Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling
2007; Elsevier BV; Volume: 283; Issue: 4 Linguagem: Inglês
10.1074/jbc.m702687200
ISSN1083-351X
AutoresHong Zhou, Wendy Mak, Yu Zheng, Colin R. Dunstan, Markus J. Seibel,
Tópico(s)Fibroblast Growth Factor Research
ResumoLineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and β-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymal progenitor through Wnt signaling. This glucocorticoid-dependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis. Lineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and β-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymal progenitor through Wnt signaling. This glucocorticoid-dependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis. Mesenchymal stem cells are able to differentiate to multiple connective tissue and musculoskeletal cell types under the control of specific transcription factors directing their lineage commitment. For example, osteoblasts and adipocytes share a common mesenchymal cell progenitor that can be isolated from neonatal mouse or fetal rat calvaria (1Bellows C.G. Heersche J.N. J. Bone Miner. Res. 2001; 16: 1983-1993Crossref PubMed Scopus (36) Google Scholar). Primary cell cultures derived from these tissues can differentiate into osteoblasts or adipocytes. The differentiation of these mesenchymal progenitors into osteoblasts or adipocytes is governed by the expression of the master transcription factors runt-related gene 2 (Runx2) (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar) or peroxisome proliferator-activated receptor γ (PPARγ) 2The abbreviations used are: PPARγperoxisome proliferator-activated receptor γGCglucocorticoid11βHSD11β-hydroxysteroid-dehydrogenaseCol2.3–11βHSD211βHSD type 2 transgene is driven by 2.3-kilobase collagen type I promoterWTwild typetgtransgenicRunx2runt-related gene 2C/EBPαCCAAT/enhancer-binding protein αsFRP1secreted Frizzled-related protein 1SOSTsclerostinGFPgreen fluorescent proteinRTreverse transcriptionTCFT cell factorCMconditioned mediumBMPbone morphogenetic protein. (3Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar), respectively. However, the cellular interactions and mechanisms that must drive the expression of these transcription factors are poorly understood. peroxisome proliferator-activated receptor γ glucocorticoid 11β-hydroxysteroid-dehydrogenase 11βHSD type 2 transgene is driven by 2.3-kilobase collagen type I promoter wild type transgenic runt-related gene 2 CCAAT/enhancer-binding protein α secreted Frizzled-related protein 1 sclerostin green fluorescent protein reverse transcription T cell factor conditioned medium bone morphogenetic protein. Glucocorticoid (GC) signaling through its cognate receptor is known to influence osteoblast and adipocyte lineage commitment both in vitro and in vivo and is, thus, likely to have a role in regulating cellular interactions driving cell commitment. In addition, specific enzymes modulate GC metabolism within the cell at the pre-receptor level (4Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (448) Google Scholar, 5Draper N. Stewart P.M. J. Endocrinol. 2005; 186: 251-271Crossref PubMed Scopus (318) Google Scholar). Within certain tissues, two isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD) vary intracellular GC concentrations independent of circulating GC levels. 11βHSD type 1 (11βHSD1) predominantly converts inactive cortisone to active cortisol to increase intracellular GC concentrations; in contrast, 11βHSD type 2 (11βHSD2) unidirectionally catalyzes the conversion of active GC to their inactive metabolites (4Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Crossref PubMed Scopus (448) Google Scholar). Kream and co-workers (6Kalajzic Z. Liu P. Kalajzic I. Du Z. Braut A. Mina M. Canalis E. Rowe D.W. Bone (NY). 