Androgen receptor activation integrates complex transcriptional effects in osteoblasts, involving the growth factors TGF-ß and IGF-I, and transcription factor C/EBPδ.

Osteoblasts respond to many growth factors including IGF-I and TGF-ß, which themselves are sensitive to other bone growth regulators. Here we show that IGF-I gene promoter activity in prostaglandin E2 (PGE2) induced osteoblasts is suppressed by dihydrotestosterone (DHT) through an essential C/EBP response element (RE) in exon 1 of the igf1 gene. Inhibition by DHT fails to occur when the androgen receptor (AR) gene is mutated within its DNA binding domain. Correspondingly, DHT activated AR inhibits gene transactivation by C/EBPδ, and transgenic C/EBPδ expression inhibits AR activity. Inhibition by DHT persists when upstream Smad and Runx REs in the IGF-I gene promoter are mutated. TGF-ß also enhances IGF-I gene promoter activity, although modestly relative to PGE2, and independently of the C/EBP, Smad, or Runx REs. Still, DHT suppresses TGF-ß induced IGF-I promoter activity, but not its effects on DNA or collagen synthesis. Notably, DHT suppresses plasminogen activator inhibitor gene promoter activity, but synergistically increases Smad dependent gene promoter activity in TGF-ß induced cells, which are differentially sensitive to AR mutations and the AR co-regulator ARA55. Finally, although the PGE2 sensitive C/EBP RE in the igf1 gene is not essential for basal TGF-ß induction, C/EBPδ activity through this site is potently enhanced by TGF-ß. Thus DHT suppresses the PGE2 and TGF-ß induced IGF-I gene promoter and differentiates other aspects of TGF-ß activity in osteoblasts. Our results extend the complex interactions among local and systemic bone growth regulators to DHT, and predict complications from anabolic steroid use in other DHT sensitive tissues.

Prostaglandin dependent control of an endogenous estrogen receptor agonist by osteoblasts.

Estrogen receptor (ER) activation has complex effects on bone cells, and loss of circulating estradiol adversely affects skeletal status in women. Hormone replacement therapy effectively circumvents bone loss after menopause, but enhances disease risk in other tissues. Here we show that prostaglandin E2 (PGE2) augments the activity of an osteoblast-derived selective ER modulator, ObSERM. The stimulatory effect of PGE2 is replicated in part by either the PG receptor EP3 agonist 17-phenyl trinor PGE2 or by the PG receptor FP agonist PGF2α⋅ Whereas activation of the various PG receptors induces multiple downstream signals, the response to PGE2 was mimicked by activators of protein kinase C, and suppressed by inhibition of protein kinase C but not by inhibition of protein kinase A. Moreover, inhibition of nitric oxide synthesis and activation of the PTH and Wnt pathways increases ObSERM activity. Our studies therefore reveal that ObSERM activity is controlled in distinct ways and revise our understanding of ER activation within bone by agents or events associated with PG expression. They also predict ways to sustain or improve bone formation, fracture repair, and surgical healing without adding the risk of disease in other tissues where ER activation also has important biological functions.

Stratified control of IGF-I expression by hypoxia and stress hormones in osteoblasts.

Bone cells respond to the integrated effects of local and systemic regulation. Here we show that hypoxia and the stress hormones PGE2 and glucocorticoid interact in complex ways in osteoblasts, converging on insulin like growth factor I (IGF-I) expression. Whereas hypoxia alone rapidly increased transcription factor HIF activity, it suppressed DNA synthesis, had no significant effects on protein synthesis or alkaline phosphatase activity, and drove discrete changes in a panel of osteoblast mRNAs. Notably, hypoxia increased expression of the acute phase response transcription factor C/EBPδ which can induce IGF-I in response to PGE2, but conversely prevented the stimulatory effect of PGE2 on IGF-I mRNA. However, unlike its effect on C/EBPδ, hypoxia suppressed expression of the obligate osteoblast transcription factor Runx2, which can activate an upstream response element in the IGF-I gene promoter. Hypoxic inhibition of IGF-I and Runx2 were enforced by glucocorticoid, and continued with prolonged exposure. Our studies thus reveal that IGF-I expression is stratified by two critical transcriptional elements in osteoblasts, which are resolved by the individual and combined effects of hypoxic stress and stress hormones. In so doing, hypoxia suppresses Runx2, limits the enhancing influence of PGE2, and interacts with glucocorticoid to reduce IGF-I expression by osteoblasts.

