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Peroxisome proliferator-activated receptor gamma (PPARγ), known as the master regulator of adipogenesis, has been regarded as a promising target for new anti-osteoporosis therapy due to its role in regulating bone marrow mesenchymal stem/progenitor cell (BMSC) lineage commitment. However, the precise mechanism underlying PPARγ regulation of bone is not clear as a bone-specific PPARγ conditional knockout (cKO) study has not been conducted and evidence showed that deletion of PPARγ in other tissues also have profound effect on bone. In this study, we show that mice deficiency of PPARγ in cells expressing a 3.6 kb type I collagen promoter fragment (PPARfl/fl:Col3.6-Cre) exhibits a moderate, site-dependent bone mass phenotype. In vitro studies showed that adipogenesis is abolished completely and osteoblastogenesis increased significantly in both primary bone marrow culture and the BMSCs isolated from PPARγ cKO mice. Histology and histomorphometry studies revealed significant increases in the numbers of osteoblasts and surface in the PPARγ cKO mice. Finally, we found that neither the differentiation nor the function of osteoclasts was affected in the PPARγ cKO mice. Together, our studies indicate that PPARγ plays an important role in bone remodeling by increasing the abundance of osteoblasts for repair, but not during skeletal development.

International audience; Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

With the aging of the population, the scope of the problem of age-related bone loss and osteoporosis will continue to increase. As such, it is critical to obtain a better understanding of the factors determining the acquisition and loss of bone mass, from childhood to senescence. While there have been significant advances in recent years in our understanding of both the basic biology of aging and a clinical definition of age-related frailty, few of these concepts in aging research have been adequately evaluated for their relevance and application to skeletal aging or fracture prevention. The March 2011 “Forum on Aging and Skeletal Health”, sponsored by the NIH and ASBMR, sought to bring together leaders in aging and bone research to enhance communications among diverse fields of study so as to accelerate the pace of scientific advances needed to reduce the burden of osteoporotic fractures. This report summarizes the major concepts presented at this meeting and in each area, identifies key questions to help set the agenda for future research in skeletal aging.

The expression of inducible cAMP early repressor (ICER) has been demonstrated in cultured osteoblastic MC3T3-E1 cells, calvarial cultures and in vivo, mainly via the cAMP-PKA signaling pathway. To determine the molecular mechanism(s) for this action, a fragment of the CREM P2 promoter from -238 bp to +14 bp was linked to a luciferase reporter (CREMP2-Luc238). This region contains 2 clusters of cAMP-responsive element (CRE). The 5’ cluster contains CRE1 and CRE2 while the 3’ cluster contains CRE3 and CRE4 and the two clusters are separated by 12 bp. In order to study if any or all CREs are responsive to cAMP induction, osteoblastic MC3T3-E1 cells were stably transfected with constructs having serial deletions of each CRE or mutations in each cluster of the CREMP2-Luc construct. Agonists that stimulate the cAMP-PKA pathway induced CREMP2-Luc238 activity, and neither PKC nor Ca++ pathways did. The deletion of CRE1 and CRE2 did not significantly abolish the cAMP inducibility of CREMP2-Luc238 activity. Electrophoretic mobility shift assay showed many DNA/protein complexes when probes from either CRE1-2 or CRE3-4 were incubated with FSK-treated nuclear extract from MC3T3-E1 cells. However, only a small portion of the complexes was competed by a consensus CRE oligonucleotide. Interestingly, a major DNA/protein complex seen in the binding of CRE3-4 was not competed by the consensus CRE oligonucleotide, but by a mutated CRE oligonucleotide suggesting that other transcription factor(s) may be involved in the binding of CRE3-4. The CRE binding DNA/protein complexes were supershifted mainly by antibodies against ICER and CREB, mildly by C/EBPβ suggesting these transcription factors may be involved in the FSK induction of the P2 promoter of the CREM gene.

No one person in the world of bone has had a greater impactthan Larry on us all. To honor his memory, we asked some ofLarry’s closest friends and colleagues to reflect upon hisextraordinary life. What follows is an expression of that meaningsprinkled with Larry’s inimitable wit and reflections on life,science, and the future.