2002; 31: 654-660Crossref PubMed Scopus (120) Google Scholar) generated a Col2.3–11βHSD2 transgenic (tg) mouse in which the rat gene for 11βHSD2 was linked to the 2.3-kilobase collagen type I (Col2.3) promoter to target transgene expression to mature osteoblasts (6Kalajzic Z. Liu P. Kalajzic I. Du Z. Braut A. Mina M. Canalis E. Rowe D.W. Bone (NY). 2002; 31: 654-660Crossref PubMed Scopus (120) Google Scholar). The Col2.3 promoter has been well characterized and specifically targets gene expression to mature osteoblasts (7Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (321) Google Scholar, 8Kalajzic I. Staal A. Yang W.P. Wu Y. Johnson S.E. Feyen J.H. Krueger W. Maye P. Yu F. Zhao Y. Kuo L. Gupta R.R. Achenie L.E. Wang H.W. Shin D.G. Rowe D.W. J. Biol. Chem. 2005; 280: 24618-24626Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Wang Y.H. Liu Y. Buhl K. Rowe D.W. J. Bone Miner. Res. 2005; 20: 5-14Crossref PubMed Google Scholar, 10Cochrane R.L. Clark S.H. Harris A. Kream B.E. Genesis. 2007; 45: 17-20Crossref PubMed Scopus (14) Google Scholar). Green fluorescent protein (GFP) transgenic mice using the same fragment of the type I collagen promoter to drive GFP expression (2.3Col-GFP) demonstrated that only cells with features of mature osteoblasts show fluorescence in vivo and in vitro (7Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (321) Google Scholar, 9Wang Y.H. Liu Y. Buhl K. Rowe D.W. J. Bone Miner. Res. 2005; 20: 5-14Crossref PubMed Google Scholar). Microarray analysis data on this population of GFP-positive cells show it to be highly enriched for expression of mature osteoblast-specific genes such as osteocalcin, bone sialoprotein (BSP), and dentin matrix protein-1 (8Kalajzic I. Staal A. Yang W.P. Wu Y. Johnson S.E. Feyen J.H. Krueger W. Maye P. Yu F. Zhao Y. Kuo L. Gupta R.R. Achenie L.E. Wang H.W. Shin D.G. Rowe D.W. J. Biol. Chem. 2005; 280: 24618-24626Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Similarly, immunohistochemistry studies localized transgenic 11βHSD2 protein expression to mature osteoblasts and osteocytes in the bones of transgenic mice with no expression in bones derived from wild type littermates. Transgenic 11βHSD2 enzyme activity was confirmed in vitro by measuring the conversion of [3H]corticosterone to 11-dehydrocorticosterone (11Sher L.B. Woitge H.W. Adams D.J. Gronowicz G.A. Krozowski Z. Harrison J.R. Kream B.E. Endocrinology. 2004; 145: 922-929Crossref PubMed Scopus (106) Google Scholar). Because overexpression of HSD2 results in the inactivation of cytoplasmic GCs, GC signaling is, thus, effectively disrupted in mature osteoblasts (11Sher L.B. Woitge H.W. Adams D.J. Gronowicz G.A. Krozowski Z. Harrison J.R. Kream B.E. Endocrinology. 2004; 145: 922-929Crossref PubMed Scopus (106) Google Scholar, 12Sher L.B. Harrison J.R. Adams D.J. Kream B.E. Calcif. Tissue Int. 2006; 79: 118-125Crossref PubMed Scopus (68) Google Scholar) in Col2.3–11βHSD2 transgenic mice. Col2.3–11βHSD2 transgenic mice have been shown to exhibit vertebral osteopenia in females (11Sher L.B. Woitge H.W. Adams D.J. Gronowicz G.A. Krozowski Z. Harrison J.R. Kream B.E. Endocrinology. 2004; 145: 922-929Crossref PubMed Scopus (106) Google Scholar), reduced femoral cortical bone area and thickness, and impaired mineralized nodule formation in primary calvarial cultures. However, the mechanism underlying these phenotypic changes remained unclear (12Sher L.B. Harrison J.R. Adams D.J. Kream B.E. Calcif. Tissue Int. 2006; 79: 118-125Crossref PubMed Scopus (68) Google Scholar). Others have targeted 11βHSD2 overexpression to osteoblasts/osteocytes with the osteocalcin gene 2 promoter. These transgenic mice had no discernible bone phenotype, but osteocyte viability was protected during GC treatment (13O'Brien C.A. Jia D. Plotkin L.I. Bellido T. Powers C.C. Stewart S.A. Manolagas S.C. Weinstein R.S. Endocrinology. 