Aldosterone stimulates fibronectin synthesis in renal fibroblasts through mineralocorticoid receptor-dependent and independent mechanisms.

In addition to its role in regulation of salt transport in the kidney, the mineralocorticoid hormone aldosterone plays an independent role as a mediator of kidney injury and progression of chronic kidney disease. Studies in both animal models and patients have shown that aldosterone enhances the accumulation of extracellular matrix and progression of fibrosis in the kidney. However, the cellular mechanisms that lead to aldosterone-dependent fibrogenesis are poorly understood. In this study we find that aldosterone stimulates fibronectin synthesis through mineralocorticoid receptor (MCR) dependent activation of the c-Jun NH2-terminal protein kinase (JNK) and subsequent phosphorylation of the AP1 transcription factor c-jun, which forms a nuclear complex with the mineralocorticoid receptor in a kidney fibroblast cell line (NRK 49f). Furthermore, MCR-independent phosphorylation of Src family kinase induces IgF1 receptor phosphorylation, which leads to stimulation of the extracellular signal-regulated kinase (ERK1/2) to enhanced fibronectin synthesis. We further find that the IgF1-R-dependent signaling pathway activates fibronectin expression faster than the MCR-dependent pathway. We propose that the mechanisms described in this study are important to aldosterone-dependent progression of interstitial fibrosis in the kidney. Due to the duality of aldosterone-dependent activation of fibronectin synthesis in kidney fibroblasts, MCR-specific inhibitors may not entirely prevent the progression of fibrosis by aldosterone in the kidney.

Inorganic phosphate stimulates fibronectin expression in renal fibroblasts.

Elevated plasma phosphate levels are signifcantly associated with progression of chronic kidney disease (CKD). Interstitial fibrosis is an important factor in the progression of CKD. In this study we investigate the role of inorganic phosphate in stimulating fibronectin (FN) synthesis in a kidney fibroblast cell line (NRK-49F). We find that phosphate increases FN abundance and message in a dose-dependent fashion and that both ERK1/2 and AKT are important signaling pathways that mediate phosphate-dependent FN expression in NRK-49F cells. Moreover phosphate srimulates the expression of the transcription factors osterix and NFATc1, which form complexes and mediate FN synthesis. Another transcription factor involved in phosphate-dependent FN synthesis is the AP1 family member c-Fos. In summary we show that even mildly elevated serum phosphate levels can induce synthesis of the interstitial matrix protein fibronectin through activation of ERK1/2 and AKT signaling pathways in kidney fibroblasts and that the synthesis of fibronectin is mediated by a transcriptional complex consisting of NFATc1, osterix and c-Fos.

Estrogen receptor dependent gene expression by osteoblasts - direct, indirect, circumspect, and speculative effects.

Hormone activated estrogen receptors (ERs) have long been appreciated as potent mediators of gene expression in female reproductive tissues. These highly targeted responses likely evolved from more elemental roles in lower organisms, in agreement with their widespread effects in the cardiovascular, immunological, central nervous, and skeletal tissue systems. Still, despite intense investigation, the multiple and often perplexing roles of ERs retain significant attention. In the skeleton, this in part derives from apparently opposing effects by ER agonists on bone growth versus bone remodeling, and in younger versus older individuals. The complexity associated with ER activation can also derive from their interactions with other hormone and growth factor systems, and their direct and indirect effects on gene expression. We propose that part of this complexity results from essential interactions between ERs and other transcription factors, each with their own biochemical and molecular intricacies. Solving some of the many questions that persist may help to achieve better, or better directed, use of agents that can drive ER activation in focused and possibly tissue restricted ways.

An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.

ß-Catenin independent cross-control between the estradiol and Wnt pathways in osteoblasts.