The goal of this study was to characterize the bone phenotype and molecular alterations in Col3.6-HSD2 mice in which a 3.6-kb Col1a1 promoter fragment drives 11beta-HSD2 expression broadly in the osteoblast lineage to reduce glucocorticoid signaling. Serum corticosterone was unchanged in transgenic females excluding a systemic effect of the transgene. Adult transgenic mice showed reduced vertebral trabecular bone volume and reduced femoral and tibial sub-periosteal and sub-endosteal areas as assessed by microCT. In adult female transgenic mice, histomorphometry showed that vertebral bone mass and trabecular number were reduced but that osteoblast and osteoclast numbers and the mineral apposition and bone formation rates were not changed, suggesting a possible developmental defect in the formation of trabeculae. In a small sample of male mice, osteoblast number and percent osteoid surface were increased but the mineral apposition bone formation rates were not changed, indicating subtle sex-specific phenotypic differences in Col3.6-HSD2 bone. Serum from transgenic mice had decreased levels of the C-terminal telopeptide of alpha1(I) collagen but increased levels of osteocalcin. Transgenic calvarial osteoblast and bone marrow stromal cultures showed decreased alkaline phosphatase and mineral staining, reduced levels of Col1a1, bone sialoprotein and osteocalcin mRNA expression, and decreased cell growth and proliferation. Transgenic bone marrow cultures treated with RANKL and M-CSF showed greater osteoclast formation; however, osteoclast activity as assessed by resorption of a calcium phosphate substrate was decreased in transgenic cultures. Gene profiling of cultured calvarial osteoblasts enriched in the Col3.6-HSD2 transgene showed modest but significant changes in gene expression, particularly in cell cycle and integrin genes. In summary, Col3.6-HSD2 mice showed a low bone mass phenotype, with decreased ex vivo osteogenesis. These data further strengthen the concept that endogenous glucocorticoid signaling is required for optimal bone mass acquisition and highlight the complexities of glucocorticoid signaling in bone cell lineages.

MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicer(c/c). In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in Dicer(Deltaoc/Deltaoc) was twice that of Dicer(c/c) controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700-5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665-3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1-34) (10-8M) or forskolin (FSK; 10-5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.

Parathyroid hormone (PTH) regulates gene expression in skeletal osteoblasts mainly through the cAMP-protein kinase A (PKA) pathway. In neuroendocrine cells, activation of the cAMP-PKA signaling pathway leads to induction of the inducible cAMP early repressor (ICER), which is transcribed from an intronic promoter of the CREM gene and acts as a transcriptional repressor. To investigate whether PTH induces ICER expression in osteoblastic cells, RNA from MC3T3-E1 cells was subjected to reverse transcriptase-polymerase chain reaction using primers spanning the ICER sequence. Amplified products were subcloned, sequenced, and used as a probe for Northern blot analysis. In MC3T3-E1 cells, PTH induced ICER mRNA levels, which peaked at 2 h and declined to baseline by 8 h. Cycloheximide caused superinduction of ICER mRNA in response to PTH. In cultured mouse calvariae, PTH also induced ICER mRNA accumulation, which peaked at 2 h and returned almost to baseline by 10 h. Overexpression of ICER IIgamma decreased both baseline and PTH-stimulated prostaglandin G/H synthase 2 promoter activity in MC3T3-E1 cells. The induction of ICER represents a novel mechanism by which PTH regulates gene expression in osteoblastic cells.

PTH increased PG synthase-2 transcription in osteoblastic MC3T3-E1 cells at 30 min, as assessed by nuclear run-on assays. To determine the signaling pathways used by PTH, the activity of a construct containing the PG synthase-2 gene between nucleotides -963 and +70 linked to a luciferase reporter was analyzed in stably transfected MC3T3-E1 cells. Agents that activate the cAMP-protein kinase A or protein kinase C pathways increased PG synthase-2 promoter activity. In contrast, the calcium ionophore ionomycin was ineffective. The protein kinase A inhibitor H89 blocked PTH stimulation of PG synthase-2 promoter activity, whereas an overnight pre-incubation with phorbol ester to down-regulate protein kinase C did not. PTH-(3-34), a peptide that has greatly reduced ability to activate the cAMP-protein kinase A pathway, did not increase PG synthase-2 transcription or promoter activity. PTH could induce PG synthase-2 messenger RNA accumulation and PG synthase-2 transcription in the presence of cycloheximide. In addition, PTH-stimulated PG synthase-2 transcription was maintained at a high level at 2 h in the presence of cycloheximide. We conclude that PTH rapidly increases PG synthase-2 transcription in MC3T3-E1 cells, mainly through a cAMP-protein kinase A-mediated pathway without the need for protein synthesis. In contrast, the attenuation of increased PG synthase-2 transcription by PTH requires de novo protein synthesis.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.