2004; 145: 1835-1841Crossref PubMed Scopus (637) Google Scholar). In the present study using primary calvarial cell cultures derived from the Col2.3–11βHSD2 tg mice, we discovered a novel mechanism in which fully differentiated osteoblasts control lineage commitment and differentiation of mesenchymal progenitor cells. We find that GC-regulated Wnt signaling is a strong candidate for mediating this function. Transgenic Mice—Col2.3–11βHSD2 transgenic mice were generated as described previously (11Sher L.B. Woitge H.W. Adams D.J. Gronowicz G.A. Krozowski Z. Harrison J.R. Kream B.E. Endocrinology. 2004; 145: 922-929Crossref PubMed Scopus (106) Google Scholar) and were provided as a gift by Dr. Barbara Kream (Dept. of Medicine, University of Connecticut Health Center, Farmington, CT). Col2.3-GFP mice (7Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (321) Google Scholar) were provided as a gift by Dr. David Rowe (Dept. of Genetics and Developmental Biology, University of Connecticut Health Center). Mice were maintained at the animal facilities of the ANZAC Research Institute (Sydney, Australia) in accordance with Institutional Animal Welfare Guidelines and according to an approved protocol. Primary Calvaria Cell Culture—Primary calvaria cells were generated from 1-day-old wild type (WT) or Col2.3–11βHSD2 tg littermates. Cells were released by 4 sequential 10-min digestions with 0.1% collagenase (Worthington Biomedical Co., Lakewood, NJ) and 0.2% dispase (Invitrogen). Cell populations from digestions 2 to 4 were collected and pooled (P2–4). For mineralized nodule formation assays, cells were cultured in 24-well plates at a density of 1 × 105/well in α-minimum essential medium (Invitrogen) containing 10% fetal bovine serum (FBS) and cortisol at a concentration of 8 × 10–9 m (derived from the FBS). Cells were allowed to attach for 24 h, and then osteogenic conditions were provided to induce osteoblast differentiation (designated day0) by adding ascorbic acid (50 μg/ml; Sigma) and β-glycerophosphate (10 mm; Sigma). Cells were incubated for 14 days with fresh medium provided three times weekly. Transwell Co-culture—Calvaria cells were isolated by sequential enzyme digestion yielding four populations (1Bellows C.G. Heersche J.N. J. Bone Miner. Res. 2001; 16: 1983-1993Crossref PubMed Scopus (36) Google Scholar). Cells collected from the first digestion, designated population 1 (P1), were cultured in 24-well plates. Cells collected from third and fourth digestions were pooled and designated as populations 3 and 4 (P3–4). This population or cells was cultured in transwell inserts (pore size 3 μm, BD Biosciences) overnight and then were transferred in the inserts to the 24-well plates containing P1 cells. Osteogenic conditions were then provided in the cultures by adding ascorbic acid and β-glycerophosphate as above, and cultures were maintained for 21 days. To evaluate the potential role for secreted Wnt proteins in the transwell system, secreted frizzled-related protein 1 (sFRP1) (R&D Systems) was added at a concentration of 200 ng/ml to the media, which was changed every second day. Cell inserts were then removed from plates, and P1 cells were fixed and stained for mineralized nodules and adipocyte lipids. Staining—Mineralized nodules were assessed by staining with 1% alizarin red (Sigma) after fixation in 70% ethanol (14Stanford C.M. Jacobson P.A. Eanes E.D. Lembke L.A. Midura R.J. J. Biol. Chem. 1995; 