Osteoblasts are controlled by the individual and combined effects of systemic and local growth regulators. Here we show functional and physical interactions between estradiol (17ßE) and Wnt activated pathways in osteoblasts. 17ßE increased gene promoter activity by the Wnt pathway transcriptional effector T cell factor (TCF) in an estrogen receptor (ER) dependent way. This occurred independently of its activity through traditional estrogen response elements and was not replicated by androgen receptor activation. 17ßE also increased the stimulatory effect of LiCl on TCF activity, LiCl increased the stimulatory effect of 17ßE through estrogen response elements, and both were further enhanced by a noncanonical Wnt receptor agonist (WAg) that functions independently of ß-catenin stabilization. In contrast to LiCl, WAg increased DNA synthesis and reduced relative collagen synthesis and alkaline phosphatase activity in otherwise untreated or 17ßE stimulated cells. In addition, WAg suppressed Runx2, osterix, and alkaline phosphatase mRNA levels, and potently induced osteoprotegerin mRNA, whereas LiCl was ineffective alone and inhibitory in combination with 17ßE. A definitive intersection between the 17ßE and Wnt pathways occurred at the protein level, where ERα physically associated with TCF-4 independently of its ß-catenin binding domain. This interaction required ligand-dependent exposure of a TCF binding region that mapped to ERα domain E and was further enhanced by Wnt pathway activation. Our studies reveal highly focused co-regulatory effects between the 17ßE and Wnt pathways in osteoblasts that involve activated ERα and TCF-4 and downstream changes in gene expression, osteoblast proliferation, and differentiated cell function.

Novel links among Wnt and TGF-beta signaling and Runx2.

Osteoblasts exhibit complex Wnt-induced effects that increase T cell factor (TCF)/lymphoid enhancing factor-dependent transcription in parallel with beta-catenin stabilization and nuclear factor binding to TCF response element DNA. Here we show that Wnt-dependent gene expression increases during the early phase of osteoblast differentiation in vitro, is enhanced by prostaglandin E(2) activation of transcription factor Runx2 (runt homology domain transcription factor 2), and is specifically suppressed in Runx2 antisense-depleted osteoblasts. Moreover, Wnt pathway induction increases expression of the Runx2-sensitive gene, TGF-beta type I receptor, without increasing nuclear Runx2 levels or Runx2 binding to DNA. Rather, despite an increase in beta-catenin levels, Wnt pathway induction enhances Runx2 transcriptional potential in a beta-catenin-independent way. Runx2 functionally associates with TCF-4 that lacks a beta-catenin-binding domain and is more fully activated in response to both prostaglandin E(2) and Wnt pathway induction. Wnt pathway induction increases TGF-beta type I receptor expression, yet regulates, both positively and negatively, TGF-beta signaling. Furthermore, TGF-beta signaling enhances TCF-4 and lymphoid enhancing factor-1 mRNA expression and increases TCF-4 transcriptional activity. Therefore, we propose that cross talk between the Wnt and TGF-beta pathways, which converge on Runx2, both promotes and attenuates individual aspects of osteoblast maturation.

Effects of cyclic strain on rat tail tenocytes.

Cyclical mechanical strain is considered an important component in flexor tendon cell activation to prevent adhesions and enhance the healing process after tissue injury or surgery, but the biochemical events associated with this remain unclear. To address this, we examined the effects of cyclic tension on the expression of hyaluronic acid, an important lubricant and signal transducer in tendon, on its receptor (CD44), and on total glycosaminoglycan content in rat tail derived tendon fibroblasts in vitro. Tenocytes were plated on fibronectin coated silastic membranes and the cultures were held static or subjected to vacuum induced deformation for a period of 5 min once every 8 h as a model of cyclic mechanical stress. After 24 h medium and cell layers were collected for analyses by product specific ELISA, Western blot, and colorimetric dye-binding assays. Strained tenocytes produced a nearly two-fold increase in hyaluronic acid and a greater than 60% increase in CD44, but had an insignificant effect on total glycosaminoglycan content. Our results predict that high levels of strain may therefore rapidly enhance the expression of hyaluronic acid and cause, albeit still unresolved, downstream effects on CD44 activation, to influence tendon cell activity and enhance the process of tendon repair.

Expression of an estrogen receptor agonist in differentiating osteoblast cultures.

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-beta, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100 degrees C and separable from the bulk of the remaining alcohol- and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a M(r) of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.

3-ketosteroid reductase activity and expression by fetal rat osteoblasts.