While the epidermal growth factor receptor (EGFR)–mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro–computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfrf/f), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre EgfrWa5/f mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit–fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, EgfrDsk5/+ mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism. © 2011 American Society for Bone and Mineral Research.

To directly compare the patterns of collagen promoter expression in cells and tissues, the activity of COL1A1 fusion genes in calvariae of neonatal transgenic mice and in primary bone cell cultures derived by sequential digestion of transgenic calvariae was measured. ColCAT3.6 contains 3.6 kb (positions -3521 to +115) of the rat COL1A1 gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. ColCAT2.3 and ColCAT1.7 are 5' deletion mutants which contain 2,296 and 1,672 bp, respectively, of COL1A1 DNA upstream from the transcription start site. ColCAT3.6 activity was 4- to 6-fold lower in primary bone cell cultures than in intact calvariae, while ColCAT2.3 activity was at least 100-fold lower in primary bone cells than in calvariae. These changes were accompanied by a threefold decrease in collagen synthesis and COL1A1 mRNA levels in primary bone cells compared with collagen synthesis and COL1A1 mRNA levels in freshly isolated calvariae. ColCAT3.6 and ColCAT2.3 activity was maintained in calvariae cultured in the presence or absence of serum for 4 to 7 days. Thus, when bone cells are removed from their normal microenvironment, there is parallel downregulation of collagen synthesis, collagen mRNA levels, and ColCAT3.6 activity, with a much greater decrease in ColCAT2.3. These data suggest that a 624-bp region of the COL1A1 promoter between positions -2296 and -1672 is active in intact and cultured bone but inactive in cultured cells derived from the bone. We suggest that the downregulation of COL1A1 activity in primary bone cells may be due to the loss of cell shape or to alterations in cell-cell and/or cell-matrix interactions that normally occur in intact bone.

We examined the effect of basic fibroblast growth factor (bFGF) on alpha 1(I) procollagen mRNA levels, alpha 1(I) collagen gene transcription, and alpha 1(I) collagen promoter activity in osteoblastic MC3T3-E1 cells. Cells were stably transfected with ColCAT 3.6, containing 3521 base pairs of alpha 1(I) collagen promoter DNA, fused to the CAT reporter gene, or an upstream deletion mutant of ColCAT 3.6 designated ColCAT 2.3. After 48 h, bFGF (0.1-10 nM) inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP). Indomethacin did not alter the inhibitory effect of bFGF on CDP labeling. Aphidicolin, an inhibitor of DNA synthesis, did not block the inhibitory effect of bFGF on CDP. bFGF (1-10 nM) decreased alpha 1(I) procollagen mRNA levels, with maximal inhibition, nearly 99% of control, caused by 10 nM bFGF. After 48 h, bFGF (1 nM) reduced alpha 1(I) procollagen gene transcription by about 92%. ColCAT 3.6 activity was inhibited with 0.1-10 nM bFGF and was maximally repressed by about 83% with 10 nM bFGF. In contrast, bFGF (1 and 10 nM) caused a stimulation of ColCAT 2.3 activity. These data show that bFGF inhibits collagen synthesis by a transcriptional mechanism and the alpha 1(I) collagen promoter contains DNA sequences which mediate bFGF inhibition of type I collagen gene expression in bone.

Background Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. Methodology/Principal Findings We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem−/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. Conclusion/Significance Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

Murine MC3T3-E1 and MC-4 cells were stably transfected with −371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at −57/−52 bp, (2) the activating protein-1 (AP-1)–binding site at −69/−63 bp, (3) the nuclear factor of activated T-cells (NFAT)–binding site at −77/−73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)–stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts. © 2010 American Society for Bone and Mineral Research

During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre, the BMP4 floxed and BMP4 knock-out mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process lead to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10–12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, are also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine role of odontoblasts derived BMP4 postnatally on the proper amelogenesis and formation of the enamel.

