciferase gene. 3xMEF2-Luc contains three copies of the MEF2 site fused to the luciferase gene. LipofectAMINE Plus reagent (Life Technologies, Inc.) was used for all transfection experiments. Unless stated otherwise, cells were first grown in GM for 36 h after transfection, then shifted to DM for another 24 h prior to cell harvest. Luciferase units were determined in a Monolight 2010 luminometer (Analytical Luminescence laboratory) using luciferase reporter gene assay kits from Roche Molecular Biochemicals and normalized against total protein amount present in each sample extract. Protein concentrations were determined using protein assay solution from Bio-Rad. Total RNA was extracted from C2C12 cells using Trizol reagent from Life Technologies, Inc. following the manufacturer's suggestion. 20 μg of total RNA was separated on a 1% formaldehyde-agarose gel, transferred to a Hybond-N+membrane (Amersham Pharmacia Biotech) and cross-linked to the membrane in a UV cross-linker (Stratagene). The mouse myogenin probe and the glyceraldehyde-3-phosphate dehydrogenase control probe were labeled using random labeling kits (Stratagene). Hybridization was carried out at 60 °C in the Church-Gilbert hybridization solution overnight and washed three times with 2 × SSC, 0.1% SDS buffer before being subjected to autoradiography. Cells were lysed in the lysis buffer (50 mm Hepes, pH 7.6, 1% Triton X-100, 150 mmNaCl, 1 mm EGTA, 1.5 mm MgCl2, 10% glycerol, 100 mm NaF, 20 mm p-nitrophenylphosphate, 20 mmβ-glycerolphosphate, 50 μm sodium vanadate, 2 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin) and whole cell lysates were prepared after removing insoluble debris. 30 μg of whole cell lysates was separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon-P (Millipore) membrane, and probed with various antibodies. The protein bands were visualized using enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech). SB202190, PD98059, LY294002, and rapamycin (Calbiochem) were added to cells at the time of medium change from GM to DM at a final concentration of 10 μm, 25 μm, 25 μm, and 20 nm, respectively. After 24 h treatment, cells were harvested for either Western blot analysis or luciferase assays. It has been demonstrated that IGF elicits dual effects in rat L6 myogenic cells (17Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar, 18Rosenthal S.M. Cheng Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10307-10311Crossref PubMed Scopus (145) Google Scholar, 19Engert J.C. Berglund E.B. Rosenthal N. J. Cell Biol. 1996; 135: 431-440Crossref PubMed Scopus (252) Google Scholar). In the first 24 h after IGF treatment, a mitogenic response takes place in L6 cells followed (around 24 h after IGF1 treatment) by a potent myogenic response indicated by a sharp increase in myogenin mRNA and protein levels (18Rosenthal S.M. Cheng Z.Q. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10307-10311Crossref PubMed Scopus (145) Google Scholar, 19Engert J.C. Berglund E.B. Rosenthal N. J. Cell Biol. 1996; 135: 431-440Crossref PubMed Scopus (252) Google Scholar). Indeed, in our experiment, no myogenin could be detected in L6 cells at either 4- or even the 9-h time point after IGF1 treatment (Fig. 1 B). However, myogenin was detected 24 h after IGF1 treatment. In contrast to L6 cells, a much faster myogenic response to IGF1 was seen in C2C12 cells. As shown in Fig. 1 A, 50 ng/ml IGF1 potently induced myogenin protein level as early as 4 h after IGF1 treatment. 24 h after IGF1 treatment, the myogenin expression reached the peak level. In agreement with previous findings (20Florini J.R. Ewton D.Z. Roof S.L. Mol. Endocrinol. 1991; 5: 718-724Crossref PubMed Scopus (237) Google Scholar), we showed that IGF-mediated myogenin induction occurred at the mRNA level (Fig. 1 C). As expected, early induction of myogenin in C2C12 was accompanied by a faster appearance of multinucleated myotubes of increased size when the morphology of the cells was examined by microscopy (Fig.1 D). The above results indicate that IGF1 can transcriptionally induce myogenin expression with a much faster kinetics in C2C12 cells than in L6 cells.Figure 1IGF1 treatment leads to a faster myogenin induction in C2C12 cells than in L6 cells. When near confluent, C2C12 or L6 cells were shifted from GM to DM in the presence (50 ng/ml) or absence of IGF1. For A (C2C12) and B (L6), cells were harvested at the indicated time points and subjected to Western blot analysis using a monoclonal anti-myogenin antibody (F5D). The same blot was also re-probed with a monoclonal anti-β-actin (Sigma) antibody to monitor the loading difference. C, total RNA was extracted from C2C12 cells and subjected to Northern blot analysis using a myogenin cDNA probe. The same blot was stripped and re-probed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to monitor the loading difference.D, C2C12 cells were fixed and subjected bright field microscopy analysis. GM, growth medium; SF,serum-free medium; Myog, myogenin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been well established that PI3K/Akt can serve as downstream mediators of IGF action in various cell types. Both PI3K and Akt have been implicated in muscle differentiation (22Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 27Kaliman P. Vinals F. Testar X. Palacin M. Zorzano A. J. Biol. Chem. 1996; 271: 19146-19151Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 28Jiang B.H. Zheng J.Z. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14179-14183Crossref PubMed Scopus (117) Google Scholar, 29Jiang B.H. Aoki M. Zheng J.Z. Li J. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2077-2081Crossref PubMed Scopus (228) Google Scholar). Since IGF1 could transcriptionally up-regulate myogenin (Fig. 1 C) (20Florini J.R. Ewton D.Z. Roof S.L. Mol. Endocrinol. 1991; 5: 718-724Crossref PubMed Scopus (237) Google Scholar), we tested whether PI3K/Akt could mediate IGFs stimulatory effect on the myogenin promoter. A 1-kilobase mouse proximal myogenin promoter fused to a promoterless luciferase gene was constructed and used as a reporter (GBBS-Luc). This 1-kilobase myogenin promoter was shown to contain sufficient regulatory elements that are responsible for somite-restricted expression of a lacZ transgene (9Cheng T.C. Hanley T.A. Mudd J. Merlie J.P. Olson E.N. J. Cell Biol. 1992; 119: 1649-1656Crossref PubMed Scopus (67) Google Scholar, 10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar). When GBBS-Luc was co-transfected into C2C12 cells along with either an empty expression vector or various PI3K/Akt constructs, significant activation of the myogenin promoter was detected only with the constitutively active PI3K or Akt, but not with the empty vector or the dominant negative mutants of PI3K and Akt (Fig.2 A). Furthermore, a promoterless luciferase vector pXP2 (into which the 1-kilobase myogenin promoter was inserted to make the GBBS-Luc) could not be activated by either the constitutively active PI3K or Akt, indicating it is the sequences in the myogenin promoter that specifically respond to PI3K/Akt signaling. Activation of the myogenin promoter reporter by either PI3K or Akt requires a functional kinase domain, as the kinase-dead PI3K/Akt mutants (PI3K-dn/Akt-dn) failed to activate the reporter (Fig. 2 A). To further delimit the region(s) in the myogenin promoter responsive to PI3K/Akt signaling, a 133-bp proximal myogenin promoter fragment fused to the luciferase gene was also constructed and used as a reporter (G133-Luc). It was previously shown that this 133-bp myogenin promoter fragment could also specifically target a lacZ transgene to somites (10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar). As shown in Fig.2 A, the constitutively active PI3K/Akt-activated G133-Luc to the similar extent as they did to GBBS-Luc, suggesting most if not all PI3K/Akt-responsive elements reside within the 133-bp proximal myogenin promoter. To further prove that both PI3K and Akt were involved in regulating endogenous myogenin expression, we transiently transfected C2C12 cells with either the dominant negative or the constitutively active PI3K/Akt and analyzed the endogenous myogenin expression by Western blotting. We reasoned that, if PI3K/Akt indeed