In addition to reproductive tissue, sex hormones induce transcriptional events in many connective tissue cells, including osteoblasts. Some sex hormone receptor modulators with bone sparing effects selectively target estrogen or androgen receptors, whereas others appear more promiscuous, in part through enzymatic metabolism. Rat osteoblasts express significant oxidative 3alpha-hydroxysteroid dehydrogenase activity, which can convert precursor substrates to potent androgen receptor agonists. Here we show that they also express 3-ketosteroid reductase activity, exemplified by 7-methyl-17-ethynyl-19-norandrostan-5 (10)en-3-one (tibolone) conversion to potent estrogen receptor alpha agonists. Conversion was rapid and quantitative, with 3alpha-hydroxytibolone as the primary metabolite. Consistently, tibolone induced estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increased the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhanced prostaglandin E2-induced activity of transcription factor Runx2. Rat osteoblasts express the 3-ketosteroid reductase AKR1C9, an aldo-keto reductase gene family member. Exposure to prostaglandin E2 increased AKR1C9 gene promoter activity and mRNA expression. AKR1C9 promoter activity was also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, and the effect of PGE2 was reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression. Moreover, prostaglandin E2 increased the amount of functional endogenous nuclear C/EBPdelta that could bind specifically to a distinct domain approximately 1.8-kb upstream from the start site of AKR1C9 transcription. In summary, in addition to 3alpha-hydroxysteroid dehydrogenase, rat osteoblasts express significant and regulatable 3-ketosteroid reductase activity. Through these enzymes, they may selectively metabolize precursor compounds into potent steroid receptor agonists locally within bone.

Prostaglandin E2 increases transforming growth factor-beta type III receptor expression through CCAAT enhancer-binding protein delta in osteoblasts.

Variations in individual TGF-beta receptors (TbetaRs) may modify TGF-beta activity and significantly alter its effects on connective tissue growth or repair. Differences in the amount of TbetaR type III (TbetaRIII) relative to signal transducing TbetaRI occur on bone cells during differentiation or in response to other growth regulators. Here we investigated prostaglandin (PG) E2, a potent effector during trauma, inflammation, or mechanical load, on TbetaR expression in primary osteoblast-enriched cultures. PGE2 rapidly increased TbetaRIII mRNA and protein expression and enhanced TbetaRIII gene promoter activity through a discrete region within 0.4 kb of the transcription start site. PGE2 alters osteoblast function through multiple signal-inducing pathways. In this regard, protein kinase A (PKA) activators, PGE1 and forskolin, also enhanced gene expression through the TbetaRIII gene promoter, whereas protein kinase C activators, PGF2alpha and phorbol myristate acetate, did not. The stimulatory effect of PGE2 on TbetaRIII promoter activity was suppressed by a dominant negative PKA-regulatory subunit, but not by dominant negative protein kinase C. PGE2 specifically increased nuclear factor CCAAT enhancer-binding protein delta (C/EBPdelta) binding to a half-binding site upstream of the basal TbetaRIII promoter region, and promoter activity was sensitive to C/EBPdelta overexpression and to dominant-negative C/EBPdelta competition. In parallel with their effect on TbetaRIII expression, activators of PKA decreased TGF-beta-induced activity. In summary, high levels of PGE2 that occur with inflammation or trauma may, through PKA-activated C/EBPdelta, preferentially increase TbetaRIII expression and in this way delay TGF-beta-dependent activation of osteoblasts during the early stabilization phase of bone repair.

Repetitive exposure to TGF-beta suppresses TGF-beta type I receptor expression by differentiated osteoblasts.

Transforming growth factor-beta (TGF-beta) has potent, cell phenotype restricted effects. In bone, it controls multiple activities by osteoblasts through three predominant receptors. Of these, the relative amounts of TGF-beta receptor I (TbetaRI) vary directly with TGF-beta sensitivity. The rat TbetaRI gene promoter includes cis-acting elements for transcription factor Runx2. Here we show conservation and selective partitioning of TbetaRI and retention of TGF-beta activity with osteoblast differentiation, Runx2 binding to the TbetaRI gene promoter on osteoblast chromatin, and decreased promoter activity by Runx2 binding site mutation. Furthermore, in contrast to the stimulatory effects induced by single or limited exposure to TGF-beta, we found that osteoblasts became resistant to TGF-beta after high dose and repetitive treatment. TbetaRI protein, mRNA, and gene promoter activity all decreased after three daily TGF-beta treatments, in parallel with a reduction in Runx2 protein and Runx dependent gene expression. In this way, sustained TGF-beta exposure can limit its own effectiveness by suppressing the expression of its primary signaling receptor. This tightly controlled system may constitute a feedback loop to protect against TGF-beta excess, and impose important limitations that are required for the progression of events during skeletal growth, remodeling and repair.