The cAMP response element modulator gene (Crem) encodes a variety of transcriptional regulators including the inducible cAMP early repressor, ICER. We previously showed that Crem knockout mice, which are deficient in CREM and ICER factors, display slightly increased long bone mass and decreased osteoclast number. These data are consistent with the notion that Crem regulates bone mass in part through an effect on osteoclast formation and/or function. Since ICER is strongly induced by cAMP, we asked whether the calcium-regulating hormone calcitonin, which stimulates cAMP production and inhibits osteoclastic bone resorption, could induce ICER in osteoclasts. The monocytic cell line RAW264.7 was treated with receptor activator of NF-kappaB ligand (RANKL) to induce osteoclast formation. Calcitonin caused a time- and dose-dependent induction of ICER mRNA and an increase in ICER protein abundance in RANKL-treated RAW264.7 cells. Calcitonin also induced ICER mRNA and protein in osteoclasts derived from primary mouse bone marrow cell cultures. Calcitonin-treated osteoclasts showed immunoreactivity with an anti-CREM antibody. Calcitonin decreased the activity of wild-type and Crem knockout osteoclasts in vitro, and this inhibitory effect was greater in Crem knockout osteoclasts. Furthermore, calcitonin decreased calcitonin receptor mRNA expression in wildtype osteoclasts, but not in Crem knockout osteoclasts. These data suggest that calcitonin induction of ICER in osteoclasts might regulate osteoclast activity.

CREM belongs to the ATF/CREB family of basic leucine zipper transcription factors. We previously showed that PTH induces ICER (inducible cAMP early repressor) in osteoblasts. ICER proteins, which are transcribed from the P2 promoter of the Crem gene, act as transcriptional attenuators. The objective of this study was to determine whether the Crem gene plays a role in the response of bone to intermittent PTH. Adult Crem knockout (KO) and wild type (WT) male mice were given daily subcutaneous injections of vehicle or hPTH(1–34) (160 μg/kg) for 10 days. Bone mineral content and density (BMC and BMD, respectively) were measured in femur and tibia by dual energy X-ray absorptiometry (DEXA). Bone morphometry was analyzed by X-ray computed microtomography (microCT) and histomorphometry. Serum bone turnover markers were measured. In vitro osteoclast formation assays were performed in bone marrow cultures treated with PTH or the combination of RANKL and M-CSF. KO mice had slightly higher basal bone mass than wild type mice. PTH treatment increased tibial BMC and BMD to a greater extent in WT mice compared to KO mice. PTH increased both cortical area and trabecular bone area in WT but not in KO femurs. PTH increased the bone formation rate and percent osteoblast surface to the same extent in femurs of WT and KO mice but increased osteoclast parameters and calvarial porosity to a greater extent in KO mice. PTH increased serum osteocalcin levels to the same extent in WT and KO mice. PTH-induced osteoclast formation was 2-fold greater in bone marrow cultures from KO mice. Collectively, our data suggest that the CREM deficiency in mice alters the response of bone to intermittent PTH treatment such that osteoclastogenesis is increased. Crem gene may specify the anabolic response to intermittent PTH treatment by restraining PTH-induced osteoclastogenesis.

The mechanism of androgen action on bone was studied in male mice with the AR deleted in mature osteoblasts. These mice had decreased trabecular bone volume associated with a decrease in trabecular number, suggesting that androgens may act directly on osteoblasts to maintain trabecular bone. Introduction: Androgens modulate bone cell activity and are important for the maintenance of bone mass. However, the mechanisms by which they exert these actions on bone remain poorly defined. The aim of this study was to investigate the role of androgens acting through the classical androgen receptor (AR) signaling pathways (i.e., DNA-binding dependent pathways) in osteoblasts using male mice in which exon 3 of the AR gene was deleted specifically in mature osteoblasts. Materials and Methods: Mice with a floxed exon 3 of the AR gene were bred with Col 2.3-cre transgenic mice, in which Cre recombinase is expressed in mineralizing osteoblasts. The skeletal phenotype of mutant mice was assessed by histomorphometry and quantitative μCT at 6, 12, and 32 weeks of age (n = 8 per group). Wildtype, hemizygous exon 3 floxed and hemizygous Col 2.3-cre male littermates were used as controls. Data were analyzed by one-way ANOVA and Tukey's posthoc test. Results: μCT analysis of the fifth lumbar vertebral body showed that these mice had reduced trabecular bone volume (p < 0.05) at 32 weeks of age compared with controls. This was associated with a decrease in trabecular number (p < 0.01) at 12 and 32 weeks of age, suggesting increased bone resorption. These effects were accompanied by a reduction in connectivity density (p < 0.01) and an increase in trabecular separation (p < 0.01). A similar pattern of trabecular bone loss was observed in the distal femoral metaphysis at 32 weeks of age. Conclusions: These findings show that inactivation of the DNA binding–dependent functions of the AR, specifically in mature osteoblasts in male mice, results in increased bone resorption and decreased structural integrity of the bone, leading to a reduction in trabecular bone volume at 32 weeks of age. These data provide evidence of a role for androgens in the maintenance of trabecular bone volume directly through DNA binding–dependent actions of the AR in mature osteoblasts.