controlled myogenin expressionin vivo and the effect of PI3K/Akt was strong enough, we might detect the difference in myogenin expression simply by assaying the level of the endogenous myogenin in the total cell population including both the transfected and untransfected cells. Indeed, compared with the empty vector transfected control cells, both the dominant negative PI3K (PI3K-dn) and Akt (Akt-dn) transfected cells had reduced levels of myogenin expression in response to IGF1 stimulation (Fig. 2 B). In contrast, both the constitutively active PI3K (PI3K-ca) and Akt (Akt-ca) could recapitulate the stimulatory effect of IGF1 and resulted in significantly enhanced myogenin expression compared with the control cells (Fig. 2 B). Considering an average transfection efficiency of 30–40% in C2C12 cells, the effect of these transfected PI3K/Akt mutants on endogenous myogenin expression was quite significant. Previous studies on transcriptional regulation of myogenin revealed the single MEF2 site and the E box within the 133-bp proximal myogenin promoter are critical for induction of myogenin and correct targeting of a lacZ transgene to somites (9Cheng T.C. Hanley T.A. Mudd J. Merlie J.P. Olson E.N. J. Cell Biol. 1992; 119: 1649-1656Crossref PubMed Scopus (67) Google Scholar, 10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar, 11Edmondson D.G. Cheng T.C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (257) Google Scholar, 12Buchberger A. Ragge K. Arnold H.H. J. Biol. Chem. 1994; 269: 17289-17296Abstract Full Text PDF PubMed Google Scholar, 13Malik S. Huang C.F. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar, 14Johanson M. Meents H. Ragge K. Buchberger A. Arnold H.H. Sandmoller A. Biochem. Cell Biol. 1999; 265: 222-232Google Scholar). We set out to test whether one or both of these sites are also required to mediate the stimulatory effect of PI3K/Akt. The single E box and the MEF2-binding site in G133-Luc were mutated either individually or at the same time. These mutations have been shown to disrupt MyoD and MEF2 binding and correct somite targeting by the lacZ transgene (10Yee S.P. Rigby P.W. Genes Dev. 1993; 7: 1277-1289Crossref PubMed Scopus (350) Google Scholar). The responsiveness of these mutant myogenin promoter reporters to PI3K/Akt was tested by reporter assays. As shown in Fig.3 A, mutation of either the E box or the MEF2 site partially inhibited the reporter activation by PI3K/Akt. Simultaneous disruption of both sites did not significantly reduce the stimulatory effect of PI3K/Akt further. Since a residual 2–3-fold activation by PI3K/Akt could still be detected in the reporter harboring double mutations (G133(2+E)), we reasoned that another cis-element within the 133-bp proximal myogenin promoter might also be under the control of the PI3K/Akt signaling pathway. It was recently demonstrated that a conserved MEF3 site (Fig.3 B), present in the 133-bp proximal myogenin promoters of chick, mouse, and human origin, also plays an important role in regulating myogenin expression (15Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (184) Google Scholar). Homeodomain-containing Six proteins were shown to bind the MEF3 site. To test whether the MEF3 site was responsible for the residual activation of G133(2+E)-Luc by PI3K/Akt, we mutated the MEF3 site and generated a myogenin promoter reporter with triple mutations (G133TM-Luc). As shown in Fig.3 A, this triple mutant myogenin promoter completely lost its ability to be activated by either the constitutively activated PI3K or Akt. We also mutated the MEF3 site (G133MEF3) either alone or in conjunction with the E box (G133(E+3)) or the MEF2 site (G133(2+3)). We found that mutation of the MEF3 site alone had minimal effect on myogenin promoter activation in response to PI3K/Akt signaling (Fig.3 A). In contrast, mutation of the MEF3 site together with either the E box or the MEF2 site severely compromised the ability of the myogenin promoter to be activated by PI3K/Akt. The above results indicate that the E box, MEF2, and MEF3 sites in the 133-bp proximal myogenin promoter are critical cis-elements under the control of the IGF/PI3K/Akt signaling pathway. It has been shown that p70 S6K functions downstream of PI3K (26Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref
Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma. Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma. Rhabdomyosarcoma is a malignant tumor of childhood that is thought to derive from muscle precursor cells. Rhabdomyosarcoma-derived RD cells are originated from the embryonal type of rhabdomyosarcoma. The RD cells are defective in myogenic differentiation even though they express both MyoD and myogenin (1Bouche M. Senni M.I. Grossi A.M. Zappelli F. Polimeni M. Arnold H.H. Cossu G. Molinaro M. Exp. Cell Res. 1993; 208: 209-217Crossref PubMed Scopus (36) Google Scholar). However, once treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 4The abbreviations used are: TPA, 12-O-tetradecanoyl-phorbol 13-acetate; CAT, chloramphenicol acetyltransferase; RD cells, rhabdomyosarcoma-derived cells; HAT, histone acetyltransferase; ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; SB, p38 inhibitor SB203580; MEF, myocytes enhancer binding factor; MAPK, mitogen-activated protein kinase; RT, reverse transcription. a phorbol ester, RD cells become refractory to growth signals and undergo cell cycle arrest (2Aguanno S. Bouche M. Adamo S. Molinaro M. Cancer Res. 1990; 50: 3377-3382PubMed Google Scholar). Meanwhile, the TPA-treated RD cells display a morphology resembling that of differentiated myotubes (2Aguanno S. Bouche M. Adamo S. Molinaro M. Cancer Res. 1990; 50: 3377-3382PubMed Google Scholar). Several protein kinases including protein kinase Cα, extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) have been implicated in the TPA-induced differentiation of RD cells (3Mauro A. Ciccarelli C. De Cesaris P. Scoglio A. Bouche M. Molinaro M. Aquino A. Zani B.M. J. Cell Sci. 2002; 115: 3587-3599Crossref PubMed Scopus (92) Google Scholar). As one of the excellent paradigms to study cellular differentiation, myogenic differentiation has been extensively studied in the past decade. Much has been learned about the myogenic regulatory factors (MRFs) of the basic helix-loop-helix protein family, such as MyoD, that govern myogenic differentiation. To mediate muscle-specific gene transcription, MRFs have to dimerize with E2A products (i.e. E12 and E47), which are also members of the basic helix-loop-helix. Together, the heterodimers bind efficiently to the consensus E box (i.e. 5′-CANNTG-3′) in the promoter regions of many muscle-specific genes including the myogenin gene. In addition to MRFs, myocytes enhancer binding factor 2 (MEF2s) are also essential for myogenic differentiation (4Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (856) Google Scholar). Four distinct members of the MEF2 family, namely MEF2A, 2B, 2C, and 2D, bind to a consensus AT-rich sequence (i.e. MEF2 site) in promoters of many muscle-specific genes as either homo- or heterodimers. Previous studies showed that a segment of the proximal mouse myogenin gene (–184/+1) containing an E box, a MEF2, and MEF3 sites is ∼88% homologous to that of the humans and is indispensable in myogenin gene expression (5Edmondson D.G. Cheng T.C. Cserjesi P. Chakraborty T. Olson E.N. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (257) Google Scholar, 6Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (184) Google Scholar). Myogenin, and other members of the MyoD family (MyoD, Myf5, and MRF4), possess the ability to convert some of the non-muscle cells into the myogenic lineage. Expression of myogenin has been considered as one of the earliest molecular markers for cells committed to differentiation in vitro. Depending on the nature of the stimuli, myogenin is either induced or repressed, this in turn determines whether the differentiation program is enhanced or aborted (7Zhang J.Z. Gao W. Yang H.B. Zhang B. Zhu Z.Y. Xue Y.F. Stem Cells. 