270: 9420-9428Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). The nodules were then imaged by scanner and were analyzed using image analysis software (ImageJ, National Institutes of Health) to determine the number and area of mineralized nodules. Adipocyte lipid was assessed by staining cells with Oil Red O (Sigma) after fixation in 4% paraformaldehyde. The Oil Red O-stained adipocytes were counted in each well in a standard area of 157 mm2. Staining by Oil Red O was further quantified by spectrophotometry. Briefly, the stained dishes were rinsed free of residue stain and eluted with 1 ml of 100% isopropanol for 20 min at room temperature. Three aliquots (100 μl) of the eluate were transferred to a 96-well plate and quantified by absorbance measurement at 490 nm (PerkinElmer Life Sciences). The linear range of detection was determined by standard solutions of Oil Red O. Wnt3a-conditioned Medium—Cell lines of murine subcutaneous connective tissue-derived L-fibroblasts permanently transfected with Wnt3a (L-Wnt3a) or an empty vector (L) were obtained from American Type Culture Collection (ATCC; Manassas, VA). Control and Wnt3a-conditioned media were prepared from Land L-Wnt3a cells, respectively, as described by the ATCC. Briefly, cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum for 3 days, after which conditioned medium was collected, filter-sterilized, and stored at –70 °C until further use. RT-PCR and Real-time RT-PCR—Total RNA was isolated from primary mouse calvaria cell cultures of populations P1 and P2–4 using NucleoSpin (Machery-Nagel, Easton, PA) according to the manufacturer's instructions. First-strand cDNA was synthesized from 2 μg of total RNA by incubating for 1 h at 50°C with Superscript III reverse transcriptase (Invitrogen) after oligo(dT) priming. The sequences of primers were used are listed in Table 1. PCR was conducted for 20–30 cycles, each of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Ten microliters of each reaction mixture was analyzed by 2% agarose gel electrophoresis. Real-time RT-PCR was carried out using IQ SYBR Green Supermix (Bio-Rad) according to manufacturer's instructions using a Bio-Rad iCycler iQ5 real-time PCR detection system. 18 S was used for cDNA normalization. All primers sequences are listed in Table 1.TABLE 1Mouse-specific primer pairs used for RT-PCR and real-time RT-PCRGeneForward PrimerReverse primer11βHSD25′-TTTGGCAAGGAGACAGCTAA-3′5′-TTCTCCCAGAGGTTCACATT-3′11βHSD15′-AGTTTGCTCTGGATGGGTTC-3′5′-TGAGGTAAAGCCGATACAACC-3′SGK5′-GTTCTACCATCTCCAGAGGGA-3′5′-TGCCACAGAAGGTAGATGTTG-3′ALP5′-CCAGCAGGTTTCTCTCTTGG-3′5′-CTGGGAGTCTCATCCTGAGC-3′BSP5′-AAAGTGAAGGAAAGCGACGA-3′5′-TGAAACCCGTTCAGAAGGAC-3′Osteocalcin5′-CCTTCATGTCCAAGCAGGA-3′5′-TGCTGTGACATCCATACTTGC-3′Runx-25′-CCTGAACTCTGCACCAAG-TC-3′5′-CCCAGTTCTGAAGCACCTG-3′PPARγ5′-TTTTCCGAAGAACCATCCGAT-3′5′-ACAAATGGTGATTTGTCCGTTG-3′C/EBPα5′-GATAAAGCCAAACAACGCAACG-3′5′-CTAGAGATCCAGCGACCCGAA-3′Wnt10b5′-CATCTTTATCGATACCCACAACC-3′5′-CACATAACAGCACCAGTGGAA-3′Wnt7b5′-TCCAAGGTCAACGCAATG-3′5′-GGGAAGGGTGTCCTCAAATAG-3′TCF75′-AGCACACTTCGCAGAGACTTT-3′5′-GTGGACTGCTGAAATGTTCG-3′BMP25′-AGATGAACACAGCTGGTCACA-3′5′-GTTCAGGTGGTCAGCAAGG-3′BMP45′-TCAGAATCAGCCGATCGTT-3′5′-GCAGTAGAAGGCCTGGTAGC-3′SOST5′-ACCGCCTAGAAGAAGCTGTTT-3′5′-CAGTTTCCTCCAACGACCTT-3′GR5′-GTTCATGGCGTGAGTACCTC-3′5′-AGAGTTTGGGAGGTGGTCC-3′18 S5′-CATGATTAAGAGGGACGGC-3′5′-TTCAGCTTTGCAACCATACTC-3′ Open table in a new tab Western Blotting—The total cellular protein levels of β-catenin were detected as described (15Young C.S. Kitamura M. Hardy S. Kitajewski J. Mol. Cell. Biol. 1998; 18: 2474-2485Crossref PubMed Google Scholar) with minor modifications. Briefly, primary calvaria cells derived from 1-day-old WT and tg mice were cultured in osteogenic media for 7 days. Total cellular protein was isolated from cell lysates in lysis buffer (1% Triton X-100, 40 mm KCl, 25 mm Tris, pH 7.4, protease inhibitor, Roche Applied Science). To isolate nuclear extract (16van den Brink G.R. Bleuming S.A. Hardwick J.C. Schepman B.L. Offerhaus G.J. Keller J.J. Nielsen C. Gaffield W. van Deventer S.J. Roberts D.J. Peppelenbosch M.P. Nat. Genet. 