The aim of this paper is identification of regulatory sequences downstream of -1683 base pairs (bp) in the rat Col1a1 promoter important for expression in osteoblasts. Previous findings suggest that a rat Col1a1 gene fragment extending from -1719 to + 115 bp linked to the chloramphenicol acetyl transferase (CAT) reporter gene (ColCAT1719) is highly and selectively expressed in osteoblasts. Three internal deletions within the ColCAT1719 construct were generated and stably transfected into ROS 17/2.8 cells. CAT activity was measured in cell extracts. An internal deletion of ColCAT1719 from -1637 to -504 bp caused an almost complete loss of CAT activity, whereas deletions of -1284 to -905 bp and -1284 to -451 bp had little effect on CAT activity. We hypothesized that removal of a Runx2/Cbfa1 consensus site at -1376 bp may have caused the loss of activity produced by the -1637 to -504 bp deletion. To test this hypothesis, we produced a more restricted internal deletion of ColCAT1719 from -1418 to -1284 bp, which removes this site. This deletion did not affect promoter activity. Our results suggest that the Runx2 site at -1376 bp by itself does not influence Col1719 promoter activity. Future studies will focus on the region between -1637 to 1418 bp, which contains several potentially interesting transcription factor binding sites.

We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) × C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre ; R26R and Col 3.6-Cre ; R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre ; R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.

Glucocorticoid excess leads to bone loss, primarily by decreasing bone formation. However, a variety of in vitro models show that glucocorticoids can promote osteogenesis. To elucidate the role of endogenous glucocorticoids in bone metabolism, we developed transgenic (TG) mice in which a 2.3-kb Col1a1 promoter fragment drives 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression in mature osteoblasts. 11β-HSD2 should metabolically inactivate endogenous glucocorticoids in the targeted cells, thereby reducing glucocorticoid signaling. The inhibitory effect of 300 nm hydrocortisone on percent collagen synthesis was blunted in TG calvariae, demonstrating that the transgene was active. Collagen synthesis rates were lower in TG calvarial organ cultures compared with wild-type. Trabecular bone parameters measured by microcomputed tomography were reduced in L3 vertebrae, but not femurs, of 7- and 24-wk-old TG females. These changes were also not seen in males. In addition, histomorphometry showed that osteoid surface was increased in TG female vertebrae, suggesting that mineralization may be impaired. Our data demonstrate that endogenous glucocorticoid signaling is required for normal vertebral trabecular bone volume and architecture in female mice.

The NAD-dependent enzyme, 11beta-hydroxysteroid dehydrogenase type II (11 beta HSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. In vivo, 11 beta HSD2 protects the mineralocorticoid receptor from activation by glucocorticoids in mineralocorticoid target tissues such as kidney. The goal of the present study was to use targeted overexpression of 11 beta HSD2 as a novel means of disrupting glucocorticoid signaling in osteoblastic cells. Rat 11 beta HSD2 complementary DNA was cloned downstream of a 2.3- and 3.6-kb alpha 1(I)-collagen (Col1a1) promoter fragment to produce the expression plasmids Col2.3-HSD2 and Col3.6-HSD2, respectively, which were transiently and/or stably transfected in osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Transgene messenger RNA and protein were detected in transfected cells by Northern blot analysis and immunostaining, respectively. Transfection of 11 beta HSD2 led to higher rates of conversion of [(3)H]corticosterone to [(3)H]dehydrocorticosterone and reduced glucocorticoid-dependent regulation of a mouse mammary tumor virus promoter-reporter construct, cell growth, and messenger RNA markers compared with transfection of a control vector. Expression of 11 beta HSD2 under the control of Col1a1 promoter fragments may provide a novel model to study the role of glucocorticoid signaling in osteoblastic cells.

