Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.
Vitamin D promotes differentiation of cells either by simply enhancing phenotypic gene expression and/or by suppressing expression of inhibitors of differentiation. Previously, we reported that expression of a gene encoding Id1, a negative type helix-loop-helix transcription factor, was transcriptionally suppressed by 1,25-dihydroxyvitamin D3(1,25(OH)2D3) (1). To identify the sequence required for the negative regulation by 1,25(OH)2D3, a 1.5-kilobase 5′-flanking region of murine Id1 gene was examined by transiently transfecting luciferase reporter constructs into ROS17/2.8 osteoblastic cells. The transcriptional activity of this construct was repressed by 10−8m 1,25(OH)2D3. Deletion analysis revealed that a 57-base pair (bp) upstream response sequence (URS) (−1146/−1090) was required for the suppression by 1,25(OH)2D3. This sequence conferred negative responsiveness to 1,25(OH)2D3 to a heterologous SV40 promoter. The 57-bp URS contained not only Egr-1 consensus sequence (2) but also four direct repeats of a heptamer sequence (C/A)CAGCCC. Electrophoresis mobility shift assay revealed that the 57-bp URS formed specific nuclear protein-DNA complexes, which were neither competed by previously known positive and negative vitamin D response elements nor supershifted by anti-vitamin D receptor antibody, suggesting the absence of vitamin D receptor in these complexes. These results indicate the involvement of the novel 57-bp sequence in the vitamin D suppression of Id1 gene transcription. Vitamin D promotes differentiation of cells either by simply enhancing phenotypic gene expression and/or by suppressing expression of inhibitors of differentiation. Previously, we reported that expression of a gene encoding Id1, a negative type helix-loop-helix transcription factor, was transcriptionally suppressed by 1,25-dihydroxyvitamin D3(1,25(OH)2D3) (1). To identify the sequence required for the negative regulation by 1,25(OH)2D3, a 1.5-kilobase 5′-flanking region of murine Id1 gene was examined by transiently transfecting luciferase reporter constructs into ROS17/2.8 osteoblastic cells. The transcriptional activity of this construct was repressed by 10−8m 1,25(OH)2D3. Deletion analysis revealed that a 57-base pair (bp) upstream response sequence (URS) (−1146/−1090) was required for the suppression by 1,25(OH)2D3. This sequence conferred negative responsiveness to 1,25(OH)2D3 to a heterologous SV40 promoter. The 57-bp URS contained not only Egr-1 consensus sequence (2) but also four direct repeats of a heptamer sequence (C/A)CAGCCC. Electrophoresis mobility shift assay revealed that the 57-bp URS formed specific nuclear protein-DNA complexes, which were neither competed by previously known positive and negative vitamin D response elements nor supershifted by anti-vitamin D receptor antibody, suggesting the absence of vitamin D receptor in these complexes. These results indicate the involvement of the novel 57-bp sequence in the vitamin D suppression of Id1 gene transcription. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol), 1The abbreviations used are: 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3; LUC, luciferase; EMSA, electrophoresis mobility shift assay(s); URS, upstream regulatory sequence(s); OPN, osteopontin; VDR, nuclear vitamin D receptor; VDRE, vitamin D response element; PTH, parathyroid hormone; BSP, bone sialoprotein; kb, kilobase; bp, base pair(s); PCR, polymerase chain reaction. is not only a major calcitrophic hormone that controls systemic calcium metabolism but also a potent modulator of differentiation in several types of cells including osteoblasts (2Tournay O. Benezra R. Mol. Cell Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar, 3Minghetti P.P. Norman A.W. The FASEB Journal. 1988; 2: 3043-3053Crossref PubMed Scopus (460) Google Scholar). Many studies have revealed that the molecular mechanisms of vitamin D actions, including its promotion of cell differentiation, could be explained mainly by its genomic actions via the vitamin D receptor (VDR) as a ligand-dependent transcription factor (4Haussler M.R. Jurutka P.W.J.C. Thompson P.D. Selznick S.H. Haussler C.A. Whitfield G.K. Bone (NY). 1995; 17: 33-38Crossref PubMed Scopus (110) Google Scholar, 5Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Shütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans M. Cell. 1996; 83: 835-839Abstract Full Text PDF Scopus (6166) Google Scholar, 6Bouillon R. Okamura W.H. Norman A.W Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 7Freedman L.P. Endocr. Rev. 1992; 13: 129-145Crossref PubMed Scopus (246) Google Scholar). VDR binds to vitamin D response elements (VDREs) within the promoter regions of the target genes to activate or suppress their expression. Several types of differentiation-related genes are regulated through this type of vitamin D action during cell differentiation. In addition, recent studies also showed the involvement of the nongenomic action of vitamin D in regulation of cell differentiation (9Bhatia M. Kirkland J.B. Meckling-Gill K.A. J. Biol. Chem. 1995; 270: 15962-15965Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 10Saunders N.A. Bernacki S.H. Vollberg T.M. Jetlen A.M. Mol. Endocrinol. 1993; 7: 387-398Crossref PubMed Scopus (66) Google Scholar, 11Bikle D.D. Pillais S. Endocr. Rev. 1993; 14: 13-19Google Scholar). For instance, monocyte differentiation was reported to be mediated by vitamin D without requiring binding to VDR (9Bhatia M. Kirkland J.B. Meckling-Gill K.A. J. Biol. Chem. 1995; 270: 15962-15965Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and keratinocyte differentiation-related genes were shown to be stimulated by 1,25(OH)2D3 without the presence of VDRE (6Bouillon R. Okamura W.H. Norman A.W Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar, 10Saunders N.A. Bernacki S.H. Vollberg T.M. Jetlen A.M. Mol. Endocrinol. 1993; 7: 387-398Crossref PubMed Scopus (66) Google Scholar, 11Bikle D.D. Pillais S. Endocr. Rev. 1993; 14: 13-19Google Scholar). Therefore, vitamin D could promote cell differentiation via both genomic and nongenomic actions (3Minghetti P.P. Norman A.W. The FASEB Journal. 1988; 2: 3043-3053Crossref PubMed Scopus (460) Google Scholar, 6Bouillon R. Okamura W.H. Norman A.W Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). We have been interested in the molecular mechanism of the differentiation of osteoblasts as one of the target cells of 1,25(OH)2D3 (3Minghetti P.P. Norman A.W. The FASEB Journal. 1988; 2: 3043-3053Crossref PubMed Scopus (460) Google Scholar). Similarly to other types of cells, expression of various phenotype-related genes is enhanced by 1,25(OH)2D3 in osteoblasts (11Bikle D.D. Pillais S. Endocr. Rev. 1993; 14: 13-19Google Scholar, 12Noda M. Vogel R.L. Craig A.M. Prhal J. DeLuca H.F. Denhardt D.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9995-9999Crossref PubMed Scopus (451) Google Scholar, 13Demay M.B. Gerardi J.M. DeLuca H.F. Kronenberg H.M Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 369-3737Crossref PubMed Scopus (304) Google Scholar, 14Kerner S.A. Scott R.A. Pike J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 84: 4455-4459Crossref Scopus (376) Google Scholar). In parallel to its direct control of the genes encoding phenotype-related proteins in osteoblasts, we hypothesized that vitamin D may regulate higher order regulatory genes to modulate osteoblastic differentiation. We have shown that Id1, a dominant negative regulator of helix-loop-helix-type transcription factors (15Benezra R. Yan W. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1974) Google Scholar), is expressed in osteoblasts and that its level is transcriptionally suppressed by 1,25(OH)2D3 (1Kawaguchi N. DeLuca H.F. