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

Osteoblasts respond to many growth factors including IGF-I and TGF-β, 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.

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.

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.

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.

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).

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.

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.

β-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 (17βE) and Wnt activated pathways in osteoblasts. 17βE increased gene promoter activity by the Wnt pathway transcriptional effector T cell factor (TCF) in an estrogen receptor (ER) dependent way.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Estren (4-estren-3alpha,17beta-diol) is a prohormone that regulates both androgenic and estrogenic transcriptional effects through the androgen receptor.

Alternative mechanisms of steroid action, through both traditional nuclear receptors and indirect pathways of gene activation, are emerging. Recent studies suggest that the synthetic steroid, 4-estrene-3alpha,17beta-diol (estren), has nongenotropic as well as sex-nonspecific osteogenic effects in ovariectomized and orchidectomized mice. We found limited estrogen receptor-dependent effects by estren on gene expression in primary osteoblast cultures and showed that it binds poorly to estrogen and androgen receptors in vitro.

Runx2 integrates estrogen activity in osteoblasts.

Steroids significantly effect skeletal integrity. For example, bone mass decreases with glucocorticoid excess or with estrogen depletion after menopause. Glucocorticoid suppresses gene expression by an essential skeletal tissue transcription factor, Runx2, in rat osteoblasts.

Activation domains of CCAAT enhancer binding protein delta: regions required for native activity and prostaglandin E2-dependent transactivation of insulin-like growth factor I gene expression in rat osteoblasts.

In osteoblasts, hormones such as prostaglandin E2 that activate protein kinase A increase the translocation of transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta) from the cytoplasm to the nucleus where it rapidly induces IGF-I gene expression. In this study, we identified activation and suppression domains in C/EBPdelta using native and heterologous gene promoter assay systems. We demonstrated functional interactions between C/EBPdelta and trans-gene-expressed cAMP response element binding protein-binding protein, and showed that the ability of C/EBPdelta to promote gene expression was suppressed by viral protein E1A, which blocks the activity of native cAMP response element binding protein-binding protein.

Transcriptional and post-transcriptional regulation of transforming growth factor beta type II receptor expression in osteoblasts.

Variations in transforming growth factor beta (TGF-beta) activity depend on the expression of specific receptors in normal as well as transformed cells. For example, in addition to mutations in TGF-beta type II receptor (TbetaRII) that abrogate normal TGF-beta function, its expression decreases during the transition from replication to extracellular matrix production, or in response to other growth regulators in bone. Therefore, to understand how TbetaRII expression is controlled, we cloned the rat TbetaRII gene promoter and defined basic aspects of its structure and activity.

Betaglycan inhibits TGF-beta signaling by preventing type I-type II receptor complex formation. Glycosaminoglycan modifications alter betaglycan function.

Transforming growth factor (TGF)-beta is a multifunctional growth factor with important roles in development, cell proliferation, and matrix deposition. It signals through the sequential activation of two serine/threonine kinase receptors, the type I and type II receptors. A third cell surface receptor, betaglycan, serves as a co-receptor for TGF-beta in some cell types, enhancing TGF-beta-mediated signaling.