The goals of this study were to examine the role of insulin-like growth factor I (IGF-I) on bone formation and to test the hypothesis that the inhibitory effects of glucocorticoids on bone formation are independent of the IGF-I pathway. In serum-free organ cultures of 18-day fetal mouse calvariae derived from Igf1 null mice (Igf1-/-) and their wild-type (Igf1+/+) and heterozygous (Igf1+/-) littermates, we measured the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), percent collagen synthesis (PCS), the incorporation of [3H]thymidine into DNA, and messenger RNA (mRNA) levels of osteoblast markers in the presence or absence of dexamethasone. After 24 h of culture, calvariae of all genotypes had similar levels of PCS. However, after 48-96 h of culture, PCS was significantly lower in Igf1-/- calvariae compared with Igf1+/+ calvariae. Treatment of calvariae with 100 nM of dexamethasone for 48-96 h decreased PCS in all genotypes. After 72 h of culture, [3H]thymidine incorporation was similar in all genotypes and 100 nM dexamethasone caused a significant reduction in [3H]thymidine incorporation in all genotypes. Dexamethasone at 100 nM decreased alpha1(I)-collagen (Colla1) mRNA and increased alkaline phosphatase, bone sialoprotein, and osteopontin mRNA in all genotypes after 72 h of culture. Type I IGF receptor mRNA levels were highest in Igf1-/- calvarial cultures. Dexamethasone at 100 nM increased Igf2 and type I IGF receptor mRNA levels in all genotypes. We conclude that one intact allele for Igf1 is sufficient to maintain normal rates of collagen synthesis in fetal mouse calvarial cultures. Moreover, the inhibitory effects of glucocorticoids on collagen synthesis and cell replication are at least partially independent of the IGF-I pathway in this model.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

To test the hypothesis that insulin-like growth factors (IGFs) play a role in the response of bone to glucocorticoids, we determined the effects of cortisol on the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), the percent collagen synthesis, and the incorporation of [3H]thymidine into DNA of 21-day fetal rat calvariae cultured in the presence and absence of recombinant human insulin-like growth factor binding protein-2 (IGFBP-2). At 24 h, cortisol (100 nM) increased CDP labeling and the percent collagen synthesis, and these effects were blocked by IGFBP-2 (1000 nM). At 24 h, cortisol decreased the incorporation of [3H]thymidine into bone, which was not affected by the addition of IGFBP-2. At 48 h, cortisol (1000 nM) decreased CDP labeling, which was maintained in the presence of IGFBP-2. At 48 h, IGFBP-2 alone decreased basal levels of CDP and NCP labeling and the percent collagen synthesis. Our data suggest that endogenous IGFs maintain basal levels of collagen synthesis and mediate the early stimulatory effect of glucocorticoids on collagen synthesis in fetal rat calvariae. However, blocking endogenous IGFs does not abrogate the inhibitory effect of glucocorticoids on DNA synthesis and the later inhibition of collagen synthesis in calvariae.

Prostaglandin G/H synthase (PGHS), a central enzyme for PG synthesis, is encoded by the constitutively expressed PGHS-1 and the inducible PGHS-2. The goal of this project was to study the regulation of PGHS-2 gene expression by PTH and its possible signaling pathways in osteoblastic MC3T3-E1 cultures. Bovine PTH-(1-34) at 0.01-10 nM increased PGHS-2, but not PGHS-1, messenger RNA (mRNA) levels. The effect of PTH was maximal at 1 h and decreased almost to control levels by 6 h. Phorbol myristate acetate (PMA), forskolin, and 8-bromo-cAMP increased PGHS-2 mRNA levels, whereas ionomycin had no effect. PTH, forskolin, and PMA increased the release of PGE2 into the culture medium. Pretreatment of cells with 0.1 microM PMA for 16 h blocked the induction of PGHS-2 mRNA levels by PMA, but did not alter the effects of PTH and forskolin. However, treatment of cells with 30 microM H-89, a protein kinase A inhibitor, significantly reduced the ability of PTH and forskolin to induce PGHS-2 mRNA levels. Moreover, PTH-(3-34) at 0.1-100 nM did not induce PGHS-2 mRNA levels. Our results show that PTH can rapidly and transiently induce PGHS-2 mRNA levels in osteoblastic MC3T3-E1 cells, primarily via the cAMP-protein kinase A signal transduction pathway. Induction of PGHS-2 may play a key role in mediating some actions of PTH on bone metabolism and gene expression.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.

