SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1–/– mkk7–/– ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1–/– ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1–/– mkk7–/– ES cells. Instead, interleukin-1β (IL-1β)-induced IL-6 gene expression was greatly suppressed in sek1–/– mkk7–/– fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages. SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1–/– mkk7–/– ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1–/– ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1–/– mkk7–/– ES cells. Instead, interleukin-1β (IL-1β)-induced IL-6 gene expression was greatly suppressed in sek1–/– mkk7–/– fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages. Apoptosis or programmed cell death is critical for many biological events such as embryonic development, immune responses, and tissue homeostasis in multicellular organisms. In mammalian cells, apoptotic signaling cascades can be divided into two broad categories: the intrinsic (mitochondria-dependent) and the extrinsic (death receptor-mediated) pathways. The initiation of mitochondria-dependent pathway requires a change in the organella membrane permeability that is prevented by anti-apoptotic molecules such as Bcl-2 and Bcl-XL and promoted by pro-apoptotic molecules including Bax and Bak, and the permeability change results in the release of mitochondrial proteins. One of the released proteins, cytochrome c, associates with Apaf1 and caspase 9 to activate the effector caspase 3 (1.Peng L. Deepak N. Imawati B. Srinivasa M.S. Manzoor A. Emad S.A. Xiaodong W. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6219) Google Scholar, 2.Orrenius S. Zhivotovsky B. Nicotera P. Nature Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2406) Google Scholar). Cellular stresses such as UV irradiation and heat shock mediate apoptosis through the mitochondria-dependent pathway (3.Yoshida H. Kong Y.-Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (997) Google Scholar). However, upstream signaling that regulates the pro-apoptotic molecules remains to be elucidated. Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), 1The abbreviations used are: SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase; ES, embryonic stem; MEF, mouse embryonic fibroblast; IL, interleukin; TNF-α, tumor necrosis factor α; E, embryonic day; ERK, extracellular signal-regulated kinase.1The abbreviations used are: SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase; ES, embryonic stem; MEF, mouse embryonic fibroblast; IL, interleukin; TNF-α, tumor necrosis factor α; E, embryonic day; ERK, extracellular signal-regulated kinase. which belongs to the family of mitogen-activated protein kinase (MAPK), is activated not only by many types of cellular stresses including UV irradiation, heat shock, cisplatinum, etoposide, thapsigargin, and tunicamycin but also by inflammatory cytokines, interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α). The activated SAPK/JNK phosphorylates a number of substrates including the c-Jun component of the activator protein-1 transcription factor to regulate gene expression for the stress responses. Activation of SAPK/JNK requires the dual phosphorylation of Tyr and Thr residues located in a Thr-Pro-Tyr motif of the MAPK, and two kinases, SEK1 (also known as MKK4) and MKK7 (SEK2), are responsible for the phosphorylation (4.Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3633) Google Scholar, 5.Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4382) Google Scholar, 6.Manning A.M. Davis R.J. Nature Rev. Mol. Drug Disc. 2003; 2: 554-565Crossref PubMed Scopus (535) Google Scholar). SEK1 and MKK7 preferentially phosphorylate the Tyr and Thr residues of SAPK/JNK, respectively (7.Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar). Interestingly, the Tyr phosphorylation by SEK1 is sequentially followed by the Thr phosphorylation by MKK7 in stress-stimulated mouse embryonic stem (ES) cells (8.Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9.Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Targeted gene-disruption experiments in mice demonstrate that both SEK1 and MKK7 are required for embryonic development. Sek1–/– embryos die between embryonic day 10.5 (E10.5) and E12.5 with impaired liver formation and massive apoptosis (10.Ganiatsas S. Kwee L. Fujiwara Y. Perkins A. Ikeda T. Labow M.A. Zon L.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6881-6886Crossref PubMed Scopus (177) Google Scholar, 11.Nishina H. Vaz C. Billia P. Nghiem M. Sasaki T. Pompa J.L. Furlonger K. Paige C. Hui C.-C. Fischer K.D. Kishimoto H. Iwatsubo T. Katada T. Woodgett J.R. Penninger J.M. Development. 