2004; 36: 277-282Crossref PubMed Scopus (318) Google Scholar), cells were lysed in ice-cold lysis buffer, cell lysates were passed through a 27-gauge needle and centrifuged at 8000 × g for 3 min, and the supernatant was recovered as the cytoplasmic fraction. The remaining pellet containing the nuclear fraction was spun for an additional 15 min at 15,000 × g, washed with lysis buffer, and resuspended in sucrose buffer (250 mm sucrose, 20 mm Tris, pH 7.4, and protease inhibitor). Both cellular and nuclear proteins were separated by SDS-PAGE (7.5% polyacrylamide) and transferred onto nitrocellulose filters. The filters were immunoblotted with anti-β-catenin antibody (BD Biosciences) at a 1:500 dilution or anti-β-actin antibody (BD Biosciences) at a 1:2000 dilution as an internal control. Protein expression was visualized with a peroxidase-labeled sheep antimouse secondary antibody by enhanced chemiluminescence detection reagents (Amersham Biosciences) and by exposure to x-ray film. Statistical Analysis—Data are represented as the means ± S.E. of the mean, and statistical analysis was performed with Student's t test. For multiple comparisons the p value was adjusted using the Bonferroni method. A p value of less than 0.05 was considered statistically significant. Osteoblast Differentiation Is Inhibited, and Adipocyte Differentiation Is Increased in Col2.3–11βHSD2 tg Culture—Primary osteoblast cultures were generated from the calvaria of 1-day-old Col2.3–11βHSD2 tg mice and WT littermates and grown under osteogenic conditions (see "Experimental Procedures"). Cultures derived from Col2.3–11βHSD2 tg mice exhibited a 60% reduction in mineralized nodule formation when compared with WT cultures (Fig. 1, A and B). In the same cultures, a significant increase in adipocyte numbers was observed when compared with WT cultures, in which only a few adipocytes formed (Fig. 1C). The Oil Red O content of cultures was found to be at least 2 times higher in tg cultures compared with WT cultures (Oil Red O elution A490: WT 0.21 ± 0.01, tg 0.47 ± 0.03, p < 0.001). In light of the fact that the transgene is exclusively expressed in mature osteoblasts (7Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (321) Google Scholar, 11Sher L.B. Woitge H.W. Adams D.J. Gronowicz G.A. Krozowski Z. Harrison J.R. Kream B.E. Endocrinology. 2004; 145: 922-929Crossref PubMed Scopus (106) Google Scholar), these results indicate that early precursor cells derived from transgenic, 11βHSD2-overexpressing animals experience a major shift in lineage commitment from osteoblast to adipocyte. The Shift in Lineage Commitment Is Due to Disrupted GC Signaling in Mature Osteoblasts—11βHSD2 mRNA expression was only detected in Col2.3–11βHSD2 tg cultures. WT cultures expressed neither rat transgene or native mouse 11βHSD2 (primers used could detect both rat transgene and native mouse 11βHSD2). Increased 11βHSD2 expression levels were observed in tg cultures from day 3 to day 7; this finding is consistent with the expected increase in the number of differentiated osteoblasts during culture (Fig. 2, A and B). mRNA for 11βHSD1 was expressed at very low levels and similarly in tg and WT cultures, with a minor increase at day 7. Thus, 11βHSD2 is the major enzyme regulating GC signaling in cultures derived from Col2.3–11βHSD2 