High concentrations of prostaglandin E2 (PGE2) inhibit collagen synthesis and reduce alpha 1(I)procollagen messenger RNA (mRNA) levels in cultured fetal rat calvariae. To examine the mechanism of this effect, we used the immortalized rat osteoblastic clonal cell line, Py1a. PGE2 at 1 microM inhibited the incorporation of [3H]proline into collagenase-digestible protein (CDP) and increased incorporation into noncollagen protein, whereas 0.1 microM PGE2 increased both CDP and noncollagen protein labeling. Because insulin-like growth factor-I (IGF-I) is an anabolic hormone in bone and PGE2 can increase its production, we added exogenous IGF-I (10 nM) to Py1a cultures. In the presence of IGF-I, PGE2 from 10 nM to 1 microM had only an inhibitory effect on CDP labeling and alpha 1(I)procollagen mRNA levels. PGE2 at 1 microM decreased the rate of alpha 1(I)procollagen gene transcription in the presence or absence of IGF-I, determined by a nuclear run-on assay. Py1a cells were stably transfected with chimeric genes containing varying lengths of the 5'-upstream region of the rat alpha 1(I)procollagen promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. In cells transfected with ColCAT 3.6, which contains 3520 base pairs of 5'-upstream DNA, CAT activity was inhibited by PGE2, but the inhibition was less than that observed for CDP labeling. With smaller 5'-upstream regions, there was no inhibitory effect of PGE2. These results demonstrate that PGE2 inhibits alpha 1(I)procollagen gene transcription and the activity of a region between -3.5 and -2.3 kilobases of the 5'-upstream collagen gene promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24–72 h with 0.1 and 1 μM cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30 μM aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [3H]proline into collagenase-digestible protein (CDP) and [3H]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by >90%, and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of α-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC. The inhibitory effect of cortisol on CDP labeling was only partially reversed 48 h after removing cortisol from calvariae treated with hormone for 48 h. These data show that DNA synthesis inhibitors by themselves do not mimic the effects of cortisol on collagen synthesis nor do they prevent the early stimulation and the late inhibition of collagen synthesis by cortisol. Thus we conclude that the long-term inhibitory effect of cortisol on collagen synthesis in calvariae is largely independent of cell replication. The component of collagen synthesis that is reversible may represent the direct inhibitory effect of cortisol on differentiated osteoblasts. The nonreversible component of inhibition may be due to a more permanent effect of cortisol on cell populations within the calvariae. One possibility is that cortisol inhibits differentiation of osteoprogenitor cells to the osteoblast lineage leading to decreased osteoblast number and a subsequent inhibition of collagen production.

The effects of recombinant human interleukin-1 alpha (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid. Collagen synthesis was assessed as [3H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a 32P-labeled alpha 1(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1-1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75-80%, and a parallel reduction of procollagen alpha 1(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1-100 nM, which inhibited CDP and reduced procollagen alpha 1(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 microM), PTH (10 nM) and prostaglandin E2 (1 microM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 microM) and (Bu)2cAMP (100 microM) failed to alter CDP or procollagen alpha 1(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the alpha 1(I) procollagen gene by 70% and 80%, respectively, while alpha 2(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the beta-actin or beta-tubulin genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone decreased the incorporation of [3H]proline into collagenase-digestible protein in cultured 21-day fetal rat calvaria but had little effect on the labeling of noncollagen protein. After 24 hr of culture, there was a 50% reduction in collaghen synthesis and a 40% decrease in the level of functional procollagen mRNA as measured in a reticulocyte lystate translation assay. The effect of parathyroid hormone on both parameters was detectable after 6 hr of treatment. In these cultures, there was also a substantial degradation or release of newly synthesized collagen from the calvaria, but parathyroid hormone had little effect on the release of collagen into the medium. These results suggest that parathyroid hormone inhibits collagen synthesis primarily by decreasing the steady-state level of procollagen mRNA.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited type I collagen synthesis in the central bone but not the periosteum of fetal rat calvaria maintained in organ culture. The central bone synthesized primarily type I collagen, whereas the periosteum synthesized both types I and III collagen. Enhanced degradation of newly synthesized collagen seemed an unlikely mechanism for the observed decrease in collagen synthesis since 1,25-(OH)2D3 reduced both the labeling of intracellular procollagen and total [3H]hydroxyproline formation in fetal rat calvaria. As measured by cell-free translation of RNA extracted from calvaria, 1,25-(OH)2D3 decreased the level of functional procollagen mRNA and collagen synthesis to the same extent. Both collagen synthesis and procollagen mRNA levels were decreased as early as 3 h after exposure to 1,25-(OH)2D3 and were 50% of the control level by 24 hr. Upon removal of 1,25-(OH)2D3 from the cultures, collagen synthesis and procollagen mRNA remained depressed for 24 h but returned to control levels by 48 h. A reduction in collagen synthesis and procollagen mRNA was also observed in calvaria excised from 6-day-old rat pups given a single subcutaneous injection of 1,25-(OH)2D3 (1.6 ng/g body weight). We conclude that 1,25-(OH)2D3 inhibits the synthesis of type I collagen in the differentiated osteoblast by reducing the level of functional procollagen mRNA.