1999; 126: 505-516Crossref PubMed Google Scholar). We have recently shown that SEK1-mediated SAPK/JNK pathway downstream TNF-α receptor 1 participates in embryonic hepatoblast proliferation and survival via a pathway different from NF-κB-induced anti-apoptosis. On the other hand, mkk7–/– embryos die between E11.5 and 12.5 with similar defects in liver formation. These results indicate that SAPK/JNK activation mediated through SEK1 plus MKK7 plays indispensable roles in hepatoblast proliferation and survival during mouse embryogenesis (12.Watanabe T. Nakagawa K. Ohata S. Kitagawa D. Nishitai G. Seo J. Tanemura S. Shimizu N. Kishimoto H. Wada T. Aoki J. Arai H. Iwatsubo T. Mochita M. Watanabe T. Satake M. Ito Y. Matsuyama T. Mak T.W. Penninger J.M. Nishina H. Katada T. Dev. Biol. 2002; 250: 332-347Crossref PubMed Google Scholar). However, the physiological role of SAPK/JNK activation in cell survival and apoptosis is still controversial, being suggested to have an anti-apoptotic, pro-apoptotic, or no function in these processes (13.Lin A. BioEssays. 2002; 25: 17-24Crossref Scopus (369) Google Scholar). Mice lacking both JNK1 and JNK2 (Jnk1–/– Jnk2–/–) die around E11 with defective neural tube morphogenesis and altered apoptosis (14.Kuan C.-Y. Yang D.D. Roy D.R.S. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 15.Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (301) Google Scholar). These results assign both pro- and anti-apoptotic functions to JNK1 and JNK2 in the development. It also appears that the SAPK/JNK pathway functions in a manner dependent on the types of cells and stimulus, and its different components can sometimes play opposing roles in apoptosis. The most convincing evidence to date that shows that the involvement of SAPK/JNK activation in pro-apoptotic function comes from Jnk1–/– Jnk2–/– and mkk4–/– mkk7–/– mouse embryonic fibroblasts (MEFs). Both Jnk1–/– Jnk2–/– and mkk4–/– mkk7–/– MEFs exhibited profound defects in stress-induced apoptosis (16.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar, 17.Tournier C. Dong C. Turner T.K. Jones S.N. Flavell R.A. Davis R.J. Genes Dev. 2001; 15: 1419-1426Crossref PubMed Scopus (301) Google Scholar). Furthermore, it has been reported that active JNK causes the release of apoptogenic factors such as cytochrome c and Smac from isolated mitochondria in a cell-free system (18.Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 19.Chauhan D. Li G. Hideshima T. Podar K. Mitsiades C. Mitsiades N. Munshi N. Kharbanda S. Anderson K.C. J. Biol. Chem. 2003; 278: 17593-17596Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). These results strongly indicate that the SAPK/JNK activation directly regulates mitochondria-dependent apoptosis in pro-apoptotic direction. To evaluate the exact role of SAPK/JNK activation in mitochondria-dependent apoptosis, we here utilized mouse ES cells in terms of the following advantages. 1) ES cells are a prototype of all cell lineages and can be differentiated into MEF-like cells with retinoic acid. 2) ES cells do not express death receptors including Fas and TNF-α receptor 1 but have stress-induced mitochondria-dependent apoptotic pathway. 3) The molecular mechanism of SAPK/JNK activation is well characterized in ES cells. The present results clearly show that SAPK/JNK activation is not required for the stress-induced mitochondria-dependent apoptosis in ES and MEF-like cells. Instead, we found that IL-1-induced IL-6 gene expression was greatly impaired in MEF derived from sek1–/– mkk7–/– ES cells. Cell Culture and Materials—The murine ES cell line E14K (wild type) was maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum and leukemia inhibitory factor as described previously (20.Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar). The generation of apaf1–/– ES cells was described previously (3.Yoshida H. Kong Y.-Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (997) Google Scholar). Sek1–/– mkk7–/– ES cells were newly generated as described in Fig. 2. MEF-like cells are prepared from ES cells by culture with 10 μm retinoic acid for 14 days without leukemia inhibitory factor. For thermotolerance, cells were incubated at 44 °C for 10 min and further cultured for 6 h. Antibodies against SAPK/JNK1 (C-17 and FL), p38 (C-20), ERK2, and Bax were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-SAPK/JNK (number 9251) and anti-phospho-p38 (number 9211) antibodies were from New England BioLabs, Inc. Anti-mitochondrial heat shock protein 70 was from Affinity BioReagents. Rat anti-SEK1 (KN-001) and anti-MKK7 (KN-004) antibodies applicable to immunoprecipitation and immunoblotting were prepared in our laboratory (8.Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). SB203580 and SP600125 were from Calbiochem and Biomol, respectively. Adenovirus-encoding Cre recombinase (number 1748) was from DNA Bank, BioResource Center, RIKEN (Ibaraki, Japan) (21.Kanegae Y. Lee G. Sato Y. Tanaka M. Nakai M. Sasaki T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar). Generation of ES Cells Lacking Both SEK1 and MKK7—sek1 and mkk7 genes were disrupted as shown in Fig. 2a. Steps 1 and 2 were done by using a sek1 (neomycin)-targeting vector and gene-dosage effect, respectively (20.Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar). Steps 3 and 4 were performed by using a mkk7 (loxP-hygromycin)-targeting vector and an adenovirus-encoding Cre recombinase, respectively (9.Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21.Kanegae Y. Lee G. Sato Y. Tanaka M. Nakai M. Sasaki T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar). Step 5 was done by using a novel mkk7 (puromycin)-targeting vector as shown in Fig. 2a. Assay of SAPK/JNK Activity—ES cells were plated at 1.5 × 106 cells/35-mm dish and cultured for overnight. The cells were stimulated by anisomycin (Sigma, 5 μg/ml), cisplatinum (Sigma, 50 μm), etoposide (Sigma, 2 μg/ml), thapsigargin (Sigma, 200 nm), tunicamycin (Sigma, 10 μg/ml), UV (1 kJ/m2, UV Stratalinker 1800, Stratagene), and heat shock (44 °C for 20 min). SAPK/JNK proteins were immunoprecipitated at 4 °C for 2 h using the anti-SAPK/JNK antibody (C-17, Santa Cruz Biotechnology, Inc.). The SAPK/JNK activity in the precipitated fractions was measured with glutathione S-transferase-c-Jun as an in vitro substrate in the presence of 60 μm [γ-32P]ATP (8.Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9.Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The amount of the precipitated SAPK/JNK that had been monitored by immunoblotting with the anti-SAPK/JNK (FL) antibody was almost constant in a series of the present experiments. Immunoprecipitation and Immunoblotting—ES cells (2 × 106 cells) were suspended at 4 °C in 0.2 ml of a lysis buffer consisting of 1% Nonidet P-40, 80 mm Tris-HCl (pH 7.5), 10 mm EDTA, and 4 μg/ml aprotinin. The cell lysates were incubated for 30 min and centrifuged at 15,000 rpm for 15 min. The supernatants were analyzed by SDS-PAGE and immunoblotting. Proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) and probed with antibodies against anti-SAPK/JNK1, anti-ERK2, anti-p38, and anti-phospho-SAPK/JNK. The bands were visualized by SuperSignal West Pico chemiluminescent substrate for the development of immunoblots utilizing a horseradish peroxidase-conjugated second antibody according to the manufacturer's instructions (Pierce). For detection of phospho-p38, cells were lysed directly in Laemmli sample buffer and sonicated by Ultrasonic Disruptor (TOMY) followed by SDS-PAGE and immunoblotting. Endogenous SEK1 and MKK7 were immunoprecipitated with anti-SEK1 (KN-001) and anti-MKK7 (KN-004) and detected with anti-SEK1 (C-20) and anti-MKK7 (T-19) antibodies, respectively (8.Wada T. Nakagawa K. Watanabe T. Nishitai G. Seo J. Kishimoto H. Kitagawa D. Sasaki T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2001; 276: 30892-30897Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9.Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). DNA Fragmentation Assay—ES cells (2 × 106 cells), which had been attached to and detached from culture dishes after stimuli, were collected by means of incubation with trypsin/EDTA and centrifugation, respectively. The cells were mixed, washed twice with phosphate-buffered saline, and collected by centrifugation. After removing the supernatants, the cells were lysed in 0.33 ml of a buffer consisting of 5 mm Tris-HCl (pH 7.4), 20 mm EDTA, and 0.5% Triton X-100. After centrifugation at 27,000 × g for 20 min at 4 °C to remove nuclei and insoluble fraction, the cell lysates were subjected to phenol extraction and ethanol precipitation for DNA purification. The precipitated DNA was suspended in 20 μl of H2O and treated with 50 μg/ml RNase for 30 min at 37 °C. The DNA samples (10 μl) were subjected to electrophoresis on 2% agarose gels and visualized by a UV illuminator (22.Kitagawa D. Tanemura S. Ohata S. Shimizu N. Seo J. Nishitai G. Watanabe T. Nakagawa K. Kishimoto H. Wada T. Tezuka T. Yamamoto T. Nishina H. Katada T. J. Biol. Chem. 2002; 277: 366-371Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Assay of Caspase 3 Activity—ES cells were harvested as described above and washed twice with phosphate-buffered saline. The cells, after being resuspended in 50 μl of a buffer consisting of 10 mm Tris-HCl (pH 7.5), 5 mm dithiothreitol, and 1 mm phenylmethanesulfonyl fluoride, were frozen in liquid nitrogen and thawed at 37 °C three times. The cell lysates (50 