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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.

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.

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.

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.

To determine the effects of locally-expressed insulin-like growth factor (IGF-I) on bone remodeling, a transgene was produced in which murine IGF-I cDNA was cloned downstream of a gene fragment comprising 3.6 kb of 5′ upstream regulatory sequence and most of the first intron of the rat Col1a1 gene. The construct was expressed at the mRNA and protein level in transfected osteoblasts. Five lines of transgenic mice were generated by embryo microinjection. Transgene mRNA levels were highest in calvaria, long bone and tendon, and lower in skin. Serum IGF-I and body weight were increased in males and females only in the highest expressing line. Histomorphometry showed that transgenic calvaria were wider and had greater marrow area and bone area. Transgenic calvaria had increased osteoclast number per bone surface. Percent collagen synthesis and cell replication were increased in transgenic calvaria. Femur length, cortical width and cross-sectional area were increased in transgenic femurs of the highest expressing line, while femoral trabecular bone volume was little affected. Thus, broad overexpression of IGF-I in cells of the osteoblast lineage increased indices of bone formation and resorption.

CREM, the cyclic adenosine monophosphate (cAMP) responsive element modulator, belongs to a multigene family of cAMP-responsive transcription factors. CREM encodes a variety of different isoforms by utilizing four promoters and a complex pattern of alternative messenger ribonucleic acid (mRNA) splicing. We showed previously that parathyroid hormone induces the CREM P2 promoter products known as ICER (inducible cAMP early repressor) in osteoblasts. Herein we report that osteoblasts also express at least 15 CREM transcripts initiated from the P1 promoter, including 7 novel transcripts that result from alternative splicing. It is of interest that we found that CREM-X contains both exon theta1, previously identified only in P3 promoter products, and a new exon termed L, which is located upstream of exon theta1.

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.

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.

The regulation of COL1A1 gene expression in bone was studied by measuring the activity of type I collagen promoter fusion genes (ColCAT) in permanently transfected osteoblastic cells and calvariae from transgenic animals. The basal activity of ColCAT fusion genes in transfected cells is mediated by DNA sequences between -3.5 to -2.3 kb while expression in vivo requires sequences between -2.3 and -1.7 kb. Parathyroid hormone, 1,25-dihydroxyvitamin D3 and interleukin-1 decrease the activity of ColCAT fusion genes in osteoblastic cells and transgenic calvariae. Because there may be differences between the expression of ColCAT fusion genes in cultured cells and intact bone, it will be important to compare data obtained from transfected cells with an in vivo model such as calvariae from transgenic mice.

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.

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.

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.

We compared the effects of four vitamin D metabolites, 1 alpha,25 dihydroxy vitamin D3 (1 alpha,25(OH)2D3), 1 alpha hydroxy vitamin D3 (1 alpha OH D3), 25 hydroxy vitamin D3 (25 OH D3), and 24R,25 dihydroxy vitamin D (24R,25(OH)sD3) on resorption and collagen synthesis in fetal rat bone maintained in organ culture. Resorption was quantitated by measuring the release of previously incorporated 45Ca from long bone shafts of 19-day fetal rats, and collagen synthesis was assessed by measuring the incorporation of 3H-proline into collagenase digestible protein (CDP) in calvaria from 21-day fetal rats. All four compounds stimulated bone resorption and inhibited collagen synthesis, but 1 alpha,25(OH)2D3 was approximately 1000 times more potent in both organ culture systems. Although the differences were small among the other three compounds, the order of potency was 1 alpha OH d325 OH D3 greater than or equal to 24R,25(OH)2D3. These results suggest that the receptor for 1 alpha 25(OH)2D3 in both bone resorbing and bone forming cells has similar affinities for several vitamin D metabolites.

The endocrine control of skeletal growth involves not only calcium regulating hormones but also several systemic hormones and other factors. Many direct effects of these agents on skeletal growth have been demonstrated in vitro. While these direct effects may also predominate in vivo, and certainly are important in helping us understand how skeletal growth is controlled, it is likely that skeletal growth in vivo is determined by the complex interactions of a concert of hormones that play both permissive and regulatory roles. Additionally the effect of hormones may be modified by local or intrinsic responses of the skeleton or by changes in ion concentration. Phosphate may have a hormone-like function in the skeleton since high serum concentrations are associated with increased growth and low serum concentrations with impaired growth and mineralization (2, 5, 7, 37, 89). Because of these interactions, in vivo and in vitro studies on hormonal control of skeletal growth are often contradictory. A careful distinction between indirect and direct effects will help to interpret the information now available and to devise better studies in the future.

Glucocorticoids Glucocorticoids have complex effects on calcium regulation and bone metabolism. Clinically, glucocorticoid excess causes impaired skeletal growth and decreased bone mass.25 Yet it seems likely that glucocorticoids have a physiologic role in regulating bone growth. Glucocorticoid excess decreases the intestinal absorption of calcium and phosphate and increases their renal excretion. These effects can produce secondary hyperparathyroidism and might be expected to produce osteomalacia. However, the osteopenia associated with glucocorticoids is termed osteoporosis because there is no obvious defect in mineralization. This may be because glucocorticoids also inhibit the formation of new matrix, so that wide osteoid seams do not . . .