Beta1 integrins modulate cell adhesion by regulating insulin-like growth factor-II levels in the microenvironment.

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.

Strain-dependent control of transforming growth factor-beta function in osteoblasts in an in vitro model: biochemical events associated with distraction osteogenesis.

BACKGROUND: Distraction osteogenesis is an important clinical method for increasing bone mass, but its effects on bone-forming cells are not well understood. In this study, the authors asked how the mechanical forces that occur during this procedure alter specific osteoblast activities such as matrix synthesis, the rate of cell replication, and enzyme activities. The authors further asked whether these changes relate to differences in the biochemical response of osteoblasts to transforming growth factor-beta (TGF-beta), a potent regulator of bone formation. METHODS: Osteoblasts were plated on flexible, collagen-coated membranes. One group was unstrained, a second group experienced a single maximum strain load once every 6 hours to simulate intermittent force associated with a distraction protocol of four screw turns per day, and a third group was strained continuously for 24 hours. In the third group, some cell cultures were allowed to recover from strain before analysis. Subsequently, each group was treated with vehicle or TGF-beta at 12 pM (0.3 ng/ml) or 120 pM (3 ng/ml). Data were collected from a minimum of 15 replicate cell culture wells obtained from at least three separate primary culture preparations. Results were assessed with statistical software. Differences were considered significant with values of p < 0.05. RESULTS: Both strain protocols increased basal osteoblast DNA synthesis but suppressed the relative stimulatory effect of TGF-beta on this event. However, neither intermittent nor continuous strain significantly altered collagen or noncollagen protein synthesis or the relative effect of TGF-beta on these processes in osteoblasts. Basal alkaline phosphatase activity, an intermediate marker of osteoblast differentiation and an early marker of matrix mineralization, decreased significantly in response to continuous strain or to TGF-beta treatment, and even more so in response to both conditions. In addition, TGF-beta binding to the type III TGF-beta receptor was increased in proportion to strain intensity. CONCLUSIONS: This study shows that cyclic strain can alter osteoblast activity in multiple ways and predicts that TGF-beta has different effects during the distraction process on osteoblasts and therefore on their ability to effect bone formation. They further indicate that mechanical load permits early aspects of osteoblast activation but delays in part later biochemical parameters associated with mineralization to allow new bone growth before consolidation.

Interactions between CCAAT enhancer binding protein delta and estrogen receptor alpha control insulin-like growth factor I (igf1) and estrogen receptor-dependent gene expression in osteoblasts.

Although ambient levels of estradiol and synthesis of the osteoblast growth factor IGF-I are inversely related in vivo, estradiol has little or no direct effect on igf1 gene expression in rat osteoblasts in vitro. Rather, estradiol suppresses the effect of hormones that enhance igf1 expression through protein kinase A dependent activation of CCAAT enhancer binding protein (C/EBP) transcription factors. We show here that inhibition of C/EBP activity by estradiol relates to the level of estrogen receptor alpha (ERalpha) expression, and that a complex between hormone-activated ERalpha and C/EBPdelta inhibits transcription by each factor. Protein fragmentation, co-immunoprecipitation, and gene expression studies identified domains for physical and functional interactions between ERalpha and C/EBPdelta. Whereas ERalpha and fragments comprising its various domains associated with C/EBPdelta, only ERalpha fragment A/B alone replicated the suppressive effect of intact ERalpha on endogenous C/EBPdelta activity. Complementary studies showed that several carboxyl regions of C/EBPdelta cooperatively inhibit ERalpha dependent transcription. Therefore, multiple domains of C/EBPdelta and ERalpha can physically interact to alter gene expression in osteoblasts in selective ways that depend on variations in the local hormone environment. Their combined effects on one important gene target, igf1, may help to determine the balance in the overall rates of bone formation.