μg) were incubated at 37 °C for 1 h with 20 μm acetyl-Asp-Glu-Val-Asp α-(4-methyl-coumaryl-7-amide) (DEVD-MCA; Peptide Institute, Inc.) in the final volume of 50 μl in 20 mm Hepes-NaOH (pH 7.4), 2 mm dithiothreitol, and 10% glycerol. The reaction was terminated by adding 450 μl of ice-cold H2O, and substrate cleavage leading to the release of free MCA was monitored (excitation 355 nm and emission 460 nm) at room temperature (22.Kitagawa D. Tanemura S. Ohata S. Shimizu N. Seo J. Nishitai G. Watanabe T. Nakagawa K. Kishimoto H. Wada T. Tezuka T. Yamamoto T. Nishina H. Katada T. J. Biol. Chem. 2002; 277: 366-371Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Sub-G1 Assay—For flow-cytometric analysis, cells were first fixed with 70% ethanol and further incubated with 10 μg/ml RNase A at 37 °C for 1 h. The cells were stained in a solution (50 μg of propidium iodide/ml, 0.1% sodium citrate, and 0.1% Nonidet P-40) for 30 min at 4 °C. The apoptotic sub-G1 population was determined by FACScan flow cytometer (BD Biosciences) with Cell Quest software (23.Nishina H. Bachmann M. Oliveira-dos-Santos A.J. Kozieradzki I. Fischer K.D. Odermatt B. Wakeham A. Shahinian A. Takimoto H. Bernstein A. Mak T.W. Woodgett J.R. Ohashi P.S. Penninger J.M. J. Exp. Med. 1997; 186: 941-953Crossref PubMed Scopus (116) Google Scholar). Subcellular Fractionation—ES cells (1 × 107 cells) were harvested by cell scraper (SUMILON) and washed twice with phosphate-buffered saline. The cells, after being resuspended in 100 μl of buffer A consisting of 250 mm sucrose, 20 mm HEPES-KOH (pH 7.4), 10 mm KCl, 1.5 mm Na-EGTA, 1.5 mm Na-EDTA, 1 mm MgCl2, 1 mm dithiothreitol, and 2 μg/ml aprotinin, were homogenized 10 strokes by a 27-gauge syringe. After homogenization, cells were centrifuged at 600 × g for 10 min at 4 °C to remove nuclei, unbroken cells, and large debris. Supernatants containing mitochondria were transferred to a new tube and further centrifuged at 10,000 × g for 10 min at 4 °C. Mitochondrial pellets were washed in 100 μl of buffer A followed by centrifugation at 10,000 × g for 10 min at 4 °C and lysed in 1.5× Laemmli sample buffer. The samples (10 μl) were analyzed by SDS-PAGE, and immunoblotting was probed with the anti-Bax and mitochondrial heat shock protein 70 antibodies. Northern Blotting—Total RNA was prepared from differentiated ES cells by TRIzol reagent (Invitrogen), separated by formamide agarose gel, and transferred to a Hybond-XL (Amersham Biosciences). IL-6 and β-actin were detected by Northern blotting using the specific DNA probes. The cDNA fragments corresponding to mouse IL-6 and β-actin were amplified using the following primers: IL-6, 5′-ATG AAG TTC CTC TCT GCA AGA GAC T-3′ and 5′-CAC TAG GTT TGC CGA GTA GAT CTC-3′, and β-actin, 5′-CAT CAC TAT TGG CAA CGA GC-3′ and 5′-ACG CAG CTC AGT AAC AGT CC-3′. All of the experiments were repeated at least three times with different batches of the cell samples, and the results were fully reproducible. Hence, most of the data shown are representative of several independent experiments. Various Stresses Induce Apoptosis through Mitochondria in Mouse ES Cells—We first investigated the thermotolerance effect on apoptosis in ES cells, which protects cells against successive stress by the pretreatment of mild heat shock. For this mechanism, a recent study revealed that HSP70 and HSP27 induced by thermotolerance suppress mitochondria-dependent apoptosis by directly associating with Apaf1 and cytochrome c and blocking the assembly of a functional apoptosome (24.Beere H.M. Wolf B.B. Cain K. Mosser D.D. Mahboubi A. Kuwana T. Tailor P. Morimoto R.I. Cohen G.M. Green D.R. Nat. Cell Biol. 2000; 2: 469-475Crossref PubMed Scopus (1284) Google Scholar, 25.Saleh A. Srinivasula S.M. Balkir L. Robbins P.D. Alnemri E.S. Nat. Cell Biol. 2000; 2: 476-483Crossref PubMed Scopus (734) Google Scholar, 26.Bruey J.-M. Ducasse C. Bonniaud P. Ravagnant L. Susin S.A. Diaz-Latoud C. Gurbuxani S. Arrigo A.-P. Kroemer G. Solary E. Garrido C. Nat. Cell Biol. 2000; 2: 645-652Crossref PubMed Scopus (832) Google Scholar). As shown in Fig. 1, various stresses could induce DNA ladder formation (Fig. 1a) and caspase 3 activation, which was measured by DEVD cleavage (Fig. 1b) in wild-type ES cells. However, these apoptotic responses were markedly attenuated by the thermotolerance treatment. To confirm the involvement of Apaf1 in the stress-induced apoptosis, ES cells lacking Apaf1 (apaf1–/–) (3.Yoshida H. Kong Y.-Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (997) Google Scholar) were subjected to the same experiments. As shown in Fig. 1c, apoptosis in response to various stresses including heat shock, UV, cisplatinum, etoposide, thapsigargin, and tunicamycin was almost completely blocked in apaf1–/– ES cells. These results clearly show that the stress-induced apoptosis is mediated through mitochondria in ES cells. Stress-induced SAPK/JNK Activation Is Impaired in ES Cells Lacking both MKK4 and MKK7—To examine the role of SAPK/JNK activation in the stress-induced and mitochondria-dependent apoptosis, ES cells lacking both MKK4 and MKK7 were generated. For generation of the null mutant, we constructed a novel mkk7-targeting vector containing puromycin-resistant cassette (Fig. 2a), and disrupted sek1 and mkk7 genes as described previously (9.Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20.Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar). The mutant sek1(neo/neo) mkk7(puro/del) clones thus generated lack both SEK1 and MKK7 completely (Fig. 2b), and these clones are used as sek1–/– mkk7–/– ES cells in this study. As shown in Fig. 2c, not only stress-induced activation but also the basal level of SAPK/JNK was completely lost in sek1–/– mkk7–/– ES cells. Stress-induced Apoptosis Is Still Observed in sek1–/– mkk7–/– ES Cells—To examine the SAPK/JNK-inactivation effect, various apoptotic responses including caspase 3 activation, DNA fragmentation, and sub-G1 population were further analyzed in sek1–/– mkk7–/– ES cells in comparison with wild-type and apaf1–/– ES cells (Fig. 3). Surprisingly, the apoptotic responses were still observed clearly in sek1–/– mkk7–/– ES cells at the same levels as wild-type ES cells. In sharp contrast, such responses were almost completely abolished in apaf1–/– cells. The dose dependence and time course of etoposide-induced apoptosis were also the same between sek1–/– mkk7–/– and wild-type ES cells (Fig. 3d and data not shown). Thus, the SAPK/JNK inactivation appears to exert no influence on the stress-induced apoptosis in ES cells. No Involvement of p38 Activation in Stress-induced Apoptosis in sek1–/– mkk7–/– ES Cells—Since it has been reported that another stress-responsive MAPK, p38, plays a pro-apoptotic role under certain conditions (27.Xia Z.G. Dickens M. Raigeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5032) Google Scholar), we examined the effect of a p38 inhibitor (SB203580) on UV stress-induced apoptosis. As shown in Fig. 4, SB203580 markedly suppressed the phosphorylation of p38 but failed to inhibit the stress-induced apoptosis both in wild-type and sek1–/– mkk7–/– ES cells. These results indicate that p38 activation is not responsible for the stress-induced apoptosis in ES cells. No Requirement of SAPK/JNK Activation for Bax Translocation in ES Cells—The above results shown in Figs. 1, 2, 3 suggest that SAPK/JNK activation is not required for mitochondria-dependent apoptosis in ES cells. We next measured the initiation step of mitochondria-dependent apoptosis, the translocation of Bax from cytoplasm to mitochondrial membranes, since it has been recently reported that SAPK/JNK activation potentiates Bax-dependent apoptosis in neuronal cells (28.Putcha G.V. Le S. Frank S. Besirli C.G. Clark K. Chu B. Alix S. Youle R.J. LaMarche A. Maroney A.C. Johnson Jr, E.M. Neuron. 2003; 38: 899-914Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Fig. 5 shows the time courses of the Bax translocation in response to UV irradiation in sek1–/– mkk7–/–, apaf1–/–, and wild-type ES cells. There were no significant differences among the three types of ES cells. These results clearly show that SAPK/JNK activation is not required for the initiation of mitochondria-dependent apoptosis in ES cells. Stress-induced Apoptosis Is Also Observable in sek1–/– mkk7–/– MEF-like Cells—Since it has recently been reported that SAPK/JNK is crucial for stress-induced apoptosis in MEF (16.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar, 17.Tournier C. Dong C. Turner T.K. Jones S.N. Flavell R.A. Davis R.J. Genes Dev. 2001; 15: 1419-1426Crossref PubMed Scopus (301) Google Scholar), ES cells were treated with retinoic acid and induced to differentiate into MEF-like cells. As shown in Fig. 6, stress-induced apoptosis measured by DNA fragmentation was also observed at the same level between sek1–/– mkk7–/– and wild-type MEF-like cells as had been observed in non-differentiated ES cells (see Fig. 3b). These results indicate that SAPK/JNK inactivation does not exert its influence on the stress-induced apoptosis in MEF-like cells as well as in ES cells. IL-1-induced IL-6 Gene Expression Is Impaired in sek1–/– mkk7–/– MEF-like Cells—To understand the physiological role of SAPK/JNK activation in ES or MEF-like cells, we examined gene expression induced by an inflammatory cytokine, IL-1. As shown in Fig. 7a, IL-6 mRNA was induced at 1 and 6 h in wild-type cells. However, this induction was greatly inhibited in sek1–/– mkk7–/– MEF-like cells, which express IL-1 receptors. Furthermore, the IL-1-dependent IL-6 expression was greatly suppressed by adding the JNK inhibitor SP600125 in wild-type MEF-like cells (Fig. 7b). Thus, SAPK/JNK activation appears to play an important role in the inflammatory cytokine-induced gene expression rather than the apoptotic responses at least in ES or MEF-like cells. In the previous study, we have reported that sek1–/– thymocytes are susceptible to Fas/CD95- and CD3-mediated apoptosis and that apoptosis is increased in sek1–/– hepatoblasts (11.Nishina H. Vaz C. Billia P. Nghiem M. Sasaki T. Pompa J.L. Furlonger K. Paige C. Hui C.