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4569-4572Crossref PubMed Scopus (57) Google Scholar). Because Id1 has been shown to be a negative modulator of positive regulatory transcription factor(s) that modulate cell differentiation, 1,25(OH)2D3could exert its effects on osteoblasts by suppressing expression of Id1. In the previous study, we have also shown that the suppression was specific to 1,25(OH)2D3 and was mediated at the level of gene transcription without requiring new protein synthesis (1Kawaguchi N. DeLuca H.F. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4569-4572Crossref PubMed Scopus (57) Google Scholar). However, the mechanism with which 1,25(OH)2D3 suppresses Id1 gene transcription was still unknown. Ligand-dependent or -independent repression by nuclear hormone receptor superfamilies has been investigated (8Beato M. Herrlich P. Shütz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1642) Google Scholar, 16Hanna-Rose W. Hansen U. Trends Genet. 1996; 12: 229-234Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 17Johnson A.D. Cell. 1995; 81: 655-658Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 18Carr F.C. Wong N.C.W. J. Biol. Chem. 1994; 269: 4175-4179Abstract Full Text PDF PubMed Google Scholar, 19Piedrafita F.J. Ortiz M.A. Pfahl M. Mol. Endocrinol. 1995; 9: 1533-1548PubMed Google Scholar, 20Diamond M.J. Miner J.N. Yoshinaga S.K. Yamamoto K.R. Science. 1990; 249: 1266-1272Crossref PubMed Scopus (1074) Google Scholar, 21Drouin J. Sunn Y.L. EMBO J. 1993; 12: 145-156Crossref PubMed Scopus (277) Google Scholar, 22Hörlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1724) Google Scholar, 23Yen P.M. Liu Y. Sugawara A. Chin W.W. J. Biol. Chem. 1996; 271: 10910-10916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, the molecular mechanisms of transcriptional repression appear to be more complicated than those of transcriptional activation (22Hörlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1724) Google Scholar,23Yen P.M. Liu Y. Sugawara A. Chin W.W. J. Biol. Chem. 1996; 271: 10910-10916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and the mechanisms for steroidal or nonsteroidal ligand-dependent repression have also been found to be variable. In some cases, to suppress expression of the target genes, hormones utilize the same or similar response elements as those used for transactivation (18Carr F.C. Wong N.C.W. J. Biol. Chem. 1994; 269: 4175-4179Abstract Full Text PDF PubMed Google Scholar), whereas in other cases, sequences different from the classical hormone response elements are utilized for negative regulation (16Hanna-Rose W. Hansen U. Trends Genet. 1996; 12: 229-234Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 17Johnson A.D. Cell. 1995; 81: 655-658Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 19Piedrafita F.J. Ortiz M.A. Pfahl M. Mol. Endocrinol. 1995; 9: 1533-1548PubMed Google Scholar). Although vitamin D receptor also acts as both a transcriptional activator and a repressor similar to other members of the nuclear hormone receptor superfamilies, little is known about the mechanisms of transcriptional repression by 1,25(OH)2D3, which is also capable of utilizing nongenomic action. Five cases of negative VDRE sequence were reported such as parathyroid hormone (PTH) gene, parathyroid hormone-related protein gene, interleukine-2 gene, bone sialoprotein (BSP) gene, and vitamin D receptor binding fragment-5, a negative vitamin D responsible gene isolated from rat genomic DNA (24Liu S.M. Koszewski N. Lupez M. Malluche H.H. Olivera A. Russel J. Mol. Endocrinol. 1996; 10: 206-215Crossref PubMed Google Scholar, 25Demay M.B. Kiernan M.S. DeLuca H.F. Kronenberg H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8097-8101Crossref PubMed Scopus (383) Google Scholar, 26Mackey S.L. Heymont J.L. Kronenberg H.M. Demay M.B. Mol. Endocrinol. 1996; 10: 298-305Crossref PubMed Scopus (103) Google Scholar, 27Falzon M. Mol. Endocrinol. 1996; 10: 672-681Crossref PubMed Scopus (86) Google Scholar, 28Li J.J. Sodek J. J. Biochem. (Tokyo). 1993; 289: 625-629Crossref Scopus (88) Google Scholar, 29Sakoda K. Fujiwara M. Arai S. Suzuki A. Nishikawa J. Imagawa M. Nishihara T. Biochem. Biophys. Res. Commun. 1996; 219: 31-35Crossref PubMed Scopus (8) Google Scholar, 30Alroy I. Towers T.L. Freedman L.P. Mol. Cell. Biol. 1995; 15: 5789-5799Crossref PubMed Scopus (371) Google Scholar). However, because these repressive sequences are quite variable, consensus sequences have not been defined yet. Furthermore, molecular mechanisms of vitamin D repression of the genes encoding key molecules such as transcription factors involved in cell differentiation has not yet been clarified. To understand the molecular mechanisms of the 1,25(OH)2D3 suppression of Id1 gene expression, which could be an important step in cell differentiation, we investigated the 1,25(OH)2D3 effect on the transcriptional activity of the 1.5-kb promoter region of the Id1 gene and identified a sequence that is required for the 1,25(OH)2D3action. The promoter region of the Id1 gene(-1574/+88; 1.5BV) or PCR-generated 5′ deletion sequences (−1372/+88; 5′del-1), (−1147/+88; 5′del-2), (−927/+88; 5′del-3), (−527/+88; 5′del-5), (−327/+88; 5′del-6), (−127/+88; 5′del-7), and (−52/+88; 5′del-8) were subcloned by Tournay and Benezra intoHindIII site of pGL2-Basic vector (BV) (Promega Corp., Madison, WI) as described previously (2Tournay O. Benezra R. Mol. Cell Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar). We generated by PCR further deletion constructs, 5′del-1100, 5′del-1050, 5′del-1000 containing the 5′ upstream regions of the Id1 promoter corresponding to −1100/+88, −1050/+88, and −1000/+88 by using 20-mer oligonucleotides as primers.SacI (5′) sites and BglII (3′) sites were introduced on each end of the PCR products, respectively, and the fragments were inserted into SacI and BglII sites of the pGL2-Basic vector. A double-stranded 63-bp oligonucleotide containing a 57-bp sequence corresponding to the −1146/−1090 region (URS) of Id1 promoter andMluI sites (underlined below) on both of its ends was synthesized. The top strand (5′-CGCGTCCAGCCCAGTTTGCCGTCTCCATGGCGACCGCCCGCGCGGCGCCAGCCTGACAGCCCA-3′) and the bottom strand (5′-CGCGTGGGCTGTCAGGCTGGCGCCGCGCGGGCGGTCGCCATGGAGACGGCAAACTGGGCTGGA-3′) were annealed and subcloned into the MluI site 11 bp upstream of SV40 promoter in PGV-P vector (Toyo Ink MFG. Co., Ltd., Tokyo, Japan) in a normal or a reverse orientation to generate 57-PGVP, 57R/PGVP (R stands for reverse orientation), and 57*2-PGVP (containing two copies of the 57-bp URS), respectively. Similarly, four types of double-stranded mutated oligonucleotides of the 57-bp URS were also inserted into the same vector to generate mutated URS constructs, 57M-PGVP, 16A16-PGVP, 57A-3-PGVP, and 57-5A-PGVP (see Table I). The top strands of the 57M, 16A16, 57A-3, and 57-5A are as follows with mutations indicated by double underlines: 57M, 5′-CGCGTCCAGCCCAGTTTGCCGTCTCCATGGCGACCGCAAAAGCGGCGCCAGCCTGACAGCCCA-3′; 16A16, 5′-CGCGTCCAGCCCAGTTTGCCGAAAAAAAAAAAAAAAAAAAAAAAAAGCCAGCCTGACAGCCCA-3′; 57A-3, 5′-CGCGTCCAGCCCAGTTTGCCGTATCCATGGCGACCGCCCGCGCGGCGCCAGCCTGACAGCCCA-3′; 57-5A, 5′-CGCGTCCAGCCCAATTTGACGTATCCATGGCGACCGCCCGCGCGGAGCCAGCCTAACAGCCCA-3′. Two copies of the 38-bp VDRE sequence previously identified in the upstream region of the osteopontin promoter (−768/−731) (10Saunders N.A. Bernacki S.H. Vollberg T.M. Jetlen A.M. Mol. Endocrinol. 1993; 7: 387-398Crossref PubMed Scopus (66) Google Scholar) were also inserted into PGV-P vector to generate OPN*2-PGVP as positive control vectors. The integrity of the constructs was confirmed by restriction analysis and dideoxy sequencing.Table ISummary of competition and luciferase assays of the mutated URS fragments† The results of the competition assay performed by using the 57-bp URS probe and the nuclear extracts prepared from ROS17/2.8 cells are evaluated based on the intensity of each group of the bands (H and L complexes: +, competed out; −, not competed; +/−, weakly competed).