Functional cooperation between CCAAT/enhancer-binding proteins and the vitamin D receptor in regulation of 25-hydroxyvitamin D3 24-hydroxylase.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces the synthesis of 25-hydroxyvitamin D(3) 24-hydroxylase [24(OH)ase], an enzyme involved in its catabolism, thereby regulating its own metabolism. Here we demonstrate that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and in osteoblastic cells and is a potent enhancer of vitamin D receptor (VDR)-mediated 24(OH)ase transcription. Transfection studies indicate that 1,25(OH)(2)D(3) induction of 24(OH)ase transcription is enhanced a maximum of 10-fold by C/EBPbeta. Suppression of 1,25(OH)(2)D(3)-induced 24(OH)ase transcription was observed with dominant negative C/EBP or osteoblastic cells from C/EBPbeta(-/-) mice. A C/EBP site was identified at positions -395 to -388 (-395/-388) in the rat 24(OH)ase promoter. Mutation of this site inhibited C/EBPbeta binding and markedly attenuated the transcriptional response to C/EBPbeta. We also report the cooperation of CBP/p300 with C/EBPbeta in regulating VDR-mediated 24(OH)ase transcription. We found that not only 1,25(OH)(2)D(3) but also parathyroid hormone (PTH) can induce C/EBPbeta expression in osteoblastic cells. PTH potentiated the induction of C/EBPbeta and 24(OH)ase expression in response to 1,25(OH)(2)D(3) in osteoblastic cells. Data with the human VDR promoter (which contains two putative C/EBP sites) indicate a role for C/EBPbeta in the protein kinase A-mediated induction of VDR transcription. From this study a fundamental role has been established for the first time for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in 1,25(OH)(2)D(3)-induced transcription. These findings also indicate a novel role for C/EBPbeta in the cross talk between PTH and 1,25(OH)(2)D(3) that involves the regulation of VDR transcription.

Skeletal hormones and the C/EBP and Runx transcription factors: interactions that integrate and redefine gene expression.

Systemic hormones and local growth factors have significant and often complex roles in normal tissue development, growth, remodeling, and repair. Early efforts in skeletal tissue attempted to define active panels of these agents and their direct effects on cell proliferation, matrix production, and secretion of other soluble mediators of differentiated cell function. Initial results resolved many of these questions and began to unveil functional interactions between specific hormones and growth factors. More recent evidence suggests that interactions between individual hormone systems also occur in less anticipated but probably not less meaningful ways. In some cases, these interactions may help to define a spectrum of effects on gene expression by focusing, refocusing, or integrating the activity of previously recognized transcription regulators. Other studies in isolated osteoblasts predict that certain steroid hormones have distinctive effects on specific transcription factors with important roles in bone growth and repair. In this review, we focus on studies that define functional and physical interactions between molecular mediators of hormone activity that could directly effect skeletal growth factor biology.

Fos-related antigen 2 controls protein kinase A-induced CCAAT/enhancer-binding protein beta expression in osteoblasts.

Transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) plays an important role in hormone-dependent gene expression. In osteoblasts C/EBPbeta can increase insulin-like growth factor I (IGF-I) transcription following treatment with hormones that activate protein kinase A, but little is known as yet about the expression of C/EBPbeta itself in these cells. We initially showed that prostaglandin E2 (PGE2) rapidly enhances C/EBPbeta mRNA and protein expression, and in this study we identified a 3'-proximal region of the C/EBPbeta promoter containing a 541-bp upstream sequence that could account for this effect. PGE2-dependent activation of C/EBPbeta was blocked by expression of a mutated regulatory subunit of protein kinase A or by mutation of two previously identified cAMP-sensitive cis-acting regulatory elements within the promoter between bp -111 and -61. Nuclear protein binding to these elements was induced by PGE2, required new protein synthesis, and was sensitive to antibody to the transcription factor termed Fos-related antigen 2 (Fra-2). Fra-2 cDNA generated from rat osteoblasts by reverse transcriptase PCR was 95% homologous to human Fra-2, and PGE2 rapidly induced Fra-2 mRNA and protein expression. Consistent with these findings, over-expression of Fra-2 significantly increased C/EBPbeta promoter activity in PGE2-induced osteoblasts, whereas expression of Fra-2 lacking its activation domain had a dominant negative inhibitory effect. Together, these results reveal a significant, hormone-dependent role for Fra-2 in osteoblast function, both directly, through its ability to increase new C/EBPbeta gene expression, and indirectly, through downstream C/EBP sensitive genes.