2006; 24: 2661-2668Crossref PubMed Scopus (41) Google Scholar). The mammalian SWI/SNF chromatin remodeling complex is required for myogenin gene expression (8Gerber A.N. Klesert T.R. Bergstrom D.A. Tapscott S.J. Genes Dev. 1997; 11: 436-450Crossref PubMed Scopus (233) Google Scholar, 9de la Serna I.L. Ohkawa Y. Berkes C.A. Bergstrom D.A. Dacwag C.S. Tapscott S.J. Imbalzano A.N. Mol. Cell. Biol. 2005; 25: 3997-4009Crossref PubMed Scopus (223) Google Scholar, 10de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar). The SWI/SNF complex depends upon either BRG1 or BRM, the catalytic subunits with ATPase activity, to hydrolyze ATP to alter chromatin conformation. p38 MAPK accelerates myogenic differentiation by activating both MyoD- and MEF2-dependent gene transcription (11Wu Z. Woodring P.J. Bhakta K.S. Tamura K. Wen F. Feramisco J.R. Karin M. Wang J.Y. Puri P.L. Mol. Cell. Biol. 2000; 20: 3951-3964Crossref PubMed Scopus (400) Google Scholar). Recently, p38 MAPK was also shown to be required for the recruitment of the SWI/SNF complex to the myogenin promoter (12Simone C. Forcales S.V. Hill D.A. Imbalzano A.N. Latella L. Puri P.L. Nat. Genet. 2004; 36: 738-743Crossref PubMed Scopus (323) Google Scholar). The role of the p38 MAPK here is to phosphorylate BAF60, a component in the SWI/SNF complex, which further recruits the ATPase subunit (BRG1 or BRM) of the complex to the myogenin promoter (12Simone C. Forcales S.V. Hill D.A. Imbalzano A.N. Latella L. Puri P.L. Nat. Genet. 2004; 36: 738-743Crossref PubMed Scopus (323) Google Scholar). In addition to the SWI/SNF complex, the histone modification enzymes, such as the histone acetyltransferases (HATs), also participate in remodeling of the chromatin structure by modifying histone tails. p300, CBP, and PCAF of the HAT family are of particular interest because these enzymes cannot only modify the histone tails, but also acetylate other transcription regulatory factors including MyoD and MEF2 (13Puri P.L. Sartorelli V. Yang X.J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 14Roth J.F. Shikama N. Henzen C. Desbaillets I. Lutz W. Marino S. Wittwer J. Schorle H. Gassmann M. Eckner R. EMBO J. 2003; 22: 5186-5196Crossref PubMed Scopus (128) Google Scholar, 15Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 16Ma K. Chan J.K. Zhu G. Wu Z. Mol. Cell. Biol. 2005; 25: 3575-3582Crossref PubMed Scopus (134) Google Scholar). However, it remains unclear which chromatin remodeling factors are involved and how they coordinate with each other in TPA-induced RD cells. To address the above questions, we focused on the epigenetic regulation of the myogenin gene in TPA-treated RD cells. Our data prompted us to propose a two-step model to explain the TPA-induced Myogenin expression in RD cells: an early step in which PCAF is specifically recruited to the myogenin promoter followed by a p38 MAPK-dependent recruitment of the SWI/SNF complex. Our studies provide potential novel drug targets for treatment of rhabdomyosarcoma in the future. Cell Culture—RD cells were purchased from ATCC and maintained in Dulbecco's modified Eagles's medium (Invitrogen) supplemented with 10% fetal calf serum, 0.37% NaHCO3, and sodium penicillin and streptomycin sulfate (100 units/ml each) in a 5% CO2 humidified atmosphere at 37 °C. For inducing differentiation, stock solution of 100 μm TPA in 50% ethanol kept at –20 °C was added with a 1/1,000 dilution to the cultured cells 24 h after plating; equal amount of ethanol without TPA was added to the medium of the control cells. Plasmids—The mammalian expression plasmids were, pcDNA3-HA-p38 (wild type) from Dr. Kun Liang Guan (University of Michigan, Ann Arbor); pcDNA3-antisense p38 from Dr. Gang Pei (SIBS, Chinese Academy of Science); and pBJ5-BRG1(wild type) and pBJ5-BRG1K798R (an ATPase-defective K798R mutant, dominant negative) expression plasmids that were from Dr. Anthony N. Imbalzano (University of Massachusetts Medical School). Reagents—p38 inhibitor SB203580 and TPA were purchased from Sigma. Antibody against p300 was provided by Dr. Q. Li (National Institutes of Health, Bethesda, MD). Antiserum against hBAF60c was raised in rabbits using the full-length BAF60c as an antigen. Anti-myogenin (F5D), anti-β-actin (I-19), anti-MyoD (M-318), anti-BRG1 (H-88), anti-p38 (H-147), anti-phospho-p38 (D-8), and anti-PCAF (E-8) antibodies were purchased from Santa Cruz Biotechnology. Antibodies against pan-acetyl lysine, acetylated histone H3-K14, and acetylated histone H3-K9 were purchased from Upstate Biotechnology (Lake Placid, NY). DNA Constructs and DNA Transfection—An upstream fragment (–1088/+50) of the mouse myogenin gene was fused upstream of the CAT gene in an expression vector modified in the laboratory of Shen and designated as pREP4m-myog-CAT. The transfection control plasmid pCMV-β-gal was generated by amplifying the β-galactosidase gene from pRSV-β-gal plasmid (constructed in our laboratory) with primer pairs of the 5′ primer: 5′-CCCAAGCTTTTCGTCTGGGACTGGGTG-3′ and the 3′ primer: 5′-GCTCTAGAGGTCGGGATAGTTTTCTTGC-3′, followed by insertion into the XbaI/HindIII-digested pRc/CMV vector (Invitrogen). Transient transfection of DNA into RD cells was carried out using Lipofectamine 2000 (Invitrogen) in this study. Expression plasmids of p38 and BRG1 genes were individually co-transfected with pREP4m-myog-CAT and pCMV-β-gal into RD cells. For promoter activity assay, TPA (100 nm) was added to the medium after transient transfection for 16 h and incubated for another 24 h or the time interval indicated followed by cell harvesting. Quantitative Real-time RT-PCR Analysis—Total RNA was extracted from cells and followed by reverse transcription with a first-strand RT-PCR kit (Promega) per the manufacturer's instructions. PCR was performed with SYBR® Premix Ex Taq (TaKaRa, Biotech) using the Rotor-Gene RG-3000A (Corbett Research) Real-time PCR System. To detect the induction of the myogenin reporter, the following primers were used: for the myogenin promoter, forward primer, 5′-ACTCTTCGCCCCCGT-3′; reverse primer, 5′-CCGCCCTGCCACTCAT-3′; for the control plasmid of pCMV-β-gal, forward primer, 5′-CTTACGGCGGTGATTTTGG-3′; reverse primer, 5′-TGCTGCTGGTGTTTTGCTT-3′. The cycle quantity required to reach a threshold in the linear range (Qt) was determined and compared with a standard curve for each primer set generated by five 3-fold dilutions of the first-strand cDNA of known concentration. Data represent the mean ± S.D. of normalized promoter activities of myogenin relative to that of pCMV-β-gal in each treatment. To quantitate products generated in the chromatin immunoprecipitation assays or mRNA expression, we used the following primers: primers for myogenin gene, 5′-ATGGAGCTGTATGAGACATCCCC-3′ (forward, +1/+23) and 5′-GGACACCGACTTCCTCTTACAC-3′(reverse, +237/+216). The relative expression at each time interval was normalized against GAPDH (5′-GCTCACTGGCATGGCCTTCCG-3′ (forward, +741/+761) and 5′-GTGGGCCATGAGGTCCACCAC-3′ (reverse, +1050/+1030) using the comparative CT method recommended by the instrument producer. Experiments were repeated at least three times with statistical analyses for each individual experimental set. All values in the experiments were expressed as mean ± S.D. Nuclear Extract, Immunoprecipitation and Immunoblot Analysis—5 × 106 RD cells were collected in 8 ml of culture medium after a different treatment, 32 μl of 0.5 m dithiobis(succinimidylpropionate) were immediately added to make a final concentration of 2 mm. After shaking for 5–10 min, glycine was added to a final concentration of 10 mm to stop the cross-linking reaction. Cells were then collected by centrifugation at 1,000 × g for 5 min at room temperature and washed with phosphate-buffered saline. Following repeating this step once more, the pellets were recovered. Nuclear extract preparation was performed as described previously (17Zhang Y. Wang J.S. Chen L.L. Cheng X.K. Heng F.Y. Wu N.H. Shen Y.F. J. Biol. Chem. 2004; 279: 42545-42551Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The coimmunoprecipitation assay was carried out by using nuclear extracts (∼500 μg of protein) incubated with 2 μl of specific antibody for 2 h at 4 °C on a shaking platform. 