‡ The results of the luciferase assay with regard to the response to vitamin D treatment in the cells transfected with constructs containing corresponding oligonucleotides ligated immediately upstream of the SV40 promoter of the PGV-P. (+ indicates suppressive response to vitamin D treatment; − indicates no response to vitamin D treatment; ND, not determined.) We found that a sequence has the capacity to respond to vitamin D treatment if the mean values of relative luciferase activities of the construct was significantly different from that of PGV-P construct (p < 0.05), based on the data from more than four independent experiments. The statistical difference was evaluated by Dunnett's test for multiple comparison. Open table in a new tab † The results of the competition assay performed by using the 57-bp URS probe and the nuclear extracts prepared from ROS17/2.8 cells are evaluated based on the intensity of each group of the bands (H and L complexes: +, competed out; −, not competed; +/−, weakly competed). ‡ The results of the luciferase assay with regard to the response to vitamin D treatment in the cells transfected with constructs containing corresponding oligonucleotides ligated immediately upstream of the SV40 promoter of the PGV-P. (+ indicates suppressive response to vitamin D treatment; − indicates no response to vitamin D treatment; ND, not determined.) We found that a sequence has the capacity to respond to vitamin D treatment if the mean values of relative luciferase activities of the construct was significantly different from that of PGV-P construct (p < 0.05), based on the data from more than four independent experiments. The statistical difference was evaluated by Dunnett's test for multiple comparison. ROS17/2.8 cells (provided by Dr. G. Rodan, Merck Research Laboratories) were maintained in modified F12 medium supplemented with 5% (v/v) fetal bovine serum as described previously (31Majeeska R.J. Rodan S.B. Rodan G.A. Endcrinology. 1980; 107: 1494-1503Crossref PubMed Scopus (431) Google Scholar). ROS17/2.8 cells were plated in 6-well cluster plates (35-mm well diameter) at 5 × 105cells/wells. Three days later, the cells were transfected with plasmids (4 μg/well) by the DEAE-dextran method as described previously (32Lopata M. Cleaveland D. Sollner-Webb B. Nucleic Acids Res. 1984; 12: 5707-5717Crossref PubMed Scopus (616) Google Scholar). Following transfection, the cells were incubated in the medium supplemented with 0.5% fetal bovine serum in the presence (10−8m) or the absence of 1,25(OH)2D3. Luciferase (LUC) assay was performed as described elsewhere (33Berthold A. Biomed. Biochem. Acta. 1990; 49: 1243-1245PubMed Google Scholar). The cells were harvested after about 72 h of the transfection, and the LUC activity was measured by a luminometer (Berthold Autolumat LB953) using a Picagene kit (Toyo Ink Co.). The LUC activity was normalized against the total protein concentration measured by the Coomassie Brilliant Blue G method (34Spector T. Anal. Biochem. 1978; 86: 142-146Crossref PubMed Scopus (1388) Google Scholar). In a part of the experiments, the LUC activity was normalized against the chloramphenicol acetyltransferase activity of pSV2CAT construct cotransfected with Id1 promoter-LUC constructs, which gave the same result as LUC activity normalized against total protein content. pGL2-Control (Promega) was used as a control. A double-stranded 57-mer oligonucleotide corresponding to the sequence in the −1146/−1190 region of the Id1 promoter and seven types of double-stranded oligonucleotides with deletion or substitution mutations (d5′-37, d3′-38, 57M, 16A16, 8A8, 57A-3, and 57-5A; see Table I) were synthesized to be used as radiolabeled probes or competitors. Annealed oligonucleotides were labeled by using T4 polynucleotide kinase (Takara Shuzo Co., Ltd., Otsu, Japan) and [γ-32P]ATP (NEN Life Science Products). Eight types of double-stranded oligonucleotides containing VDREs previously reported (see legend to Fig. 7 B) were also synthesized to be used as competitors. Electrophoresis mobility shift assays were performed essentially as described elsewhere (35Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology, Unit 12-2. Wiley-Interscience, New York1987: 1-11Google Scholar). Crude nuclear extracts of ROS17/2.8 cells or MG63 cells treated with 1,25(OH)2 D3 or with vehicle were prepared according to the method described by Dignam et al. with some modifications (36Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (10033) Google Scholar). Briefly, the confluent cells were treated with either 10−8m 1,25(OH)2D3 or vehicle for 24 h before extraction. The cells were then rinsed with phosphate-buffered saline and scraped in buffer A (10 mm Hepes, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol). The nuclei were isolated by giving 10 strokes in a Dounce homogenizer and were centrifuged and resuspended in buffer C (20 mmHepes, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 25% (v/v) glycerol) and then were lysed by a Dounce homogenizer again. Supernatents of the nuclear lysates were dialyzed against buffer D (20 mmHepes, 100 mm KCl, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol, 20% glycerol), and the aliquots were stored at −80 °C. Pig intestine nuclear extracts were kindly provided by Dr. DeLuca. Aliquots of 20,000 cpm of the probes were incubated with nuclear proteins for 30 min at 30 °C in a 35-ml reaction mixture containing 3 mg of bovine serum albumin and 2 mg of poly(dI-dC) (Pharmacia Biotech Inc.). For the detection of vitamin D receptor, monoclonal antibodies (10C6 or 8C12) raised against porcine vitamin D receptor (37Dame M.C. Pierce E.A. Prahl J.M. Hayes C.E. DeLuca H.F. Biochemistry. 1986; 25: 4523-4534Crossref PubMed Scopus (114) Google Scholar) (kindly provided by Dr. DeLuca) were added to the incubation mixture containing nuclear proteins extracted from pig intestine. Statistical significance of the difference was evaluated by Dunnett's test for multiple comparison or Student's t test for per-comparison analysis. To test whether certain sequences respond to vitamin D treatment, mean values of the “relative luciferase activities” of the constructs (vitamin D(+) versus (−)) were compared with those of the control construct, pGL2-Control, or to the noninserted construct, PGV-P. The data based on more than three independent experiments for each construct were put together, and statistical significance (p < 0.05) was evaluated by Dunnett's test. In the case of comparison between 57*2-PGVP and 57-PGVP, per-comparison analysis was applied. We first examined the effect of 1,25(OH)2D3 on the transcriptional activity of the 1.5-kb promoter region of Id1 gene (−1574/+88, 1.5BV) by LUC assay and found that 1,25(OH)2D3 treatment suppressed Id1 promoter activity by 50–70% (Fig.1). The effect was first observed at 10−9m and peaked at 10−8m 1,25(OH)2D3 (data not shown). Although the levels of the luciferase activity in the control cultures continuously increased up to 72 h, similar levels of vitamin D suppression were observed during this time period (Fig. 1). Because Id1 promoter activity has been shown to be activated by serum (2Tournay O. Benezra R. Mol. Cell Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar), we examined the effect of serum concentration on the 1,25(OH)2D3 suppression. Although the basal levels of Id1 transcriptional activity was correlated to serum concentrations as reported before (2Tournay O. Benezra R. Mol. Cell Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar), the magnitude of 1,25(OH)2D3 suppression was similar (about 50–70% suppression) regardless of the concentrations of fetal bovine serum at either 0.5 or 10% (data not shown). These results were consistent with our previous observation of the effect of 1,25(OH)2D3 on Id1 expression in Northern analysis and nuclear run-on assay (1Kawaguchi N. DeLuca H.F. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4569-4572Crossref PubMed Scopus (57) Google Scholar), indicating that this 1.5-kb promoter fragment is necessary and sufficient for the 1,25(OH)2D3 suppression of Id1 gene. To examine whether any particular regions in the 1.5-kb fragment mediate the vitamin D suppression of Id1 expression, deletion analysis was carried out by transfecting 1.5BV and seven types of deletion mutant constructs of the Id1 promoter into ROS17/2.8 cells. By this deletion analysis, the response region was located within a 221-bp fragment (−1147/−927) (Fig.2 A). The downstream promoter region located within −927/+88 did not significantly contribute to the 1,25(OH)2D3 suppression. Therefore, we concentrated on analyzing the 231-bp region by further deletion analysis. Three additional deletion mutants were made by PCR and were subcloned into pGL2-Basic vector (5′del-1100, 5′del-1050, and 5′-1000). This second series of deletion analysis showed that only the activity of 5′del-2 (−1147/+88) construct but not that of any of the other constructs (5′del-1100, 5′del-1050, and 5′del-1000) was repressed by 1,25(OH)2D3, indicating that the sequence between −1147 and −1100 was essential for the 1,25(OH)2D3 suppression (Fig.2 B). To examine the negative 1,25(OH)2D3 regulation via the sequence between −1147 and −1100 identified above, we made three additional constructs, 57-PGVP, 57R-PGVP, and 57*2-PGVP. To generate these constructs, a double-stranded oligonucleotide of the 57-bp URS corresponding to the positions between −1147 and −1090, which includes an additional 10-bp sequence downstream to the position −1100, was made. Then, a single or a double copy of this sequence was inserted into the multiple cloning site immediately upstream from the SV40 early promoter in PGV-P vector in a normal (57-PGVP and 57*2-PGVP) or a reverse orientation (57R-PGVP). As shown in Fig.3, the 57-bp URS conferred repressive response to 1,25(OH)2D3 to the SV40 early promoter in a position- and orientation-independent manner. There was a small but statistically significant difference between the levels of vitamin D suppression in 57-PGVP versus 57*2-PGVP constructs when per-comparison analysis was applied (Student's t test,p < 0.01; Fig. 3). The activity of 1,25(OH)2D3 was confirmed by using a positive VDRE control vector, OPN*2-PGVP, which showed 3–4-fold enhancement of luciferase activity in response to 1,25(OH)2D3treatment (Fig. 3). The 57-bp fragment contained in its mid-portion an Egr-1 consensus sequence (5′-CGCCCGCGC-3′) (37Dame M.C. Pierce E.A. Prahl J.M. Hayes C.E. DeLuca H.F. Biochemistry. 1986; 25: 4523-4534Crossref PubMed Scopus (114) Google Scholar) at −1117 to −1109 and a YY-1 consensus sequence (5′-CCATGGCGA-3′) at −1127 to −1119 as reported before (2Tournay O. Benezra R. Mol. Cell Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar) (underlined in Fig.4). In addition, it contains a sequence 5′GGGCGG-3′ in −1118/−1113 (reverse direction), which matches the core region of Sp1 binding consensus sequence (38Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) (Fig. 4,dotted line), and an overlapping 12-bp sequence in almost the same region (−1120/−1109), which corresponds to the 10-bp sequence out of the 12-bp consensus sequence for WT1, a suppressor protein for Wilms' tumor (5′-GCGGGGGCGGTG-3′) (39Drummond I.A. Rupprecht H.D. Rohwer-Nutter P. Lopez-Guisa J.M. Madden S.L. Rauscher III, F.J. Sukhatme V.P. Mol. Cell. Biol. 1994; 14: 3800-3809Crossref PubMed Scopus (142) Google Scholar) (Fig. 4,dashed line). We referred to this 21-bp portion of the 57-bp URS, as the GC-rich region (20 bases are G or C) (Fig. 4). Moreover, there are four direct repeats of a novel heptamer sequence (5′-(A/C)CAGCCC-3′) separated by 1-, 8-, or 20-bp gaps within the 57-bp URS (Fig. 4, Hep1–Hep4), whereas no conserved VDR binding sequence that includes VDRE half-site consensus sequence RRKNSA (40Carlberg C. Bendik I. Wyss A. Meier E. Sturzenbecker l.J. Grippo J.F. Hunziker W. Nature. 1993; 361: 657-660Crossref PubMed Scopus (525) Google Scholar) was found within the 57-bp URS. To examine whether the 57-bp sequence binds to nuclear factors in ROS17/2.8 cells, electrophoresis mobility shift assays (EMSA) were performed. As shown in Fig.5 A, the 57-bp URS bound nuclear proteins forming two complexes L and H (Fig. 5 A,lane 2). The complex marked “L” (for “lower”) is a prominent one and contains one major band and a minor band, which migrates only slightly faster than the major band. The faint faster band is possibly formed due to the lack of one or more small components in forming the DNA-protein complex. We refer to these bands as L complex. The other complex, “H” (for “higher”), is a faint one that migrates slower than L complex. These L and H complexes were competed out by a 100-fold molar excess of unlabeled 57-bp URS probe (Fig. 5 A, lane 3), whereas cyclic AMP response element sequence used as a control did not compete them out even at a 200-fold molar excess (Fig. 5 A, lanes 9 and10). We further examined within the 57-bp URS the presence of possible essential subregions, which are required both for the formation of nuclear protein-DNA complexes and for the suppression by vitamin D treatment. Seven types of mutant oligonucleotides were made to be used for the competition assays (Fig.5 and Table I), as well as to construct reporter plasmids for the transcription assays (Fig.6 and Table I). To design these mutant oligonuc
Osteoporosis causes fractures that lead to reduction in the quality of life and it is one of the most prevalent diseases as it affects approximately 10% of the population. One of the important features of osteoporosis is osteopenia. However, its etiology is not fully elucidated. Dok-1 and Dok-2 are adaptor proteins acting downstream of protein tyrosine kinases that are mainly expressed in the cells of hematopoietic lineage. Although these proteins negatively regulate immune system, their roles in bone metabolism are not understood. Here, we analyzed the effects of Dok-1 and Dok-2 double-deficiency on bone. Dok-1/2 deficiency reduced the levels of trabecular and cortical bone mass compared to wildtype. In addition, Dok-1/2 deficiency increased periosteal perimeters and endosteal perimeters of the mid shaft of long bones. Histomorphometric analysis of the bone parameters indicated that Dok-1/2 deficiency did not significantly alter the levels of bone formation parameters including mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR). In contrast, Dok-1/2 deficiency enhanced the levels of bone resorption parameters including osteoclast number (N.Oc/BS) and osteoclast surface (Oc.S/BS). Analyses of individual osteoclastic activity indicated that Dok-1/2 deficiency enhanced pit formation. Systemically, Dok-1/2 deficiency increased the levels of urinary deoxypyridinoline (Dpyr). Search for the target point of the Dok-1/2 deficiency effects on osteoclasts identified that the mutation enhanced sensitivity of osteoclast precursors to macrophage colony-stimulating factor. These data revealed that Dok-1 and Dok-2 deficiency induces osteopenia by activation of osteoclasts.
Rheumatoid Arthritis (RA) associated osteopenia is a resultant of multifactorial secondary osteoporosis, including disuse-atrophy, glucocorticoid-induced osteopenia, and RA specific activation of osteolysis. In addition, primary osteoporosis, i.e. postmenopausal and senile osteoporosis may overlap with them. Pursuits for the genetic factors for the causes of RA associated osteopenia could be clarified both from the characterization of RA associated factors and the osteoporosis associated factors. Recent progress in systematic genome-wide analysis for the association studies with multiple gene polymorphisms may improve the understandings of genetic contribution of these factors for the pathogenesis of this symptom.
Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major age-related diseases in our modern world (1Riggs B.L. Hartmann L.C. N. Engl. J. Med. 2003; 348: 618-629Crossref PubMed Scopus (829) Google Scholar, 2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (40) Google Scholar, 4Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (43) Google Scholar). Especially, high fracture risk in osteoporosis patients results in not only loss of quality of life but also loss of life in a certain fraction of aged patients. The number of osteoporosis patients is estimated to be close to 10% of the whole population in many advanced countries. Among this patient population, a significant number of patients have disuse osteoporosis based on bedridden conditions caused by aging-related cardiovasucular as well as cerebrovascular diseases (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar). Bone has been known to be lost upon the removal of the mechanical stimuli, and prolonged lack of mechanical stimuli leads to disuse osteoporosis (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar, 7Bikle D.D. Sakata T. Halloran B.P. Gravit. Space Biol. Bull. 2003; 16: 45-54PubMed Google Scholar, 8Bikle D.D. Halloran B.P. J. Bone Miner. Res. 1991; 6: 527-530PubMed Google Scholar, 9Serhan C.N. N. Engl. J. Med. 2004; 350: 1902-1903Crossref PubMed Scopus (6) Google Scholar). However, the mechanisms underlying such disuse osteoporosis are largely unknown (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). Bone mass is determined by the actions of osteoblasts, which make bone, and those of osteoclasts, which resorb bone (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Augat P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google Scholar, 13Strewler G.J. N. Engl. J. Med. 2004; 350: 1172-1174Crossref PubMed Scopus (38) Google Scholar, 14Mohamed A.M. N. Engl. J. Med. 2003; 349: 1671Crossref PubMed Scopus (3) Google Scholar, 15Fuller K.E. N. Engl. J. Med. 2004; 350: 189-192Crossref PubMed Scopus (18) Google Scholar). The balance between the two activities is under the control of hormones and cytokines. Usually, simultaneous changes in the two activities are considered to be coupled to compensate bone loss. However, in the case of disuse osteoporosis, bone formation activities are not enhanced even in the presence of enhanced bone resorption. Rather, bone formation is significantly suppressed in disuse osteoporosis (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 17Harada S. Rodan G.A. Nature. 2003; 423: 349-355Crossref PubMed Scopus (1139) Google Scholar). Therefore, disuse osteoporosis is a critical pathological situation where bone mass is continuously lost without having any compensatory activity against the reduction of bone. However, how such a critical reduction in bone formation occurs in unloading-induced pathological bone loss is not yet known. Bone formation and bone resorption are under the control of the systemic hormones and local cytokines (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). However, none of these factors have been proven to be the major cause of the disuse osteoporosis. In addition to the bedridden patients, astronauts under gravity conditions also lose bone due to the loss of mechanical stress. Analysis of such astronauts returning from space indicated that sympathetic nervous tone is enhanced in their muscle (18Fu Q Levine B.D. Pawelczyk J.A. Ertl A.C. Diedrich A. Cox J.F. Zuckerman J.H. Ray C.A. Smith M.L. Iwase S. Saito M. Sugiyama Y. Mano T. Zhang R. Iwasaki K. Lane L.D. Buckey Jr., J.C. Cooke W.H. Robertson R.M. Baisch F.J. Blomqvist C.G. Eckberg D.L. Robertson D. Biaggioni I. J. Physiol. 2002; 544: 653-664Crossref PubMed Scopus (76) Google Scholar, 19Cox J.F. Tahvanainen K.U. Kuusela T.A. Levine B.D. Cooke W.H. Mano T. Iwase S. Saito M. Sugiyama Y. Ertl A.C. Biaggioni I. Diedrich A. Robertson R.M. Zuckerman J.H. Lane L.D Ray C.A White R.J. Pawelczyk J.A. Buckey Jr., J.C. Baisch F.J. Blomqvist C.G. Robertson D. Eckberg D.L. J. Physiol. 2002; 538: 309-320Crossref PubMed Scopus (73) Google Scholar). Sympathetic nervous system would regulate bone mass via bone formation by osteoblasts systemically (20Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). However, nothing has been known about how this system is related to pathophysiology in bone metabolism in the body. Therefore, we examined whether sympathetic nervous tone is involved in reduction of bone mass in a disuse osteoporosis model via osteoblastic and osteoclastic cells using hind limb unloading. Animals—Male 129 or C57BL/6J mice (10–14 weeks old) were used for the experiments. Mice were housed for at least 1 week prior to the study. The mice were subjected to either intraperitoneal injections of propranolol (20 μg/g of body weight/day) (21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), continuous administration of guanethidine via an osmotic minipump (20 μg/g of body weight/day) (22Sherman B.E. Chole R.A. J. Bone Miner. Res. 2000; 15: 1354-1360Crossref PubMed Scopus (19) Google Scholar), or intraperitoneal injections of isoproterenol (6 μg/g of body weight/day) (23Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar). In the case of osmotic minipump implantation, the pump was implanted 12 h before the start of hind limb unloading. Osmotic minipumps were implanted into the subcutaneous tissue in the back of the animals according to the manufacturer's instruction. For dopamine β-hydroxylase (DBH) 1The abbreviations used are: DBH, dopamine β-hydroxylase; BV/TV, bone volume/tissue volume; BFR, bone formation rate; TRAP, tartrate-resistant acid phosphatase; MAR, bone mineral apposition rate; MS, mineralizing surface; CT, computerized tomography. gene deletion experiments, heterozygous knockout mice with a C57BL6/129sv F2 background and wild type litter mate mice were used (13-week-old females) (24Thomas S.A. Matsumoto A.M. Palmiter R.D. Nature. 1995; 374: 643-646Crossref PubMed Scopus (468) Google Scholar). All of the mice were injected intraperitoneally with calcein at 4 mg/kg at 4 and 2 days before sacrifice. After treatment for 10 or 14 days, mice were anesthetized with tribromoethanol at 200 mg/kg and were sacrificed by cervical dislocation. Hind Limb Unloading Model—Hind limb unloading was conducted by applying a tape to the surface of the hind limb to set a metal clip (10Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. J. Bone Miner. Res. 2002; 17: 661-667Crossref PubMed Scopus (88) Google Scholar, 16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar). The end of the clip was fixed to an overhead bar. The height of the bar was adjusted to maintain the mice at an ∼30° head down tilt with the hind limbs elevated above the floor of the cage. The mice were subjected to hind limb unloading for 10 or 14 days. Loaded control mice were also housed individually under the same conditions except for hind limb unloading for the same duration. Body Weight—The body weight of the mice was monitored during the experimental period. There were no significant changes in body weight in any of the groups during the course of the study. This confirmed that stress could be considered minimal in our experiments as previously described (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Two-dimensional Micro-CT Analysis of Bone—Bone volume/tissue volume (BV/TV) was determined based on two-dimensional micro-CT analyses using a micro-CT apparatus (Musashi, Nittetsu-ELEX Co., Kita-Kyushu City, Japan). The data were quantified by using automated image analysis system (Luzex-F, Nireco). The fractional bone volume (BV/TV) was obtained in an area of 0.47 mm2 with its closest and furthest edges at 0.28 and 0.84 mm, respectively, distal to the growth plate of the proximal ends of the tibiae. The threshold level for the measurements was set at 110 for the analyses (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Histomorphometric Analysis of Bone—At the end of the experiments, the right femora of each mouse were removed and fixed in 70% ethanol. Serial 3-μm-thick sagittal sections