animals, and the expression of 11βHSD1 is not regulated by 11βHSD2. mRNA for the glucocorticoid receptor was similarly expressed in both WT and tg cultures and was not regulated by 11βHSD2 (data not shown). The increase in 11βHSD2 mRNA was accompanied by a reciprocal decrease in mRNA for serum and glucocorticoid-induced kinase, a GC target gene (17Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (639) Google Scholar), indicating that GC signaling is attenuated in Col2.3–11βHSD2 tg cultures (Fig. 2C). Thiram, a dithiocarbamate that inhibits 11βHSD2 but not 11βHSD1 enzyme activity (18Atanasov A.G. Tam S. Rocken J.M. Baker M.E. Odermatt A. Biochem. Biophys. Res. Commun. 2003; 308: 257-262Crossref PubMed Scopus (81) Google Scholar) was added to WT and tg cultures. At a concentration of 10–10 m, Thiram increased mineralized nodule formation to levels seen in WT cultures. In the same cultures, Thiram dose-dependently inhibited adipocyte formation (Fig. 2F). Thiram had no effect on WT cultures at any concentration used (Fig. 2, D–F). Hence, the Col2.3–11βHSD2 tg culture phenotype could be completely reversed by blocking 11βHSD2 enzyme activity in the culture system. Taken together, these results indicate that the shift in lineage commitment from osteoblast to adipocyte in tg cultures is due to disrupted GC signaling in mature osteoblasts overexpressing 11βHSD2. Corticosterone or dexamethasone, substrates for 11βHSD2 (19Ferrari P. Smith R.E. Funder J.W. Krozowski Z.S. Am. J. Physiol. 1996; 270: E900-E904PubMed Google Scholar, 20Woitge H. Harrison J. Ivkosic A. Krozowski Z. Kream B. Endocrinology. 2001; 142: 1341-1348Crossref PubMed Scopus (28) Google Scholar), were added to tg and WT cultures at concentrations of 10–10–10–7 m. As expected, neither steroid had any effect at low concentrations (10–10–10–8 m), but each inhibited mineralized nodule formation at a high concentrations (10–7 m) in both WT and tg cultures (data not shown). Transcription Factors for Osteoblasts and Adipocytes Are Reciprocally Regulated in Col2.3–11βHSD2 Transgenic Culture—mRNA expression of three markers for osteoblast differentiation (alkaline phosphatase, bone sialoprotein, and osteocalcin) was similar in Col2.3–11β-HSD2 tg and WT cultures at day 1, indicating that the cell populations at the start of culture are nearly identical in both WT and tg cultures. However, at day 3 expression of each of these genes was markedly reduced in tg cultures compared with WT cultures, a finding that is consistent with inhibition of osteoblast differentiation (Fig. 3A). Expression of osteoblast-associated transcription factors, such as Runx2, increased with time in the WT cultures but remained substantially lower in tg cultures (Fig. 3, B and C). The expression of other osteoblast transcription factors, Twist2, Dlx5, and Osterix, were also lower in transgenic cultures after 7 days of differentiation (Fig. 3D). Conversely, mRNA expression of the adipogenic transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and PPARγ was increased at day 3 in Col2.3–11βHSD2 tg cultures but not in WT cultures (Fig. 3, B and C). These results indicate that disrupting GC signaling in osteoblasts affects mesenchymal precursor cell differentiation at a very early stage and promotes adipogenic differentiation. We, therefore, hypothesized the existence of a GC-regulated "paracrine" signal between mature osteoblasts and their mesenchymal progenitors that influences lineage commitment. We investigated this hypothesis further in transwell co-culture experiments. Osteoblastic Cells Produce Paracrine Factor(s) That Promote Osteoblastogenesis and Inhibit Adipogenesis—To define the cell populations representing early precursor