20 μl of Protein A-agarose (Santa Cruz) was added to each tube and incubated at 4 °C overnight. Pellets were recovered, and washed three times with RIPA buffer, followed by adding 40 μl of 1× Laemmli buffer (50 mm Tris-HCl, pH 6.8, 80 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), resuspended and boiled for a 10% SDS-PAGE. Western blot assay was performed as described elsewhere (18Xiao L. Lang W. Cancer Res. 2000; 60: 400-408PubMed Google Scholar). Chromatin Immunoprecipitation (ChIP) and Quantitative PCR Analyses—ChIP assays were carried out with formaldehyde cross-linking as previously described (17Zhang Y. Wang J.S. Chen L.L. Cheng X.K. Heng F.Y. Wu N.H. Shen Y.F. J. Biol. Chem. 2004; 279: 42545-42551Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (488) Google Scholar), and detected with PCR and gel electrophoresis. ChIPed DNA was subjected to PCR amplification to yield a positive fragment of 140 bp that was separated on 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Gel images were scanned with an AlphaImager 2000™ as described previously (17Zhang Y. Wang J.S. Chen L.L. Cheng X.K. Heng F.Y. Wu N.H. Shen Y.F. J. Biol. Chem. 2004; 279: 42545-42551Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). For quantitative assay, standard curve and ChIPed DNA samples were analyzed on a Rotor-Gene RG-3000A Real-time PCR System (Corbett Research, Australia) with PCR Master Mix for SYBR Green assays (TaKaRa, Biotech). Primer pairs used for amplification of the myogenin gene were 5′-GAATCACATCTAATCCACTGTA-3′ (forward, –142/–121) and 5′-ACGCCAACTGCTGGGTGCCA-3′ (reverse, –3/–22). The cycle quantity required to reach a threshold in the linear range (Qt) was determined and compared with a standard curve for the primer set generated by five 10-fold dilutions of genomic DNA samples of known concentration. In all experiments, the following cycling parameters were used: 95 °C for 10 s, 40 cycles of 95 °C for 8 s, 60 °C for 15 s, and 72 °C for 10 s. The percentage of ChIPed DNA relative to input was calculated and shown as mean ± S.D. from three independent experiments. Enhanced Expression of Myogenin in TPA-treated RD Cells—To study the dynamic effect of TPA on RD cells, we treated the cells with TPA for various intervals of time as indicated. After 24–96 h of TPA treatment, the induced RD cells showed morphological changes including elongation of cells and formation of more multinucleated cells (Fig. 1A), which were in agreement with a previous report (2Aguanno S. Bouche M. Adamo S. Molinaro M. Cancer Res. 1990; 50: 3377-3382PubMed Google Scholar). Western blotting and quantitative real-time RT-PCR assays showed that myogenin was present at low levels in non-induced parental RD cells (Fig. 1, B and C). As differentiation progressed, the mRNA and protein levels of myogenin gradually increased and reached a peak value after 12–24 h of TPA treatment. We also analyzed the effect of TPA on promoter activity of the myogenin gene. As shown in Fig. 1D, the activity of the gene was enhanced by 2.9-fold 6 h after TPA treatment and by some 6-fold after 12 h of TPA treatment. A similar pattern of TPA-induced myogenin mRNA expression (Fig. 1C) suggested that the myogenin gene is mainly regulated at the promoter level in TPA-treated RD cells. Enhanced Histone Acetylation at the Promoter Region of Myogenin in TPA-induced RD Cells—To uncover the mechanism by which TPA promotes differentiation of RD cells, we first examined the acetylation status of lysines 9 and 14 in histone H3 (i.e. H3-K9 and H3-K14) as these residues are related to the activation status of the local chromatin (20Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7702) Google Scholar). ChIP assays were performed. Sonicated chromosome fragments were pulled down with specific antibodies against acetylated H3-K9 and H3-K14, respectively, followed by real-time PCR analysis using primers in the 5′-flanking region of the myogenin gene. We found that whereas K9 and K14 showed limited acetylation in the first 3 h of TPA induction, the acetylation of K14 reached to a higher level at 6 h post-treatment (Fig. 2B). Eventually both sites were significantly acetylated in RD cells after 12 h of TPA treatment (Fig. 2, A and B). The enhanced acetylation of histone H3 shown here indicated that HATs are involved in the early stage of TPA treatment. PCAF Is the First Histone Acetyltransferase Recruited to the Myogenin Promoter after TPA Treatment—To determine which HAT is preferentially recruited to the myogenin promoter in response to TPA treatment, we employed chromatin immunoprecipitation assays. Interestingly, we found that both p300 and MyoD consistently associated with the myogenin promoter with or without TPA induction (Fig. 3A). In contrast, PCAF was gradually recruited to the promoter only after 6 h of TPA treatment (Fig. 3A). A quantitative analysis was shown in Fig. 3B. As PCAF is known to interact with and acetylate MyoD (15Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), we next tested whether this event occurred in TPA-treated RD cells. We found that: 1) PCAF started to accumulate in the nucleus of RD cells after 6 h of TPA treatment (Fig. 3C, top panel); 2) PCAF directly interacts with MyoD, a key regulator of myogenin gene, and vice versa (Fig. 3C, second and third panels); and 3) MyoD was acetylated after 6 h of TPA treatment (Fig. 3C, bottom panel). These results suggested that PCAF is likely responsible for acetylation of both histones (e.g. H3-K9 and H3-K14) and MyoD in the early stage of TPA-induced RD cells. SWI/SNF Complex Is Recruited to the Myogenin Promoter at a Later Stage in TPA-treated RD Cells—To examine whether the TPA-induced expression of myogenin requires the participation of other chromatin remodeling molecules, we focused on the mammalian SWI/SNF complex as it has been implicated in myogenin gene expression (9de la Serna I.L. Ohkawa Y. Berkes C.A. Bergstrom D.A. Dacwag C.S. Tapscott S.J. Imbalzano A.N. Mol. Cell. Biol. 2005; 25: 3997-4009Crossref PubMed Scopus (223) Google Scholar, 10de la Serna I.L. Carlson K.A. Imbalzano A.N. Nat. Genet. 2001; 27: 187-190Crossref PubMed Scopus (277) Google Scholar). Although either BRG1 or Brm could function as the catalytic subunit of the mammalian SWI/SNF complex, only Brg1 was found to be present in RD cells (Fig. 4B, and data not shown). Expression constructs of the wild type BRG1 (wtBRG1), its dominant negative mutant (dnBRG1), and an empty vector were individually co-transfected with the myogenin reporter plasmid and pCMV-β-gal. Although the wtBRG1 slightly increased the TPA-induced myogenin promoter activity, the dominant-negative BRG1 drastically abolished the TPA induction of the gene to the basal level without affecting the constitutive promoter activity of the gene (Fig. 4A). By immunoprecipitation and Western blotting, we found that BAF60 physically interacted with Brg1 only after 12 h of TPA induction (Fig. 4B, top row). In addition, we showed by ChIP assays that BAF60 was efficiently recruited to the promoter region of myogenin in RD cells only after 12 h of TPA induction and that its levels reached a plateau after 24–48 h of TPA treatment (Fig. 4C). Our results suggested that Brg1 is recruited to the myogenin promoter via BAF60 and participates in regulation of the myogenin gene during TPA-induced RD cell differentiation. p38 Is Activated by TPA and Plays a Pivotal Role in TPA-induced Myogenin Expression—The p38 MAPK pathway plays an essential role in muscle cell differentiation (11Wu Z. Woodring P.J. Bhakta K.S. Tamura K. Wen F. Feramisco J.R. Karin M. Wang J.Y. Puri P.L. Mol. Cell. Biol. 2000; 20: 3951-3964Crossref PubMed Scopus (400) Google Scholar). Recently, it was found that p38 MAPK promotes myogenic differentiation by controlling the recruitment of the SWI/SNF complex to the myogenin promoter (12Simone C. Forcales S.V. Hill D.A. Imbalzano A.N. Latella L. Puri P.L. Nat. Genet. 