were made as undecalcified sections (right femora). For bone formation rate (BFR), metaphyseal cancellous bone in the femora was used to obtain bone fraction in a rectangular area of 0.34 mm2 (0.5 × 0.67 mm) with its closest and furthest edges at 0.3 and 0.8 mm distal to the growth plate, respectively. For decalcified sections, the left tibiae of the mice were removed at the end of the experiments and fixed in 4% paraformaldehyde and then decalcified in 20% EDTA. Serial 5-mm-thick sagittal sections were made using a microtome and stained for tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated cells attached to bone were scored as osteoclasts. Measurements were made within an area of 0.24 mm2 (0.6 × 0.4 mm), with its closest and furthest edges at 0.3 and 0.7 mm distal to the growth plate of the proximal ends of the tibiae. Histomorphometry was conducted to quantify the number of osteoclasts (Oc.N/BS) and osteoclast surface (Oc.S/BS) as defined by Parfitt et al. (11Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4920) Google Scholar). Nodule Formation Analysis—We cultured the cells obtained from the bone marrow of the animals that were subjected to hind limb unloading (12Augat P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google Scholar) or loading in combination with the treatment with adrenergic modulators. The cells were cultured in the presence of ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mm) in α-minimal essential medium supplemented with 10% FBS, 1% antibiotics. After 3 weeks in culture, alizarin red staining was conducted. This staining was used to visualize the calcified materials formed in vitro. Briefly, after the cultures were terminated, the cells were fixed in 100% ethanol and then were stained in alizarin red solution (1%) for 1 min. The cultures were then rinsed several times with water. The area of alizarin red positive nodules was measured by using an image analyzer. Urinary Deoxypyridinoline—Deoxypyridinoline levels in urine at the end of the hind limb unloading were measured by enzyme-linked immunosorbent assay (Metra Biosystems) (6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). Urine samples were collected from five mice per group, which were housed in a metabolic cage during the last 24 h and analyzed. Statistical Analysis—Data were expressed as means ± S.D., and statistical evaluation was performed based on analysis of variance, using a statistical software package for Windows, Statview version 5.0 (SAS Institute). A p value less than 0.05 was considered to be statistically significant. Hind limb unloading reduced bone volume in vehicle-treated control mice as reported previously (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar) (Fig. 1, a and b, column 1 versus column 2). In contrast, treatment with propranolol, a β-adrenergic blocker acting at the receptor levels, suppressed hind limb unloading-induced reduction in bone mass expressed as BV/TV (Fig. 1, a and b; no significant difference between columns 3 and 4). With regard to the drug effect in unloaded mice, propranolol treatment resumed the bone loss induced by unloading (Fig. 1b, column 2 versus column 4). Control-loaded mice treated with propranolol revealed significant difference in bone mass compared with unloaded vehicle-treated mice (Fig. 1b, column 2 versus column 3). If hind limb unloading suppressed the levels of bone mass and bone formation through sympathetic tone, not only the actions of the blockers for sympathetic signaling, which act at the receptor levels, but also the depletion of the presynaptic transmitter reservoir should affect the unloading-induced reduction in bone mass. Therefore, guanethidine sulfate was administrated to deplete norepinephrine at the presynaptic nerve ending levels in the animals that were subjected to hind limb unloading. As shown in Fig. 2, guanethidine treatment suppressed unloading-induced bone loss (Fig. 2, a and b). The pattern of the levels in BV/TV was similar to that in the case of propranolol (Fig. 1). Body weight was not altered significantly due to unloading or drug treatment in all of our experiments (Fig. 2, c–e).Fig. 2Guanethidine treatment suppressed unloading-induced pathological bone loss. During hind limb unloading of mice (129 strain) for 14 days, guanethidine treatment or vehicle was administered using an osmotic minipump (model 1002). The number of the mice used for the experiments represented in each bar is indicated as N. During the 14-day hind limb unloading of 129 mice, guanethidine treatment was carried out.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further test whether unloading signaling is exerted in the line of sympathetic tone, we examined the effects of activation by isoproterenol of sympathetic tone. This was in order to determine whether isoproterenol may mask the unloading-induced signals. Isoproterenol treatment reduced bone mass in control-loaded mice as reported previously (Fig. 3, a and b, column 1 versus column 3), and under this condition, hind limb unloading no longer reduced bone mass (Fig. 3, a and b; compare columns 3 and 4). Notably, the levels of bone mass reduction by isoproterenol treatment were similar to those in unloaded mice (Fig. 3b, column 3 versus column 2). Thus, the data of these three sets of experiments are in accordance with the notion that unloading signaling is suppressed by blockers for sympathetic signaling, and such signaling is no longer active in the presence of the action of β-adrenergic agonist. In order to examine the mechanisms of β-adrenergic actions in unloading-induced bone loss, we conducted dynamic histomorphometry. Hind limb unloading induced reduction in the levels of bone mineral apposition rate (MAR)), mineralizing surface (MS), and BFR (Fig. 4, a–d) after 10 days of hind limb unloading and propranolol treatment suppressed the unloading-induced decrease in MAR, MS, and BFR (Fig. 4, a–d). The patterns of the columns were similar to those in the case of bone mass (Fig. 1). Guanethidine treatment also suppressed the hind limb unloading-induced reduction in MAR, MS, and BFR (Fig. 5, a–d) in the mice subjected to hind limb unloading for 2 weeks. Again, the patterns of the columns were similar to those in the case of bone mass (Fig. 1). Thus, depletion of sympathetic neurotransmitter has effects similar to the block of β-adrenergic receptor with respect to bone formation activity in vivo. Isoproterenol treatment alone suppressed the levels of mineral apposition rate, mineralizing surface, and bone formation rate, whereas in the presence of isoproterenol treatment, 10 days of hind limb unloading failed to suppress all of these parameters (Fig. 6, a–d). It is again notable that the patterns of the columns in Fig. 6 were similar to those observed in the effects of isoproterenol on bone mass (Fig. 3). These three lines of evidence based on dynamic histomorphometry indicated that bone formation is the target of sympathetic tone in mice subjected to hind limb unloading.Fig. 5Guanethidine treatment suppressed unloading-induced reduction in bone formation in vivo. Mice were treated with guanethidine as described in the legend to Fig. 3. The number of mice in each group is indicated as N. a, calcein double-labeled surfaces of the bones at the ends of the femora after hind limb unloading (Unload) or loading (Load) in vehicle- or guanethidine-treated mice. The arrows indicate the lines of calcein labeling (light green) used to obtain data shown in b–d. b–d, in the undecalcified sections of the distal ends of the femora, MAR (b), MS (c), and BFR (d) were measured in all areas at 0.3–0.8 mm distal to the growth plate in the metaphyseal region as described under "Materials and Methods." The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before sacrifice at 2 weeks. Data are expressed as means and S.D. for five bones from each of the vehicle- and guanethidine-treated mice groups. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups). §, statistically significant difference between vehicle-treated unloaded group and drug-treated control-loaded group (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Isoproterenol treatment suppressed the level of bone formation parameters, and unloading did not further suppress these parameters in vivo. Mice were treated with isoproterenol as described in the legend to Fig. 3. The number of mice in each group is indicated as N. a, calcein double-labeled surfaces of the bones at the ends of the femora after hind limb unloading (Unload) or loading (Load) in vehicle or isoproterenol-treated mice. The arrows indicate the lines of calcein labeling (light green) used to obtain data shown in b–d. b–d, in the undecalcified sections of the distal ends of the femora, MAR (b), MS (c), and BFR (d) were measured in all areas at 0.3–0.8 mm distal to the growth plate in the metaphyseal region as described under "Materials and Methods." The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before sacrifice at 10 days. Data are expressed as means and S.D. for five bones from each of the vehicle- and isoproterenol-treated mouse groups. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further examined whether our observations can be detected at cell levels in culture. For this purpose, a nodule formation assay was conducted by using bone marrow cells obtained from the bone of the mice after they were subjected to hind limb unloading or control loading in the presence or the absence of the treatment with pharmacological agents. Hind limb unloading reduced nodule formation in the cultures of cells obtained from the animals (Fig. 7, a and b, column 1 versus column 2). Propranolol treatment in vivo suppressed the unloading-induced reduction in the mineralized nodule formation in culture (Fig. 7, a and b, column 3 versus column 4). Isoproterenol treatment suppressed the levels of nodule formation in loaded control mice (Fig. 8, a and b, column 1 versus column 3), and in the presence of isoproterenol treatment in vivo, hind limb unloading failed to further reduce the levels of nodule formation in bone marrow cells in culture (Fig. 8, a and b, column 3 versus column 4). Thus, these in vitro experiments indicated that β-adrenergic sympathetic tone mediates unloading-induced reduction in mineralization of bone marrow cell cultures.Fig. 8Isoproterenol treatment in vivo suppressed the levels of nodule formation, and unloading conditions failed to further reduce the mineralization levels in vitro. Mice were treated with isoproterenol as described in legend to Fig. 6. The number of mice in each group is indicated as N. The cells obtained from the bone marrow of the animals subjected to hind limb unloading with the isoproterenol or vehicle treatment were cultured in the presence of ascorbic acid and β-glycelophosphare. After 3 weeks, alizarin red staining was conducted, and the area of the alizarin red-positive nodule was quantified. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to examine the effects of the sympathetic nervous tone on unloading-induced reduction in bone mass and bone formation in a genetic model rather than pharmacological modulation, DBH (dopamine β-hydroxylase) knockout mice were subjected to hind limb unloading. Hind limb unloading reduced bone mass by about 68.6% in wild type littermate mice (Fig. 9a, column 1 versus column 2). Heterozygous loss for the dopamine β-hydroxylase gene attenuated the reduction in bone loss by about 29.7% after hind limb unloading (Fig. 9a, column 3 versus column 4). The rate of bone loss due to unloading (calculated as (control load – unload)/(control load × 100%) was significantly reduced from 68 ± 7% in DBH+/+ (wild type) to 30 ± 24% in DBH+/– (Fig. 9c)(p < 0.05). The nodule formation in cultures of bone marrow cells of the wild type littermate was reduced by unloading (Fig. 9b, column 1 versus column 2). The marrow cells obtained from DBH gene heterozygous knockout mice indicated suppression of hind limb unloading-induced reduction in nodule formation (Fig. 9b, column 3 versus column 4). During the course of unloading-induced bone loss, bone resorption also occurs as critical events to reduce bone mass. Unloading in tail-suspended mice caused an increase in osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS) based on histomorphometry in vivo as reported previously (Fig. 10, b–g, column 1 versus column 2). In contrast, inhibition of sympathetic tone by treatment with propranolol or guanethidine, suppressed such unloading-induced increase in osteoclast number (Oc.N/BS) (Fig. 10, a and b for propranolol, d for guanethidine; column 3 versus column 4) and osteoclast surface (Oc.S/BS) (Fig. 10, c for propranolol, e for guanethidine; column 3 versus column 4). The levels of Oc.N/BS and Oc.S/BS were enhanced by either unloading or isoproterenol treatment alone to similar levels (Fig. 10, f and g, column 2 versus column 3). The simultaneous presence of unloading conditions and isoproterenol treatment resulted in similar levels in the increase in osteoclast number (Oc.N/BS) and surface (Oc.S/BS) to those in mice subjected to either one of the two conditions alone (Fig. 10, f and g, column 4 versus columns 2 and 3). Furthermore, unloading-induced increase in deoxypyridinoline excretion into urine (Fig. 11, column 1 versus column 2) was also suppressed by guanethidine treatment (Fig. 11, column 3 versus column 4).Fig. 11Pharmacological inhibition of sympathetic tone suppresses unloading-induced systemic bone loss assessed by the levels of urinary deoxypyridinoline excretion. Mice were subjected to hind limb unloading and guanethidine treatment as described under "Materials and Methods." After 14 days, urine samples of the mice were collected, and the amount of deoxypyridinoline in the urine was measured as described under "Materials and Methods." *, statistically significant difference (p < 0.05). The number of mice in each group is indicated as N. #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups). §, statistically significant difference between vehicle-treated unloaded group and drug-treated control-loaded group (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our data reveal that sympathetic nervous tone is mediating unloading-induced bone loss via reduction in osteoblastic cell activity as well as enhancement in osteoclastic cell activity. This is the first report that sympathetic control of the bone mass is involved in the unloading-induced bone loss by controlling osteoblasts. Unloading-induced bone loss was suppressed by the treatment of the animals with propranolol, a receptor antagonist, suggesting that β-adrenergic receptors in the sympathetic nervous system are the target of unloading-induced bone loss. The peripheral sympathetic nervous targets receive signals from the proximal upper nervous ending, which releases noradrenalin as a neurotransmitter into the synaptic gap. We observed that unloading-induced bone loss was again suppressed by the treatment of the animals with guanethidine. These data indicate that the depletion of noradrenalin in the proximal ending of the synaptic gap could suppress the unloading-induced osteoporosis. Furthermore, unloading failed to further suppress the bone volume that was reduced by a β-adrenergic agonist, isoproterenol. These three series of observations further indicate that sympathetic nervous tone is involved in unloading-induced pathological bone loss. We also examined the effects of heterozygous deletion of the DBH gene. DBH is required for the sympathetic nervous tone. Therefore, we subjected the heterozygous knockout mice to hind limb unloading. Unloading-induced bone loss was attenuated by the absence of the half-dosage of dopamine β-hydroxylase gene, indicating that the presence of a full dosage of DBH gene in the animals is necessary for the complete effects of the