cells and osteoblasts, we used calvarial cells derived from Col2.3-GFP tg mice in which GFP is driven by the same promoter as used for the 11βHSD2 transgene (7Kalajzic I. Kalajzic Z. Kaliterna M. Gronowicz G. Clark S.H. Lichtler A.C. Rowe D. J. Bone Miner. Res. 2002; 17: 15-25Crossref PubMed Scopus (321) Google Scholar). Calvarial cells from the first digestion were designated as P1; these cells were negative for the GFP signal after 7 days of induction of differentiation (Fig. 4A). The cells from later digestions, designated P3–4, showed few GFP-positive cells at day 1 but numerous cells differentiating into GFP-positive, mature osteoblasts at day 7 (Fig. 4A) These findings confirm that at the start of culture induction, P1 mainly contains early mesenchymal precursors, whereas P3–4 is rich in committed osteoblastic precursors that later differentiate into osteoblasts (21Suda N. Gillespie M.T. Traianedes K. Zhou H. Ho P.W. Hards D.K. Allan E.H. Martin T.J. Moseley J.M. J. Cell. Physiol. 1996; 166: 94-104Crossref PubMed Scopus (104) Google Scholar). We then isolated the P1 cell population from Col2.3–11βHSD2 tg mice and their WT littermates and found that these cells did not form mineralized nodules in either WT or Col2.3–11βHSD2 tg cultures after 21 days (Fig. 4, B and C). In contrast, cells from P3–4 populations formed many mineralized nodules in WT and tg cultures, although numbers were significantly lower in tg cultures (data not shown). P1 cells were then co-cultured with P3–4 cells of the same or reciprocal genotype seeded in the transwell inserts (Fig. 4B). P3–4 cells from WT mice induced nodule formation in cultures of both WT and Col2.3–11βHSD2 tg P1 cells. In contrast, P3–4 cells from tg mice failed to induce mineralized nodules in either WT or tg P1 cells (Fig. 4, B and C). P1 cells, without co-culture, readily differentiated into adipocytes at comparable levels in both WT and Col2.3–11βHSD2 tg cultures (Fig. 4D). In the co-cultures, adipocyte formation in WT or tg P1 cells was inhibited by about 90% by P3–4 cells derived from WT mice (Fig. 4D). These results clearly demonstrate that osteoblastic cells from WT mice produce soluble factor(s) that act on the precursor cells to promote osteoblastogenesis and inhibit adipogenesis. Although P3–4 cells from Col2.3–11β HSD2 tg mice failed to induce osteoblast differentiation (Fig. 4, B and C), a significant reduction in the number of adipocytes was observed in both WT and Col2.3–11βHSD2 tg P1 cells (Fig. 4D), although this effect was reduced relative to that seen with WT P3–4 cells. P1 cells derived from WT or Col2.3–11βHSD2 tg mice cultured alone formed similar numbers of adipocytes and had similar numbers of mineralized nodules induced by WT P3–4 cells (Fig. 4, C and D). This again suggests that both WT and Col2.3–11βHSD2 tg cultures consisted of similar cell populations and that P1 cells from WT and tg mice had the same potential to differentiate into either adipocytes or osteoblasts, depending on the strength of the signals received. Osteoblasts Control the Fate of Their Mesenchymal Precursors via the Canonical Wnt Signaling Pathway—The Wnt family consists of a large number of secreted glycoprotein-signaling molecules that are involved in regulating cell differentiation (22Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar, 23Moon R.T. Brown J.D. Torres M. Trends Genet. 1997; 13: 157-162Abstract Full Text PDF PubMed Scopus (546) Google Scholar) and plays an important role in regulating osteoblast differentiation (24Gaur T. Lengner C.J. Hovhannisyan H. Bhat R.A. Bodine P.V. Komm B.S. Javed A. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. 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