Sandra Casinghino

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

CBFa(AML/PEBP2)-related elements in the TGF-β type I receptor promoter and expression with osteoblast differentiation

Organization of the transforming growth factor‐β (TGF‐β) type I receptor (TRI) promoter predicts constitutive transcription, although its activity increases with differentiation status in cultured osteoblasts. Several sequences in the rat TRI promoter comprise cis‐acting elements for CBFa (AML/PEBP2α) transcription factors. By gel mobility shift and immunological analyses, a principal osteoblast‐derived nuclear factor that binds to these sites is CBFa1(AML‐3/PEBP2αA). Rat CBFa1 levels parallel expression of the osteoblast phenotype and increase under conditions that promote mineralized bone nodule formation in vitro. Fusion of CBFa binding sequence from the TRI promoter to enhancer‐free transfection vector increases reporter gene expression in cells that possess abundant CBFa1, and overexpression of CBFa increase the activity of transfected native TRI promoter/reporter plasmid. Consequently, phenotype‐restricted use of cis‐acting elements for CBFa transcription factors can contribute to the high levels of TRI that parallel osteoblast differentiation and to the potent effects of TGF‐β on osteoblast function. J. Cell. Biochem. 69:353‐363.

17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2(PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 μm PGE2 increased IGF-I P1 activity by 5-fold. 17β-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 ∼0.1 nm). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17α-estradiol was 100–300-fold less effective. By contrast, 17β-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17β-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.

Multiple and essential Sp1 binding sites in the promoter for transforming growth factor-beta type I receptor.

Maximal gene expression driven by the promoter for the transforming growth factor β type I receptor (TGF-βRI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBFα binding sites among other possiblecis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBFα-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-βRI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-βRI levels that parallel changes in bone cell differentiation or activity.

RAPID FLUX IN TRANSFORMING GROWTH FACTOR-BETA RECEPTORS ON BONE CELLS

The proportion of transforming growth factor-β (TGF-β) binding among conventional membrane receptors on bone cells can vary with hormone or growth factor treatment or with the state of osteoblast-like activity and appears to determine the nature of its biological effects. Therefore, functional TGF-β receptor stability could be an important aspect of regulation. Suppression of protein synthesis reduced TGF-β binding to types I and II receptors with t1/2 of 2 h and to betaglycan with t1/2 of 6 h. In contrast, suppression of mRNA transcription reduced TGF-β binding at least 3-fold more slowly at each receptor site. Preexposure to TGF-β decreased its binding at all three sites within 4 h in osteoblast-enriched cultures. This effect was transient with lower TGF-β concentrations, where the receptor profile was nearly fully restored within 24-48 h. In contrast, less differentiated bone cells were less sensitive to ligand-dependent receptor down-regulation. Agents that alter protein kinase and phosphatase activity also modified the TGF-β binding profile in specific ways. Together, these results indicate that cell surface TGF-β receptors turn over rapidly by ligand-independent and ligand-dependent mechanisms, demonstrate that the binding capacity of TGF-β receptors is less stable than their mRNAs, and that functional receptor levels may be determined in part by post-transcriptional events.

Opposing effects by glucocorticoid and bone morphogenetic protein‐2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF‐β) on collagen synthesis in osteoblast‐enriched cultures in vitro, where it also suppresses TGF‐β binding to its type I receptors. Analogous studies with bone morphogenetic protein‐2 (BMP‐2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP‐2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF‐β binding or function. BMP‐2 activity was retained in part in osteoblast‐enriched cultures pre‐treated or co‐treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP‐2 treatment. In addition, BMP‐2 suppressed the effects of cortisol on TGF‐β activity, on TGF‐β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP‐2 also alters the function of less‐differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP‐2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid‐dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997.

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone.

Insulin‐like growth factor (IGF)‐I and IGF‐II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell‐associated, and matrix‐bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP‐1. Of these, IGFBP‐3, ‐4, and ‐5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP‐dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP‐2 and ‐5 occurred in osteoblast cultures, and more IGFBP‐3 and ‐6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP‐3, ‐4, and ‐5 mRNA and accordingly increased soluble IGFBP‐3, ‐4, and ‐5 and matrix‐bound IGFBP‐3 and ‐5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP‐5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP‐2, ‐3, and ‐5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP‐4 does not significantly accumulate in cell‐associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell‐bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP. J. Cell. Biochem. 71:351–362, 1998.

Alternate signaling pathways selectively regulate binding of insulin‐like growth factor I and II on fetal rat bone cells

Bone cells synthesize and respond to IGF‐I and IGF‐II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E2 stimulates IGF‐I but not IGF‐II synthesis through a cAMP‐dependent protein kinase A (PKA)‐related event. However, protein kinase C (PKC) activation by PGE2 enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast‐enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE2 activity has now been examined on IGF receptors in these bone cell culture models. PGE2 and other agents that activate PKA enhanced 125I‐IGF‐II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced 125I‐IGF‐I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor‐independent way, was effective in osteoblast‐enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post‐translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue. J. Cell. Biochem. 68:446–456, 1998.