2004; 36: 738-743Crossref PubMed Scopus (323) Google Scholar). To test whether p38 MAPK is also involved in TPA-induced myogenin gene expression, we employed SB203580 (SB), a specific inhibitor for p38 MAPK. We showed that SB blocked the TPA-induced myogenin expression in RD cells (Fig. 5A). In contrast, basal levels of myogenin expression were insensitive to SB203580. In addition, we found that the protein levels of p38 in both the nucleus and whole cell extracts were enhanced by TPA treatment. Most importantly, the levels of the dually phosphorylated p38 MAPK (i.e. the active form of p38) were also enhanced after 3 h of TPA treatment (Fig. 5B). Similar results were also obtained from analysis of the myogenin promoter activity in response to TPA treatment. We found that SB effectively reduced the TPA-induced myogenin activity back to the basal levels (Fig. 5C). In addition, the p38 antisense construct also completely repressed the TPA-induced promoter activity of myogenin to the basal levels in RD cells (Fig. 5D). In contrast, the wild-type p38 had no obvious effect. These results confirmed that p38 is indispensable for TPA-induced myogenin induction in RD cells. Because p38 is known to target the SWI/SNF complex to the myogenin promoter in normal myogenic differentiation, we next examined whether a similar mechanism also worked in TPA-induced RD cells. ChIP assays were performed with or without the p38 inhibitor SB. We found that recruitment of Brg1 to the myogenin promoter was detectable only after 12 h of TPA treatment (Fig. 5E). Importantly, the recruitment of Brg1 was completely dependent on p38 MAPK, in agreement with the findings by Simone et al. (12Simone C. Forcales S.V. Hill D.A. Imbalzano A.N. Latella L. Puri P.L. Nat. Genet. 2004; 36: 738-743Crossref PubMed Scopus (323) Google Scholar). In contrast, recruitment of PCAF in response to TPA induction was not affected by SB203580 treatment. The quantitative analysis was shown in Fig. 5E, FA and FB. We have shown that p300 constitutively associates with the myogenin promoter in RD cells independent of TPA treatment (Fig. 3A). In contrast, PCAF is recruited to the myogenin promoter in a TPA-inducible manner (Figs. 3A and 5D). As the binding of PCAF to the myogenin promoter correlates with the increase in acetylation of H3-K9, H3-K14, and MyoD (Figs. 2, A and B, and 3B), it suggests that PCAF is likely the HAT that acetylates both histones and MyoD at the early stage of TPA action. Although p300 is also capable of acetylating MyoD (21Polesskaya A. Duquet A. Naguibneva I. Weise C. Vervisch A. Bengal E. Hucho F. Robin P. Harel-Bellan A. J. Biol. Chem. 2000; 275: 34359-34364Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 22Polesskaya A. Naguibneva I. Fritsch L. Duquet A. Ait-Si-Ali S. Robin P. Vervisch A. Pritchard L.L. Cole P. Harel-Bellan A. EMBO J. 2001; 20: 6816-6825Crossref PubMed Scopus (101) Google Scholar), it is less likely responsible for MyoD acetylation in RD cells as both p300 and MyoD already associate with the myogenin promoter before TPA induction. However, MyoD is not acetylated until PCAF is recruited into the complex. As both p300 and PCAF can physically associate with each other (23Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar), it raises the interesting possibility that p300 facilitates TPA-induced PCAF recruitment to the myogenin promoter. Whether this is indeed the case remains to be further explored. In addition, it remains unclear how TPA induces PCAF recruitment to the myogenin promoter. Further investigation is needed to address this issue. Regarding the sequential recruitment of the SWI/SNF complex, our chromatin immunoprecipitation data clearly demonstrated that the mammalian SWI/SNF complex represented by Brg1 is also recruited to the myogenin promoter with another 6-h delay after PCAF recruitment. Presumably, an earlier recruitment of PCAF functions to acetylate both histones and MyoD, which facilitates subsequent recruitment of the SWI/SNF complex. This is consistent with a general mode of action for the SWI/SNF complex, as the SWI/SNF complex in different biological systems is normally recruited to local promoter regions after these regions are pre-acetylated by HATs (9de la Serna I.L. Ohkawa Y. Berkes C.A. Bergstrom D.A. Dacwag C.S. Tapscott S.J. Imbalzano A.N. Mol. Cell. Biol. 2005; 25: 3997-4009Crossref PubMed Scopus (223) Google Scholar). In the SWI/SNF complex, BAF60c appears to associate with the myogenin promoter earlier than the BRG1, as BAF60 can be detected to associate with the myogenin promoter as early as 6 h after TPA induction (Fig. 4C). In addition, we found that BAF60 directly interacts with MyoD (data not shown). Based on these data, we suggest that the SWI/SNF complex is recruited to the myogenin promoter via BAF60. Whether the interaction between MyoD and BAF60 is essential in the recruitment of SWI/SNF complex remains to be established. It has been reported that the p38 pathway in RD cells is defective, which could be responsible for the inability of the cells toward differentiation (11Wu Z. Woodring P.J. Bhakta K.S. Tamura K. Wen F. Feramisco J.R. Karin M. Wang J.Y. Puri P.L. Mol. Cell. Biol. 2000; 20: 3951-3964Crossref PubMed Scopus (400) Google Scholar, 24Puri P.L. Wu Z. Zhang P. Wood L.D. Bhakta K.S. Han J. Feramisco J.R. Karin M. Wang J.Y. Genes Dev. 2000; 14: 574-584PubMed Google Scholar). As the malfunction of defective p38 can be overcome by an ectopic constitutively active mutant of MKKK6 (7Zhang J.Z. Gao W. Yang H.B. Zhang B. Zhu Z.Y. Xue Y.F. Stem Cells. 2006; 24: 2661-2668Crossref PubMed Scopus (41) Google Scholar), which indicates target elements in the downstream of p38 are not involved in the defect differentiation of RD cells. By using SB to inhibit the activity of p38, we have found that TPA-induced promoter activity of myogenin is abolished, whereas the basal expression remain unaffected. Meanwhile, Brg1 recruitment at the promoter region of the myogenin gene is also repressed by SB after 12–24 h of treatment. These results indicate that p38 activity is essential for an open chromatin conformation on the TPA-induced expression of the myogenin gene. However, the direct target(s) of p38 MAPK in TPA-treated RD cells remains to be explored. Collectively, the timing for each individual regulator such as PCAF or BRG1 to bind to or modify the myogenin promoter occurred strictly at either 6 or 12 h of TPA treatment. We thus propose that there are two distinct activation steps for the induction of myogenin in the early stage of TPA-induced RD cell differentiation. First, an early step that requires PCAF activity to provide acetylation on core histones and MyoD to initiate myogenin gene expression. Second, a later oriented step, in which p38 dependent activity of the SWI/SNF remodeling complex is required to provide an open conformation for the induction of myogenin in the TPA-treated RD cells. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma. We thank Drs. K. L. Guan, A. N. Imbalzano, Gang Pei, and Qiao Li for providing antibodies and plasmids used in the paper.
The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated.Insertion of a mini-Mu molecule into certain sites in pUCl9 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition.Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type l-deficient mini-Mu molecules.Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends.This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction.Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.The transposition properties of the temperate bacteriophage Mu have been exploited by geneticists, molecular biologists, and biochemists.The ability of Mu to insert its DNA at a high frequency virtually anywhere in the chromosome of M. G.
Activation of the thermogenic brown and beige fat is an effective means to increasing whole‐body energy expenditure. In this work, a unique label‐free method was developed to quantitatively assess the metabolism and thermogenesis of mouse adipose tissues in vivo. Specifically, an optical redox ratio (ORR) based on the endogenous fluorescence of mitochondrial metabolic coenzymes (nicotinamide adenine dinucleotide and flavin adenine dinucleotide) was used to measure the metabolic state of adipocytes. Our findings demonstrate that the ORR provides a label‐free and real‐time biomarker to determine the thermogenic response of brown, beige and white adipose tissues to a variety of physiological stimulations. In addition, the redox ratio also can be used to evaluate the degree of browning in the white fat of cold‐acclimated mice. This technique is important to understand the recruitment and activation of thermogenic adipocytes in mammals and thus can help to develop therapeutic strategies against obesity.