-C. Fischer K.D. Kishimoto H. Iwatsubo T. Katada T. Woodgett J.R. Penninger J.M. Development. 1999; 126: 505-516Crossref PubMed Google Scholar, 20.Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar). These results indicate that SAPK/JNK activation is involved in cell survival. However, it has been reported that Jnk1–/– Jnk2–/– and mkk4–/– mkk7–/– MEFs are resistant to UV-induced apoptosis (16.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar, 17.Tournier C. Dong C. Turner T.K. Jones S.N. Flavell R.A. Davis R.J. Genes Dev. 2001; 15: 1419-1426Crossref PubMed Scopus (301) Google Scholar). Furthermore, there are many other reports supporting the involvement of SAPK/JNK activation both in cell survival and in apoptosis. Thus, the role of SAPK/JNK in cell survival and apoptosis is still controversial (13.Lin A. BioEssays. 2002; 25: 17-24Crossref Scopus (369) Google Scholar). Our present results do not support the concept that “SAPK/JNK activation is required for the stress-induced and mitochondria-dependent apoptosis” (4.Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3633) Google Scholar, 16.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar, 17.Tournier C. Dong C. Turner T.K. Jones S.N. Flavell R.A. Davis R.J. Genes Dev. 2001; 15: 1419-1426Crossref PubMed Scopus (301) Google Scholar), at least in ES and MEF-like cells, based on the following findings. First, SAPK/JNK activation is completely dependent on the existence of both SEK1 and MKK7 in mouse ES cells in which stress-induced apoptosis is mediated through mitochondria (Fig. 1). The mutant sek1–/– mkk7–/– ES cells had a defect in the SAPK/JNK activation in response to a variety of stimuli (Fig. 2) but displayed normal stress-induced apoptosis as the same level as wild-type cells (Fig. 3). Another stress-responsive MAPK, p38, was not involved in the stress-induced apoptosis (Fig. 4). Second, Bax was capable of translocating into mitochondrial membranes in response to UV irradiation in sek1–/– mkk7–/– ES cells as observed in the wild-type cells (Fig. 5). Third, the apoptosis in response to various stresses such as UV, etoposide, tunicamycin, and anisomycin occurred even in sek1–/– mkk7–/– MEF-like cells. Therefore, there must be a SAPK/JNK-independent signaling that is required for the initiation of stress-induced apoptosis in ES and MEF-like cells. To examine the involvement of MKK7 in cell cycle and senescence, we have recently prepared mkk7–/– MEFs from mouse embryos. Interestingly, early passaged wild-type and mkk7–/– MEFs showed comparable extent and kinetics of cell death in response to UV irradiation; however, at late time points of culture (after getting cellular immortality), mkk7–/– MEFs displayed a resistance to the UV-induced apoptosis. 2T. Wada, N. Joza, H.-Y. M. Cheng, T. Sasaki, I. Kozieradzki, M. Nghiem, K. Bachmaier, T. Katada, H. Nishina, and J. M. Penninger, submitted for publication. These results suggested that the resistance to UV response observed in Jnk1–/– Jnk2–/– and mkk4–/– mkk7–/– MEFs (16.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar, 17.Tournier C. Dong C. Turner T.K. Jones S.N. Flavell R.A. Davis R.J. Genes Dev. 2001; 15: 1419-1426Crossref PubMed Scopus (301) Google Scholar) might have some relations to cellular immortality. Because SAPK/JNK activation is crucial for hepatoblast proliferation (12.Watanabe T. Nakagawa K. Ohata S. Kitagawa D. Nishitai G. Seo J. Tanemura S. Shimizu N. Kishimoto H. Wada T. Aoki J. Arai H. Iwatsubo T. Mochita M. Watanabe T. Satake M. Ito Y. Matsuyama T. Mak T.W. Penninger J.M. Nishina H. Katada T. Dev. Biol. 2002; 250: 332-347Crossref PubMed Google Scholar), the immortalized MEFs may lose the regulation of checkpoint in cell cycle and result in resistance to stress-induced apoptosis due to impaired G1 growth arrest. Therefore, it will be interesting to find out whether re-expression of JNK1 plus JNK2 in Jnk1–/– Jnk2–/– or MKK4 plus MKK7 in mkk4–/– mkk7–/– MEFs can restore the apoptotic response to UV stress. However, recent reports with Jnk1–/– Jnk2–/– mice clearly show that SAPK/JNK activation is required not only for anti-apoptosis but also for pro-apoptosis in the development of mouse fetal brain (14.Kuan C.