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.

Glucocorticoid in excess suppresses bone formation in vivo and disrupts bone matrix protein synthesis by osteoblasts in vitro. In contrast, transforming growth factor ␤ (TGF-␤) potently enhances bone matrix apposition. The rat TGF-␤ type I receptor gene promoter contains cis-acting elements for transcription factor CBFa1, which increases in parallel with osteoblast differentiation. Here we present molecular data linking these events. We show that previously unexplained effects of glucocorticoid on bone loss may be mediated in part by suppression of CBFa1, with a resultant decrease in the expression and activity of the TGF-␤ type I receptor on matrix-producing bone cells.Glucocorticoid-dependent bone loss by disregulated hormone expression or pharmacologic excess causes clinically significant osteoporosis in approximately 50% of affected individuals. Although changes in calcium absorption and effects on nonskeletal tissues contribute to the disease, striking effects occur directly on osteoblasts and at sites of active skeletal matrix deposition and remodeling (1, 2). A chronic reduction in osteoblast activity without corresponding changes in resorption would uncouple normal bone remodeling and decrease skeletal durability. Important genes targeted by glucocorticoid and molecular mediators for these events remain uncertain.Transforming growth factor ␤ (TGF-␤) 1 enhances bone matrix synthesis and repair, and bone contains perhaps the largest store of TGF-␤ in the body (reviewed in Ref. 3). Bone cells exhibit conventional type II and type III TGF-␤ receptors (TRII and TRIII) that influence TGF-␤ binding to type I receptor (TRI) or its activation, both essential for TGF-␤-dependent events (4 -6). There are few systems where regulation of TRI expression has been examined in detail and where functional changes correlate with these variations. We found TRI levels specifically maintained on differentiated bone cells in vitro, despite decreases in TRII and TRIII in response to bone morphogenetic protein 2 (5). In contrast, high levels of glucocorticoid rapidly reduce the proportion of TGF-␤ binding to TRI on bone cells and correspondingly decrease TGF-␤ activity (6).To understand these events further, we cloned the rat TRI promoter and observed higher promoter activity in osteoblastlike cells compared with undifferentiated bone cells or dermal fibroblasts (7). The TRI promoter includes a CpG island, several transcription factor Sp1 binding sites consistent with constitutive expression by many cells, and six cis-acting elements for transcription factors, termed CBFa (7-8).2 Whereas CBFa2 and CBFa3 are important gene regulators in lymphoid cells (9), CBFa1 expression increases in parallel with osteoblast differentiation in vitro (10).2 Moreover, targeted disruption of the CBFa1 gene eliminates osteogenesis in mice, and insertion, deletion, or missense mutations in CBFa1 occur in humans with the skeletal disorder cleidocranial dysplasia (11). Genes directly affected by CBFa1, especially those important for skeletal developme...

Maximal gene expression driven by the promoter for the transforming growth factor ␤ type I receptor (TGF-␤RI) 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/CBF␣ binding sites among other possible cis-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/CBF␣-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-␤RI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-␤RI levels that parallel changes in bone cell differentiation or activity.Transforming growth factor-␤ (TGF-␤) 1 receptors occur on most cells, and a functional TGF-␤ type I receptor (TGF-␤RI) is required for all known TGF-␤-dependent effects. In some situations its activity is controlled by complex interactions with other cell surface components (1-3). However, in contrast to TGF-␤RII and the cell surface proteoglycan also termed TGF-␤RIII or betaglycan, expression of TGF-␤RI is maintained on differentiated bone cells (4). For these reasons, and because little is known about the molecular control of TGF-␤RI expression, we cloned the rat TGF-␤RI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats. The rat TGF-␤RI promoter lacks a typical TATA box, but initiates transcription at multiple sites within a 220-bp span upstream of the initial methionine codon in differentiated bone cells. The 3Ј-terminal 300-bp sequence encompassing this region contains a GC-rich CpG island, seven consensus Sp1 binding sites, and two CCAAT boxes. Transfection studies using different fragments of TGF-␤RI promoter cloned upstream of the reporter gene luciferase demonstrated maximal activity by a 1.0-kb fragment that encompassed these and other possible cis-acting elements. Importantly, several dispersed elements appeared to cooperate for maximal reporter gene expression in osteoblast-enriched cultures (5). Coincident with this work, the human TGF-␤RI promoter was cloned, and its sequence reveals a similar organization with identically spaced CCAAT box motifs (6).These features suggested that the TGF-␤RI gene is driven by a constitutively active promoter that maintains expression of TGF-␤RI in many cells. Nevertheless, this promoter is part...