Hibernation is an example of extreme hypometabolic behavior. How mammals achieve such a state of suspended animation remains unclear. Here we show that several strains of type 2 diabetic mice spontaneously enter into hibernation-like suspended animation (HLSA) in cold temperatures. Nondiabetic mice injected with ATP mimic the severe hypothermia analogous to that observed in diabetic mice. We identified that uric acid, an ATP metabolite, is a key molecular in the entry of HLSA. Uric acid binds to the Na+ binding pocket of the Na+/H+ exchanger protein and inhibits its activity, acidifying the cytoplasm and triggering a drop in metabolic rate. The suppression of uric acid biosynthesis blocks the occurrence of HLSA, and hyperuricemic mice induced by treatment with an uricase inhibitor can spontaneously enter into HLSA similar to that observed in type 2 diabetic mice. In rats and dogs, injection of ATP induces a reversible state of HLSA similar to that seen in mice. However, ATP injection fails to induce HLSA in pigs due to the lack of their ability to accumulate uric acid. Our results raise the possibility that nonhibernating mammals could spontaneously undergo HLSA upon accumulation of ATP metabolite, uric acid. Hibernation is an example of extreme hypometabolic behavior. How mammals achieve such a state of suspended animation remains unclear. Here we show that several strains of type 2 diabetic mice spontaneously enter into hibernation-like suspended animation (HLSA) in cold temperatures. Nondiabetic mice injected with ATP mimic the severe hypothermia analogous to that observed in diabetic mice. We identified that uric acid, an ATP metabolite, is a key molecular in the entry of HLSA. Uric acid binds to the Na+ binding pocket of the Na+/H+ exchanger protein and inhibits its activity, acidifying the cytoplasm and triggering a drop in metabolic rate. The suppression of uric acid biosynthesis blocks the occurrence of HLSA, and hyperuricemic mice induced by treatment with an uricase inhibitor can spontaneously enter into HLSA similar to that observed in type 2 diabetic mice. In rats and dogs, injection of ATP induces a reversible state of HLSA similar to that seen in mice. However, ATP injection fails to induce HLSA in pigs due to the lack of their ability to accumulate uric acid. Our results raise the possibility that nonhibernating mammals could spontaneously undergo HLSA upon accumulation of ATP metabolite, uric acid. Hibernation is a survival strategy characterized by dramatic decreases in body temperature, heart rate, respiratory rate, and metabolism rate (1Heldmaier G. Ortmann S. Elvert R. Natural hypometabolism during hibernation and daily torpor in mammals.Respir. Physiol. Neurobiol. 2004; 141: 317-329Crossref PubMed Scopus (372) Google Scholar). 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Henning R.H. Schmidt M. Reversible remodeling of lung tissue during hibernation in the Syrian hamster.J. Exp. Biol. 2011; 214: 1276-1282Crossref PubMed Scopus (38) Google Scholar) and are able to suppress the apoptotic response (5Rouble A.N. Hefler J. Mamady H. Storey K.B. Tessier S.N. Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation.PeerJ. 2013; 1: e29Crossref PubMed Scopus (50) Google Scholar), maintain an immunosuppressed state to prevent general inflammation in the body, and survive a long period with no sign of organ damage (6Tøien Ø. Drew K. Chao M. Rice M. Ascorbate dynamics and oxygen consumption during arousal from hibernation in Arctic ground squirrels.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2001; 281: R572-R583Crossref PubMed Google Scholar, 7Bouma H.R. Carey H.V. Kroese F.G. Hibernation: The immune system at rest?.J. Leukoc. Biol. 2010; 88: 619-624Crossref PubMed Scopus (161) Google Scholar). The realization of human hibernation would have critical clinical applications, including reduced organ damage and long-term preservation of patients (8Aslami H. Juffermans N.P. Induction of a hypometabolic state during critical illness - a new concept in the ICU?.Neth. J. Med. 2010; 68: 190-198PubMed Google Scholar, 9Asfar P. Radermacher P. Drug-induced "suspended animation": Can a dream become true?.Crit. Care Med. 2015; 43: 1528-1530Crossref PubMed Scopus (4) Google Scholar). Torpor and hibernation are traditionally defined as two different types of hypometabolic states under natural conditions (10Ruf T. Geiser F. Daily torpor and hibernation in birds and mammals.Biol. Rev. Camb. Philos. Soc. 2015; 90: 891-926Crossref PubMed Scopus (374) Google Scholar). Different from torpor, animals under hibernation display much lower body temperatures and metabolic rates (11Geiser F. Ruf T. Hibernation versus daily torpor in mammals and birds: Physiological variables and classification of torpor patterns.Physiol. Zoology. 1995; 68: 935-966Crossref Scopus (397) Google Scholar). A prominent physiologic and behavioral characteristic of hibernation is suspended animation, associated with tolerance to lethal metabolic rate reduction, bradycardia, and profound hypothermia (12Hartmann C. Nussbaum B. Calzia E. Radermacher P. Wepler M. Gaseous mediators and mitochondrial function: The future of pharmacologically induced suspended animation?.Front. Physiol. 2017; 8: 691Crossref PubMed Scopus (18) Google Scholar). Some chemical compounds have been used to induce a hibernation-like suspended animation (HLSA) in nonhibernating animals. Hydrogen sulfide (H2S) experimentally induces suspended animation in mice, and they will return to normal temperature after H2S removal (13Blackstone E. Morrison M. Roth M.B. 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In nature, the decline in body temperature is closely related to the adenosine signal of A1 receptor in the brain (16Jinka T.R. Tøien Ø. Drew K.L. Season primes the brain in an arctic hibernator to facilitate entrance into torpor mediated by adenosine A(1) receptors.J. Neurosci. 2011; 31: 10752-10758Crossref PubMed Scopus (76) Google Scholar). The central inhibition of A1 receptor in the brain reverses the temperature rise of ground squirrels during hibernation, indicating that adenosine could play an essential role in adjusting body temperature (16Jinka T.R. Tøien Ø. Drew K.L. Season primes the brain in an arctic hibernator to facilitate entrance into torpor mediated by adenosine A(1) receptors.J. Neurosci. 2011; 31: 10752-10758Crossref PubMed Scopus (76) Google Scholar). The physiological definition of hibernation is not yet clear. During natural hibernation, glucose metabolism via glycolysis seems to be strongly suppressed. Inhibition of glycolysis readily induces hypothermia in a classic hibernator, indicating that the underlying mechanism of hibernation could be metabolic suppression (17Stamper J.L. Dark J. Zucker I. Photoperiod modulates torpor and food intake in Siberian hamsters challenged with metabolic inhibitors.Physiol. Behav. 1999; 66: 113-118Crossref PubMed Scopus (25) Google Scholar). Indeed, the ability to replicate this naturally hibernating status has been achieved using metabolic inhibitors alone in rodents such as mice and rats (18Bouma H.R. Verhaag E.M. Otis J.P. Heldmaier G. Swoap S.J. Strijkstra A.M. Henning R.H. Carey H.V. Induction of torpor: Mimicking natural metabolic suppression for biomedical applications.J. Cell. Physiol. 2012; 227: 1285-1290Crossref PubMed Scopus (60) Google Scholar). Insulin resistance is significant in the fattening period of hibernating animals. During the prehibernation period, serum insulin levels are elevated and maintain a high level in the initial months of hibernation (19Buck M.J. Squire T.L. Andrews M.T. Coordinate expression of the PDK4 gene: A means of regulating fuel selection in a hibernating mammal.Physiol. Genomics. 2002; 8: 5-13Crossref PubMed Scopus (114) Google Scholar). The actions of chronically high insulin levels are usually associated with insulin resistance in type 2 diabetes (T2DM). Lack of insulin response has also been reported in hibernating dormice and hedgehogs (20Castex C. Tahri A. Hoo-Paris R. Sutter B.C. Glucose oxidation by adipose tissue of the edible dormouse (Glis glis) during hibernation and arousal: Effect of insulin.Comp. Biochem. Physiol. A Comp. Physiol. 1987; 88: 33-36Crossref PubMed Scopus (10) Google Scholar, 21Hoo-Paris R. Castex C. Sutter B.C. Plasma glucose and insulin in the hibernating hedgehog.Diabete Metab. 1978; 4: 13-18PubMed Google Scholar). These observations imply there may be some similar metabolic regulation mechanisms between type 2 diabetic mice and hibernating animals. Here, we found that several strains of type 2 diabetic mice spontaneously enter into a hibernation-like suspended animation in cold temperatures. Nondiabetic mice with ATP injection mimic the severe hypothermia state similar to that observed in the type 2 diabetic mice. We identified that the accumulation of uric acid from ATP metabolism is an indispensable step in the induction of HLSA. Uric acid is an inhibitor of Na+/H+ exchanger, which controls the pH homeostasis of cytoplasm and influences the activities of a series of metabolic enzymes. It is well known that uric acid accumulation is a common feature of animals in long-term food shortage or fasting (22Balasubramanian T. Uric acid or 1-methyl uric acid in the urinary bladder increases serum glucose, insulin, true triglyceride, and total cholesterol levels in Wistar rats.ScientificWorldJournal. 2003; 3: 930-936Crossref PubMed Scopus (13) Google Scholar, 23Robin J.-P. Boucontet L. Chillet P. Groscolas R. Behavioral changes in fasting emperor penguins: Evidence for a "refeeding signal" linked to a metabolic shift.Am. J. Physiol. Regul. Integr. Comp. Physiol. 1998; 274: R746-R753Crossref PubMed Google Scholar, 24Jenni L. Jenni-Eiermann S. Spina F. Schwabl H. Regulation of protein breakdown and adrenocortical response to stress in birds during migratory flight.