-Y. Yang D.D. Roy D.R.S. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 15.Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (301) Google Scholar). There was a reduction of cell death in the lateral edges of hind brain prior to neural tube closure. In contrast, there was increased apoptosis and caspase activation in the forebrain in Jnk1–/– Jnk2–/– mouse embryos. Thus, SAPK/JNK activation plays a pro-apoptotic role under some circumstances. SAPK/JNK activation may be involved in regulating apoptosis indirectly rather than directly. SAPK/JNK is activated in T cells by co-stimulation with antigen and CD28 receptors and regulates the production of growth factor IL-2 and cell proliferation (29.Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Benneriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar). Previously, we reported impaired CD28-mediated IL-2 production and proliferation in sek1–/– T lymphocytes (23.Nishina H. Bachmann M. Oliveira-dos-Santos A.J. Kozieradzki I. Fischer K.D. Odermatt B. Wakeham A. Shahinian A. Takimoto H. Bernstein A. Mak T.W. Woodgett J.R. Ohashi P.S. Penninger J.M. J. Exp. Med. 1997; 186: 941-953Crossref PubMed Scopus (116) Google Scholar). It has recently been reported that activation of wild-type CD8+ T cells in the presence of different concentrations of SP600125 caused a dose-dependent inhibition of IL-2 production and cell proliferation (30.Conze D. Krahl T. Kennedy N. Weiss L. Lumsden J. Hess P. Flavell R.A. Gros G.L. Davis R.J. Rincon M. J. Exp. Med. 2002; 195: 811-823Crossref PubMed Scopus (119) Google Scholar). Thus, SAPK/JNK activation plays an important role in T cell functions. IL-1 is a pro-inflammatory cytokine in inflamed tissues, and the molecular mechanism of IL-1-induced gene expression will yield novel molecular targets for anti-inflammatory therapy. It has been reported that SAPK/JNK activation is required for the gene expression of a cytokine IL-6 and a chemokine IL-8 in a human epidermal carcinoma cell line by using overexpression of a catalytically inactive mutant or antisense RNA of SAPKβ (31.Krause A. Holtmann H. Eickemeier S. Winzen R. Szamel M. Resch K. Saklatvala J. Kracht M. J. Biol. Chem. 1998; 273: 23681-23689Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32.Holtmann H. Enninga J. Kalble S. Thiefes A. Dorrie A. Broemer M. Winzen R. Wilhelm A. Ninomiya-Tsuji J. Matsumoto K. Resch K. Kracht M. J. Biol. Chem. 2001; 276: 3508-3516Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These results indicate that IL-1-mediated activation of SAPK/JNK induces the phosphorylation of a Jun family of component(s) of activator protein-1, resulting in the gene expression of IL-6 and IL-8. In this study, we have also observed the impaired IL-1-mediated IL-6 gene expression both in sek1–/– mkk7–/– MEF-like cells and in SP600125-treated wild-type cells (Fig. 7). These results provide the first genetic link between SAPK/JNK activation and IL-6 gene expression using gene-disrupted cells and also indicate that SAPK/JNK activation provides a crucial and specific signal for immune responses. Thus, the molecular switch, SAPK/JNK activation, regulates cell proliferation and gene expression in embryonic development and immune responses rather than stress-induced apoptosis. We thank Dr. Izumu Saito and RIKEN BRC for providing us with adenovirus-encoding Cre recombinase. We also thank Dr. James R. Woodgett for suggestions and criticism.
Background The risk of transferring blood‐borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor‐transmitted viral infections is also increasing. Although nucleic acid amplification technology ( NAT ) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. Study Design and Methods We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen‐specific probes. Results We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus ( HCV ), human hepatitis B virus ( HBV ), human parvovirus B 19 ( PVB 19), and West Nile virus ( WNV ), using a single plate. We also detected all genotypes of human immunodeficiency virus ( HIV ) but sensitivity was less than for the other viruses. Conclusion We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV , HBV , HCV , PVB 19, and WNV , with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.