unloading-induced bone loss. Furthermore, DBH data excluded the possibility that pharmacological experiments might be influenced by possible artifacts due to the systemic drug administration. Thus, both pharmacological and genetic interventions of sympathetic signals supported the idea that the sympathetic nervous system causes pathological loss of bone in unloading-induced osteopenia. Since bone formation is the critical activity to determine the levels of bone loss due to unloading, it is the major target to elucidate the mechanisms required for unloading-induced loss of bone mass. Dynamic histomorphometric analyses on osteoblastic cell activity in vivo revealed that the reduction in bone formation activity in vivo due to unloading was suppressed by a series of pharmacological agents including propranolol and guanethidine. Furthermore, bone cell culture experiments using the bone marrow cells taken from the animals subjected to either pharmacological or genetic interventions of the sympathetic nervous tone indicate that these interventions suppressed unloading-induced reduction in mineralized nodule formation. The interpretation of the reduction in the formation of osteoblastic bone nodules after 3 weeks in culture would be that progenitor cell populations for osteoblastic cell lineage could be reduced at the point of harvesting the cells from animals at the end of unloading. This suppression was blocked by the treatment with propranolol. We also carried out 3-week culture experiments to form bone nodules in the presence or absence of isoproterenol or propranolol and guanethidine in culture using bone marrow cells that were taken from wild type (untreated) C57Bl6 mice. There was no effect of these agents in culture to modulate the nodule formation in the bone marrow cells from untreated mice (data not shown). These data support the idea that the progenitor population in bone marrow in vivo would be reduced by the unloading condition or by treatment with drugs such as propranolol, guanethidine, and isoproterenol during the periods of the tail suspension experiments. Therefore, the action of sympathetic nervous tone during unloading-induced bone loss renders cell level effects on osteoblastic cell activity. It is known that propranolol at doses of 10 μg/g body weight could reduce blood pressure in mice. It is also known that such blood pressure change could be enhanced by the treatment with isoproterenol. However, there has yet been no clear evidence in clinical or experimental settings in terms of a direct relationship between blood pressure and bone mass. However, at this point, we cannot fully exclude the possibility that propranolol or isoproterenol used in our experiments may have affected bone mass via regulation of blood pressure, and these points have to be elucidated in the future. It is intriguing to consider the differences in the time courses of the β-adrenergic receptor modulators. Propranolol has been known to reduce blood pressure in days, whereas isoproterenol and guanethidine alter blood pressure immediately. It is not known whether such time course differences seen in their effects on blood pressure may also affect their modulation of the bone mass. However, both propranolol and guanethidine blocked unloading-induced bone loss similarly in our experiments. This may partially be due to the nature of bone where the biological read out (i.e. bone mass) could be detected in a relatively slow manner compared with blood pressure. If these types of drugs could be proven to be effective in the treatment of unloading-induced osteoporosis, it has to be considered that side effects might occur in the treatment of the patients. In the future, we may have to identify certain windows of the dosages by which bone effects may be obtained without affecting the blood pressure, or these drugs may be used only for those patients whose side effects could be predicted to be less based on the individual genomic data. Our data indicated that sympathetic tones regulate unloading-induced enhancement in bone resorption. This was evidenced by the observations that inhibition of sympathetic tone by propranolol and guanethidine, suppressed unloading-induced bone resorption, and this leads to suppression in bone loss. Histomorphometric analyses indicated that unloading activates bone resorption through the increase in osteoclast number and osteoclast surface. These increases in the bone resorption parameters due to unloading were suppressed by treatment with propranolol and guanethidine. Such observations regarding the inhibitors for sympathetic tone effects on unloading-induced bone resorption were not limited to particular bone, since suppression of sympathetic tone by the treatment with guanethidine also suppressed unloading-induced increase in deoxypyridinoline excretion into urine, which is a systemic bone resorption maker. These data revealed that sympathetic tone regulates unloading-induced bone resorption as well. Involvement of sympathetic tone in the induction of bone resorption after unloading was further supported by the analysis on the mice subjected to simultaneous unloading and isoproterenol treatment. Either isoproterenol treatment or unloading alone could cause an equivalent increase in the levels of bone loss as well as an increase in the levels of bone resorption parameters (osteoclast number and osteoclast surface). The simultaneous presence of unloading conditions and isoproterenol treatment resulted in an increase in bone loss as well as an increase in osteoclast number and osteoclast surface to levels equivalent to those in the cases of either one of the two conditions alone. These data further support the notion that sympathetic nervous tone and the unloading condition would share signaling pathways. Unloading induces rapid bone loss and increases fracture risk significantly, especially in elderly bedridden patients. In fact, deoxypyridinoline excretion into urine was reported to increase in astronauts within a few hours after they are exposed to microgravity conditions in space. Such rapid bone loss is caused by an immediate response of osteoclastic activity when the body is subjected to unloading conditions. Although this phenomenon is so clearly observed in human (bedridden patients and astronauts) and various animal models, the mechanism for such a response of bone resorbing activity has not been identified. Since the nervous system could elicit signals at a relatively fast speed, our identification of the sympathetic tone as responsible for the unloading-induced increase in bone resorption and loss of bone mass would explain the rapid response of osteoclasts to unloading. Although we have used DBH knockout mice to see the effects of such a deletion of the gene on the bone loss due to tail suspension, this enzyme is also required to produce epinephrine in the adrenals as well as norepinephrine. The enzyme required to produce epinephrine from norepinephrine, phenylethanolamine-N-methyltransferase, has been suggested in several reports to be subjected to induction by immobilization-induced stress. As a result, the DBH heterozygous knock-out mice results may not represent conclusive proof of a prominent role for the sympathetic nervous system here. However, our data on the effects of unloading on bone in DBH knockout mice is at least in part in accordance with the idea that sympathetic tone is involved in the bone loss due to unloading. In conclusion, our data indicate that sympathetic tone is in charge of the pathological reduction in bone mass upon unloading by suppressing osteoblastic cell actions and enhancing osteoclastic actions. These data predict that identification of the involvement of systemic modulation in the bone loss in unloading conditions could give a clue to appropriate measures to treat patients with disuse osteoporosis (9Serhan C.N. N. Engl. J. 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