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2000; 278: R1182-R1189Crossref PubMed Google Scholar, 25Wilhelmi de Toledo F. Grundler F. Goutzourelas N. Tekos F. Vassi E. Mesnage R. Kouretas D. Influence of long-term fasting on blood redox status in humans.Antioxidants. 2020; 9: 496Crossref Scopus (14) Google Scholar), and natural hibernation always commences when food is absent or limited (10Ruf T. Geiser F. Daily torpor and hibernation in birds and mammals.Biol. Rev. Camb. Philos. Soc. 2015; 90: 891-926Crossref PubMed Scopus (374) Google Scholar). Our results strongly suggest that uric-acid-regulated metabolic suppression reflects not only a drop of metabolic activity during the development of T2DM, but also associates the metabolic regulation mechanism of natural hibernation. Severe hypothermia observed during hibernation is a form of suspended animation (26Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature.Physiol. Rev. 2003; 83: 1153-1181Crossref PubMed Scopus (782) Google Scholar). Accumulated evidence indicates that metabolic suppression must be a key factor to achieve hibernation (26Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature.Physiol. Rev. 2003; 83: 1153-1181Crossref PubMed Scopus (782) Google Scholar). Hypometabolic behavior is also the main feature of T2DM. Therefore, we undertook a study to determine whether there were differential responses in body temperature (Tb) and locomotive activity while type 2 diabetic mice with severe metabolic stress were exposed to cold temperatures. Dramatically, the db/db diabetic mice displayed none of the thermoregulatory defenses when maintained in the environment with ambient temperatures (Ta) around the freezing point (0 °C). Tb measurement revealed a rapid decline accompanied by distinct behavioral responses with physical inactivity (Fig. 1A, left). About 1–3 h (stage I) after cold exposure, Tb of mice dropped to 18 ± 0.5 °C, and mice entered HLSA. In this stage, mice lost the righting reflex when placed on their backs or sides. The HLSA in type 2 diabetic db/db mice would last for several hours by adjusting the Ta of 17 ± 0.5 °C (stage II). Then, mice would arouse from HLSA spontaneously or be awakened when Ta was adjusted to more than 32 °C. This stage would last for about 2–3 h until the Tb of mice was close to 36 to 37 °C, and the moving activity was recovered (stage III). Simultaneously, the Tb in control mice was within the normal range. These differences were confirmed by surface thermal imaging (Fig. 1A, right). The same HLSA has also been observed in HFD-STZ diabetic mice, while chow-fed lean control mice maintained a relatively constant Tb (Fig. 1B). Similarly, diabetic ob/ob mice entered into HLSA in cold temperatures, while nondiabetic ob/ob mice tended to reduce Tb and failed to enter into HLSA (Fig. 1C). During the HLSA in diabetic ob/ob mice, the average heart rate and respiratory rate also declined to about 69 beats per minute and 18 times per minute, respectively (Table 1). Moreover, all groups of type 2 diabetic mice showed a severe decrease in spontaneous locomotive activity compared with control mice (Fig. 1, D–F). While oxygen consumption was relatively stable in control mice in cold temperatures, all type 2 diabetic mice decreased oxygen consumption accompanied by the drop in Tb (Fig. 1, G–I). These observations indicated that laboratory type 2 diabetic mouse models are capable of entering a suspended animation state similar to that observed in hibernators.Table 1Changes in heart rate and respiratory rate in nondiabetic ob/ob mice and diabetic ob/ob mice after cold treatmentGroupHeart rate (bpmaBeats per minute.)Respiratory rate (bpmbBreaths per minute.)Nondiabetic ob/ob (N = 6)473 ± 6685 ± 21Diabetic ob/ob (N = 6)69 ± 11∗∗18 ± 7∗∗Data were presented as mean ± SD (Student's t test: ∗∗p < 0.01).a Beats per minute.b Breaths per minute. Open table in a new tab Data were presented as mean ± SD (Student's t test: ∗∗p < 0.01). The above observations could be explained by a changed metabolite that acted as a metabolic repressor in type 2 diabetic mice. We reasoned that the putative metabolic repressor, when injected into wild-type mice under cold exposure, should induce HLSA as observed in diabetic mice. Then we investigated whether endogenous energy molecules in diabetic mice were different from those in nondiabetic mice. HPLC analysis revealed that ATP metabolites, including hypoxanthine, xanthine, and uric acid, were elevated in the plasma and livers of diabetic ob/ob mice compared with nondiabetic ob/ob mice (Fig. 2, A–D). We hypothesized that the changes in endogenous energy molecules must be a key factor to achieve HLSA in diabetic mice. Therefore, we undertook a study to determine whether there were differential responses in Tb while administrating endogenous energy molecules in animals. The wild-type mice were given adenosine triphosphate (ATP 2Na salt) and then maintained at 4 °C of Ta. They displayed no thermoregulatory defenses as observed in diabetic mice under cold exposure. Tb measurement revealed a rapid decline accompanied by distinct physical inactivity (Fig. 2E). About 30 min (stage I) after ATP injection, Tb dropped to 16 ± 0.5 °C, and mice entered the HLSA (stage II). Tb drop caused by ATP was clearly detected with an infrared thermometer (Fig. 2F), and a severe decrease of locomotive activity compared with control mice was measured by a radio frequency receiver platform (Fig. 2G). HLSA would last for about 6–8 h. Then, mice would arouse from HLSA spontaneously or be awakened when Ta was adjusted to 25 °C, and the moving activity was recovered (stage III). The changes in oxygen consumption corresponded with the major decline in Tb (Fig. 2H). During the HLSA in mice, the average heart rate and respiratory rate also declined to about 85 beats per minute and 13 times per minute, respectively (Table 2). The concentrations of ATP are important for entering HLSA. With the increase of ATP dosage, the entry time of HLSA was shortened (Fig. 2I). Together, these studies showed that ATP is a signaling molecule that can activate HLSA, which is used as a mechanism for energy conservation.Table 2Changes in heart rate and respiratory rate in control mice and mice under HLSA stateGroupHeart rate (bpmaBeats per minute.)Respiratory rate (bpmbBreaths per minute.)Control (N = 10)552 ± 8791 ± 13HLSA (N = 10)85 ± 8∗∗13 ± 5∗∗Data were presented as mean ± SD (Student's t test: ∗∗p < 0.01).a Beats per minute.b Breaths per minute. Open table in a new tab Data were presented as mean ± SD (Student's t test: ∗∗p < 0.01). Next, we compared the effects of ATP and its downstream products, AMP and adenosine, on the induction of hypothermia. At a Ta of about 4 °C, ATP induced 100% of mice to HLSA, while AMP and adenosine were about 80% and 60%, respectively (Fig. 3A). The HLSA entry time of ATP was shorter than that of AMP and adenosine (Fig. 3B). In order to determine whether the specific role of ATP was performed by its receptor P2X or P2Y, we used the antagonists of P2X and P2Y. While P2X antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) had no effect on the induction of ATP, the antagonist of P2Y receptor suramin significantly prolonged the induction period of ATP (Fig. 3C). However, no matter how high the dose of suramin is, it could not completely block mice from ATP-induced HLSA. Plasma ATP can be rapidly degraded to ADP, AMP, and then adenosine. To investigate whether ATP induces HLSA via the adenosine receptors, ATP was administered to mice deficient in A1, A2a, A2b, and A3 adenosine receptors, respectively. Figure 3D showed that ATP induced HLSA in all adenosine receptor knockout mice. Next, we detected that the metabolites of ATP were elevated in the plasma and livers of ATP-treated mice at 1 h after the injection. As observed in type 2 diabetic mice, ATP led to the accumulation of a large number of intermediate and terminal metabolites, including hypoxanthine, xanthine, and uric acid, in the plasma and livers of mice (Fig. 3, E–H). These data implied that the accumulations of ATP metabolites might play a role in ATP-induced HLSA. Because ATP-induced hypothermia is immediate, we reasoned that ATP action in this process was very fast, and it could not be achieved through complex regulation of gene and protein expression, although some of them may have a rapid response. Then, we investigated whether exogenous nucleotides immediately changed the metabolic environment, such as intracellular pH. Figure 4A showed representative pseudo-colored ratio images of intracellular pH value. The results revealed that ATP metabolites, including adenosine, hypoxanthine, xanthine, and uric acid, acidified cytoplasm with different efficiency, and uric acid is the most effective for cytoplasm acidification. Then, several metabolic enzymes would be impaired or lost their catalytic activity. Glucokinase and phosphofructokinase are two rate-limiting enzymes of glycolysis. The activities of these enzymes decreased with the decline of pH (Fig. 4, B and C). Fluorescence intensity assay revealed that glucose uptake was also significantly impaired with uric acid incubation in cultural cells (Fig. 4, D and E). Since uric acid is the most effective inducer of intracellular acidification and the elevation in UA level and the decrease in body temperature occurred simultaneously (Fig. S1), we investigated whether the inhibition of uric acid accumulation influenced HLSA in mice. Xanthine oxidase is required to participate in the biosynthesis of uric acid. Surprisingly, when mice were pretreated with the inhibitors of xanthine oxidase, febuxostat, or allopurinol, ATP failed to induce mice to HLSA (Fig. 4, F and G), nor could diabetic db/db