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.

Cytosol prepared from intestinal homogenates of vitamin D3-deficient rats demonstrate a 3.2 S protein having high affinity and low capacity for 1,25-dihydroxyvitamin D3. Appearance of the protein is dependent upon the presence of 0.3 M potassium chloride and dithiothreitol in the homogenization buffer. A 6 S protein having greater affinity for 25-hydroxyvitamin D3 than 1,25-dihydroxyvitamin D3 is observed using all conditions and is increased by the addition of rat serum to cytosol. The 3.2 S protein is neither a serum contaminant nor a component of the 6 S protein and is probably not a consituent of tissues which which are not responsive to vitamin D3.

Cytosol fractions were prepared from fetal rat or embryonic chick calvaria and analyzed for binding of vitamin D3 metabolites on sucrose density gradients. Both cytosol fractions contain a 3.5S macromolecule which specifically binds 1,25-dihydroxy-[3H]vitamin D3 and in addition, a 5 to 6S macromolecule which binds 25-hydroxy-[3H]vitamin D3. In rat calvaria cytosol, 1,25-dihydroxy-[3H]vitamin D3 also binds to the 5 to 6S macromolecule but appears to have greater affinity for the 3.5S component.

Transforming growth factor alpha (TGF-alpha) and interleukin-1 (IL-1) have been shown to affect bone metabolism in vitro by prostaglandin-dependent and PG-independent mechanisms. We assessed the effects of the combination of these two agents on [3H]thymidine (TdR) incorporation into DNA, DNA content, [3H]proline incorporation into collagenase-digestible (CDP), noncollagen protein (NCP), and PGE2 production in 21 day fetal rat calvaria cultured for 24-96 h. We also determined whether TGF-alpha plus IL-1 altered procollagen mRNA levels at 96 h. TGF-alpha, 1-30 ng/ml, produced a 41-59% increase in TdR incorporation into DNA, but the effect was partially blocked by human recombinant IL-1. At 96 h TGF-alpha alone or in combination with IL-1 significantly increased the DNA content of calvaria. At 96 h, TGF-alpha inhibited CDP labeling and the addition of IL-1 further enhanced this inhibitory effect. The enhanced inhibitory effect of TGF-alpha plus IL-1 on collagen synthesis was associated with a synergistic increase in prostaglandin accumulation in the medium. Addition of indomethacin blocked PGE2 accumulation and partially reversed the inhibitory effect of TGF-alpha alone or in combination with IL-1 on collagen synthesis. TGF-alpha decreased procollagen mRNA levels by 55%, but the combination of TGF-alpha plus IL-1 decreased procollagen mRNA levels by 82%. Our results show that TGF-alpha and IL-1, which are both produced by certain tumors as well as activated macrophages, appear to act synergistically to increase prostaglandin synthesis and inhibit collagen synthesis in vitro. Thus these agents may have a regulatory role on bone formation in vivo.

A competitive binding assay for 1,25-dihydroxyvitamin D [1,25-(OH2D] in plasma has been developed in which intestinal cytosol preparations from rachitic chicks are used as the binding protein. A new method of extraction and two new chromatographic procedures are used for this assay. The method is sensitive to as little as 10 picograms of 1,25-(OH)2D, and triplicate assays can be done on 5 milliliters of plasma. This assay shows that in the plasma of normal adult subjects there is a 1,25-(OH)2D concentration of 29 +/- 2 picograms per milliliter, while none can be detected in the plasma of nephrectomized subjects and end-stage renal failure patients.