Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

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.

Insulin-Like Growth Factor I Does Not Drive New Bone Formation in Experimental Arthritis.

INTRODUCTION: Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively. METHODS: Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324-7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts. RESULTS: 90-100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels. CONCLUSION: Overexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.

Impact of targeted PPARγ disruption on bone remodeling.

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 (PPAR(fl/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.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA-CREM/ICER: a potential role for Epac.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

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.

Forum on aging and skeletal health: summary of the proceedings of an ASBMR workshop.

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 evaluated adequately 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 that meeting and in each area identifies key questions to help set the agenda for future research in skeletal aging.

Selective osteoblast overexpression of IGF-I in mice prevents low protein-induced deterioration of bone strength and material level properties.

Protein deficiency is frequently observed in elderly osteoporotic patients. Undernutrition leads to decreased levels of IGF-I, an important factor in regulating bone homeostasis throughout life. IGF-I is produced in the liver and locally in the skeleton. We hypothesized that increasing IGF-I expression in the osteoblasts, the bone forming cells, would protect the skeleton from the negative effects of a low-protein diet. To test our hypothesis, we employed a mouse model in which IGF-I was overexpressed exclusively in osteoblasts and fed either a 15% (normal) or a 2.5% (low) protein isocaloric diet to the transgenic (TG) mice and their wild-type (WT) littermates for 8 weeks. Blood was collected for biochemical determinations and weight was monitored weekly. Bones were excised for microstructural analysis (µCT), as well as biomechanical and material level properties. Histomorphometric analysis was performed for bone formation parameters. A low protein diet decreased body weight, circulating IGF-I and osteocalcin levels regardless of genotype. Overexpression of IGF-I in the osteoblasts was, however, able to protect the negative effects of low protein diet on microstructure including tibia cortical thickness and volumetric density, and on bone strength. Overexpression of IGF-I in osteoblasts in these mice protected the vertebrae from the substantial negative effects of low protein on the material level properties as measured my nanoindentation. TG mice also had larger overall geometric properties than WT mice regardless of diet. This study provides evidence that while a low protein diet leads to decreased circulating IGF-I, altered microstructure and decreased bone strength, these negative effects can be prevented with IGF-I overexpression exclusively in bone cells.

Epidermal growth factor receptor plays an anabolic role in bone metabolism in vivo.

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 Egfr(f/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 Egfr(Wa5/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, Egfr(Dsk5/+) 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.

PDE8 regulates rapid Teff cell adhesion and proliferation independent of ICER.

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.

Col3.6-HSD2 transgenic mice: a glucocorticoid loss-of-function model spanning early and late osteoblast differentiation.

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.

Dicer inactivation in osteoprogenitor cells compromises fetal survival and bone formation, while excision in differentiated osteoblasts increases bone mass in the adult mouse.

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.

Parathyroid hormone induction of cyclooxygenase-2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway.

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.

Effects of global or targeted deletion of the EP4 receptor on the response of osteoblasts to prostaglandin in vitro and on bone histomorphometry in aged mice.

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre-LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E(2) (PGE(2)) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE(2) in cell culture but may not be essential for maintenance of bone remodeling in vivo.

Calcitonin induces expression of the inducible cAMP early repressor in osteoclasts.

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.

Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor.

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

CREM deficiency in mice alters the response of bone to intermittent parathyroid hormone treatment.

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 mug/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.

Rearrangement of a conditional allele regardless of inheritance of a Cre recombinase transgene.

To generate conditional gene knockouts in osteoblasts, we previously developed transgenic mice in which Cre recombinase cDNA was cloned downstream of a 3.6 or 2.3 kb fragment of the rat Col1a1 promoter (Col3.6-Cre and Col2.3-Cre, respectively). Col-Cre mice were bred with mice in which exon 4 of the Igf1 gene is flanked by loxP sites. Mating units were arranged such that either the male or the female breeder transmitted the Col-Cre transgenes. Progeny were evaluated for Cre-mediated Igf1 gene rearrangement. We found that the loxP-flanked Igf1 locus was rearranged in the absence of inheritance of the Cre transgene. The incidence was 50 and 28% with Col2.3-Cre and Col3.6-Cre females, respectively, and 15 and 18% with Col2.3-Cre and Col3.6-Cre males, respectively.

Osteoblast deletion of exon 3 of the androgen receptor gene results in trabecular bone loss in adult male mice.

UNLABELLED: 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 microCT 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: microCT 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.

Analysis of internal deletions of a rat Col1a1 promoter fragment in transfected ROS17/2.8 cells.

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.