The efficacy of vaccine adjuvants depends on their ability to appropriately enhance the immunogenicity of vaccine antigens, which is often insufficient in non-adjuvanted vaccines. Genomic analyses of immune responses elicited by vaccine adjuvants provide information that is critical for the rational design of adjuvant vaccination strategies. In this study, biomarker genes from the genomic analyses of lungs after priming were used to predict the efficacy and toxicity of vaccine adjuvants. Based on the results, it was verified whether the efficacy and toxicity of the tested adjuvants could be predicted based on the biomarker gene profiles after priming. Various commercially available adjuvants were assessed by combining them with the split influenza vaccine and were subsequently administered in mice through nasal inoculation. The expression levels of lung biomarker genes within 24 h after priming were analyzed. Furthermore, we analyzed the antibody titer, cytotoxic T lymphocyte (CTL) induction, IgG1/IgG2a ratio, leukopenic toxicity, and cytotoxicity in mice vaccinated at similar doses. The association between the phenotypes and the changes in the expression levels of biomarker genes were analyzed. The ability of the adjuvants to induce the production of antigen-specific IgA could be assessed based on the levels of Timp1 expression. Furthermore, the expression of this gene partially correlated with the levels of other damage-associated molecular patterns in bronchoalveolar lavage fluid. Additionally, the changes in the expression of proteasome- and transporter-related genes involved in major histocompatibility complex class 1 antigen presentation could be monitored to effectively assess the expansion of CTL by adjuvants. The monitoring of certain genes is necessary for the assessment of leukopenic toxicity and cytotoxicity of the tested adjuvant. These results indicate that the efficacy and toxicity of various adjuvants can be characterized by profiling lung biomarker genes after the first instance of immunization. This approach could make a significant contribution to the development of optimal selection and exploratory screening strategies for novel adjuvants.
A 50-year-old man demonstrated markedly increased number of white blood cells, anemia, severe splenomegaly, and bleeding tendency. Bone marrow analysis revealed remarkable hypercellularity; dysplasia in multilineage cells, including megakaryocytes; and fibrosis. He was eventually diagnosed with triple-negative myelofibrosis. A massive hematoma developed at the bone marrow biopsy site. A similar episode recurred after the second bone marrow biopsy. The von Willebrand factor and other coagulation factor activities were within normal ranges. Platelet aggregation analyses demonstrated highly impaired aggregation induced by ADP, collagen, and epinephrine. Treatment with hydroxyurea and ruxolitinib, a JAK inhibitor, was ineffective, and he eventually died on day 144 after hospitalization. Acquired platelet dysfunction uncommonly occurs in patients with myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), without precise elucidation of the frequency and underlying mechanism. The onset of bleeding tendency in the current patient suggested that platelet dysfunction may be caused by somatic genetic events. Here, we discuss the mechanisms of acquired platelet dysfunction in MDS or MPN with a literature review.
Adult T-cell leukemia (ATL) is a malignant disease caused by human T-lymphotropic virus type 1. In aggressive ATL, the response to chemotherapy is extremely poor. We hypothesized that this poor response is due to the existence of chemotherapy-resistant cells, such as leukemic stem cells. Previously, we successfully identified an ATL stem cell (ATLSC) candidate as the c-kit+/CD38-/CD71- cells in an ATL mouse model using Tax transgenic mice. Here, with a new ATL mouse model using HBZ-transgenic mice, we further discovered that the functional ATLSC candidate, which commonly expresses c-kit, is drug-resistant and has the ability to initiate tumors and reconstitute lymphomatous cells. We characterized the ATLSCs as c-kit+/CD4-/CD8- cells and found that they have a similar gene expression profile as T cell progenitors. Additionally, we found that AP-1 gene family members, including Junb, Jund, and Fosb, were up-regulated in the ATLSC fraction. The results of an in vitro assay showed that ATLSCs cultured with cytokines known to promote stem cell expansion, such as stem cell factor (SCF), showed highly proliferative activity and maintained their stem cell fraction. Inhibition of c-kit-SCF signaling with the neutralizing antibody ACK2 affected ATLSC self-renewal and proliferation. Experiments in Sl/Sld mice, which have a mutation in the membrane-bound c-kit ligand, found that ATL development was completely blocked in these mice. These results clearly suggest that the c-kit-SCF signal plays a key role in ATLSC self-renewal and in ATL initiation and disease progression.