mice (Fig. S2). These pretreated diabetic mice lost the ability to enter stage II and recovered to normal Tb or died within 1–2 h during stage I. Moreover, mice pretreated with a uricase inhibitor potassium oxonate for 7 days developed hyperuricemia and could spontaneously enter into HLSA under cold exposure (Fig. S3). These studies indicated that uric acid suppresses metabolic rate by acidifying cytoplasm, and uric acid is indispensable during ATP-induced HLSA in mice. To identify the target of uric acid acidifying cell, the relationship between uric acid and the Na+/H+ exchanger activity was investigated. The Na+/H+ exchanger is a key regulator of cellular pH homeostasis. Interestingly, uric acid inhibited the activity of Na+/H+ exchangers in a dose-dependent manner. (Fig. 5, A–C). Na+/H+ exchanger 1 (NHE1) is one of the most important isoforms of Na+/H+ exchanger and is ubiquitously distributed throughout the plasma membrane of virtually all tissues. We investigated whether uric acid could interact with NHE1 proteins. The structure of the transmembrane segments of NHE1 was predicted according to Phyre2 homology modeling portal. We then conducted the molecular docking by AutoDock4.2 software. According to the molecular docking prediction, uric acid displayed a high binding affinity to the extracellular Na+-binding site of NHE1 protein, and the crucial amino acids involved in the NHE1-uric acid binding are SER161, PHE164, PHE165, and PHE467 (Fig. 5D). Then, knockout of the possible uric acid-binding domain (15 amino acid residues from position 156–170) was implemented by overlap extension PCR (Fig. 5E). A microscale thermophoresis assay verified that uric acid was able to bind to NHE1 (Fig. 5F). Nontransfected NIH3T3 cells were used to verify the absence of binding with a nonrelevant mCherry-fused protein, which was undetectable in undiluted lysates. The binding between NHE1 and uric acid was virtually abolished when this putative binding region was mutated (Fig. 5G). Moreover, in the AML12 cells transfected with specific siRNA targeting NHE1, the initial pHi value was decreased, and the decrease of pHi triggered by uric acid was dampened (Fig. 5H, upper panel). The sequence-optimized NHE1-WT constructs, but not NHE1-mutant, rescued the effects of intracellular acidification reduced by NHE1-siRNA (Fig. 5H, middle and lower panels). Interestingly, the wild-type mice injected intravenously with zoniporide hydrochloride hydrate, a selective inhibitor of NHE1, were successfully induced into HLSA (Fig. S4). Together, these studies indicated that the primary role of uric acid is as an endogenous inhibitor of Na+/H+ exchanger. To determine whether the mice under HLSA or recovery from HLSA (R-HLSA) have systemic inflammation or organ damages, we evaluated plasma levels of MMP-1, IL-1β, and CRP by ELISA. There were no statistically significant differences in MMP-1, IL-1β, and CRP levels between the HLSA, R-HLSA, and control groups (Fig. 6A, upper left panel). In order to assess the heart, kidney, and liver functions, we measured the plasma levels of creatine kinase-MB (CK-MB), creatinine, blood urea nitrogen (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in all groups. Using plasma CK-MB as a marker of heart injury, we observed no significant increase in plasma CK-MB in the HLSA and R-HLSA groups compared with the control group (Fig. 6A, upper right panel). The levels of BUN and creatinine in plasma increased slightly in HLSA and dropped to the normal range in R-HLSA (Fig. 6A, lower left panel). It should be noted that the HLSA-mediated elevations in plasma AST and ALT compared with the control mice were small and likely represented no damage to liver tissue (Fig. 6A, lower right panel). Measurements of water and food consumption were performed 3 days before and 3 days after a single ATP-induced HLSA, respectively, and there was virtually no change (Fig. 6B, upper panel). Then, we utilized metabolomics to investigate the metabolic changes in the circulation and main organs during and after HLSA. The PCA analysis was performed on the metabolic profiles of the control, HLSA, and R-HLSA groups. In the PCA score plots from the plasma, liver, kidney, and brain (Fig. 6B, lower panel), the HLSA group was well separated from the control group, while the R-HLSA group and control group clustered together, revealing that with the recovery of Tb, mice can quickly recover from HLSA-induced metabolic disturbance without apparent damage. The corresponding coefficient-coded loading plots (Figs. S5–S8) also elucidated that mice could quickly recover from HLSA-induced metabolic disturbance to their normal status. To observe the influence of the ATP-induced HLSA on the immune system, flow cytometric analysis of the draining lymph nodes and spleens was performed after mice underwent HLSA once a day for 10 consecutive days. There was no change in the percentages of immature T cells in the lymph nodes (Fig. 6C) and the spleens (Fig. 6D). Together, these results demonstrated that ATP-induced HLSA has no systemic inflammation or organ damage. Next, we investigated the effects of ATP in other mammals. Rats were injected with ATP and then maintained at a Ta of about 4 °C. The behavior response of rats during HLSA was similar to that observed in mice. However, the time of entering HLSA is about 2.5–3 h (stage I) in rats, compared with 0.5 h in mice (Fig. 7A). The Tb of rats in HLSA was close to 16 °C, as same as that of mice. Then, we transferred these rats to a Ta of about 15 °C. The rats would keep HLSA for 6–8 h (stage II). The rats were then awakened by being moved to a Ta of 25 °C. After 3–4 h (stage III), the Tb of the rats would recover to close to 37 °C (Fig. 7B). The time of stage III in mice is about 2–3 h. The rats in stage II could also awaken by elevating Ta at any required time. For dogs, they must be shaved off prior to ATP injection. Shaved dogs were injected with ATP and maintained at a Ta of about 4 °C (Fig. 7C). After 1.5–2 h (stage I), the dogs would enter the HLSA similar to that observed in rats and mice. The Tb of dogs in HLSA was about 18–20 °C. Then, we transferred the dogs to a Ta of 10 °C. The dogs could maintain HLSA for 8–10 h (stage II). The dogs during stage II could not spontaneously arouse at Ta of 10 °C. However, while the dogs were transferred to a Ta of 25–30 °C, their Tb would recover to about 37 °C within 8–10 h (stage III, Table S1). Especially, when injected with ATP (0.5 μmol/g BW) intraperitoneally at 4 °C, the pigs only reduced their Tb to 32–33 °C and remained active for several hours (Fig. 7D). Pigs injected with ATP (1 μmol/g BW) at 4 °C would die within 8–12 h. ATP could not induce pig to HLSA. Type 2 diabetes and hibernation are two different pathological and physiological phenomena. However, both of them have a common metabolic characteristic that glucose utilization is seriously inhibited. Hibernators are considered a remarkable model for reversible insulin resistance (27Wu C.W. Biggar K.K. Storey K.B. Biochemical adaptations of mammalian hibernation: Exploring squirrels as a perspective model for naturally induced reversible insulin resistance.Braz. J. Med. Biol. Res. 2013; 46: 1-13Crossref PubMed Scopus (29) Google Scholar). Many hibernators naturally undergo a massive increase in body fat storage before the hibernation season and, to a large extent, rely on triglycerides in white adipose tissue as an energy source in winter (28Geiser F. Hibernation.Curr. Biol. 2013; 23: R188-193Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29Storey K.B. Out cold: Biochemical regulation of mammalian hibernation - a mini-review.Gerontology. 2010; 56: 220-230Crossref PubMed Scopus (136) Google Scholar). For fat-storing hibernators, reduced metabolic rate and body temperature are also accompanied by shifts of fuel utilization and energy-generating pathways from the utilization of carbohydrates to the utilization of fat (26Carey H.V. Andrews M.T. Martin S.L. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature.Physiol. Rev. 2003; 83: 1153-1181Crossref PubMed Scopus (782
The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.
Background: Serial analysis of cellular dynamics over time offers new insights into human skin responses to solar radiation. However, most of the previous studies are based on biopsy ex vivo analysis approaches that preclude the monitoring of the same cells and sites over time. Optical in vivo microscopy enables the possibility of real-time live cell imaging. Here we report a robust non-invasive method to achieve repeated access to the same micro-location over a long period with unprecedented precision.
Methods: The technique is based on a temporary “surface marker” as landmark to help locate the same cells or microstructures between imaging sessions. At baseline, the region-of-interest (ROI) is determined and imaged. At follow up sessions, the ROI can be automatically located. Using this method, we precisely revisited the same cells in human skin after UVB radiation over two weeks. Skin microscopic responses was studied with a multimodality in vivo microscopy system capable of co-registered video rate reflectance confocal microscopy (RCM) imaging, two-photon fluorescence (TPF) imaging and second harmonic generation (SHG) imaging.
Results: The quantitative analysis of TPF signal revealed that melanin distribution pattern changed with time after UVB exposure, suggesting that melanin migrates towards the skin surface. Blood flow was monitored in the same capillary over two weeks. Multimodal analyses enabled accurate calculation of viable epidermis, stratum corneum thickness and cell density variations over time, demonstrating the time points of tissue edema and cell proliferation.
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