Primary osteoblast-enriched (Ob) cultures from fetal rat bone synthesize insulin-like growth factor (IGF) I and IGF-II, which each enhance Ob function. While a number of agents modulate IGF-I production, IGF-II is constitutively expressed in this culture model. Independent of their expression, however, the activity of the IGFs can be modified by a small group of proteins termed IGF binding proteins (IGFBPs), but little is known about the regulation of individual IGFBPs that are synthesized by Ob cells. Northern blot analysis revealed that serum-deprived primary rat Ob cells express transcripts encoding IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, but undetectable levels of IGFBP-1 transcripts. Western ligand blots of Ob culture medium probed with 125I-IGF-I or 125I-IGF-II showed predominant IGFBPs migrating at 30/32 kDa, with minor bands at 24 and 38-47 kDa. Western antibody analysis identified IGFBP-2 and IGFBP-5 within the 30/32 kDa complex, while gel mobility shift on SDS-PAGE following deglycosylation determined that IGFBP-3 comprised the 38-47 kDa complex. By Northern analysis, 6 h treatment with prostaglandin E2 (PGE2), growth hormone (hGH), IGF-I, or IGF-II revealed a complex pattern of regulatory effects on steady-state IGFBP transcript expression. PGE2 increased the transcript levels of IGFBP-3, IGFBP-4, and IGFBP-5, (approximately 22-, approximately 2- and approximately 4-fold respectively), but had no effect on IGFBP-2 or IGFBP-6 transcripts. hGH enhanced IGFBP-3 and IGFBP-5 transcripts (each approximately twofold). IGF-I and IGF-II had no effect on IGFBP-2 steady-state transcript levels but enhanced the level of IGFBP-5 transcripts (approximately fourfold). By Western ligand blot analysis, 24 h treatment with PGE2 elevated the 24 and 38-47 kDa IGFBPs and to a lesser extent the 30/32 kDa complex, hGH elevated the 38-47 kDa IGFBPs, and IGF-I and IGF-II each increased the 30/32 kDa IGFBP complex. Therefore, a comparison of results obtained from Northern, Western ligand, and Western antibody studies indicates that multiple IGFBPs are expressed by primary rat Ob cultures. While IGFBP-2 and IGFBP-6 synthesis in Ob cultures is relatively unaffected by short-term treatment with PGE2, hGH, or the IGFs, these agents modify IGFBP-3, IGFBP-4, and IGFBP-5 expression with individual patterns of effects. In addition, some changes in IGFBP polypeptide levels that are independent of alterations in transcript expression may result from the formation of complexes between IGFs and certain IGFBPs, which could serve to store IGFs for future utilization in the formation phase of bone remodeling.

The proportion of transforming growth factor-␤ (TGF-␤) 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-␤ receptor stability could be an important aspect of regulation. Suppression of protein synthesis reduced TGF-␤ binding to types I and II receptors with t 1/2 of 2 h and to betaglycan with t 1/2 of 6 h. In contrast, suppression of mRNA transcription reduced TGF-␤ binding at least 3-fold more slowly at each receptor site. Preexposure to TGF-␤ decreased its binding at all three sites within 4 h in osteoblast-enriched cultures. This effect was transient with lower TGF-␤ 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-␤ binding profile in specific ways. Together, these results indicate that cell surface TGF-␤ receptors turn over rapidly by ligand-independent and ligand-dependent mechanisms, demonstrate that the binding capacity of TGF-␤ receptors is less stable than their mRNAs, and that functional receptor levels may be determined in part by post-transcriptional events.Transforming growth factor type ␤s (TGF-␤s) 1 are potent, ubiquitous growth regulators that are abundant in skeletal tissue. Their expression is controlled in temporal and spatial ways during skeletal tissue development, and they produce complex stimulatory and inhibitory changes in osteoblast function in vitro. The presence of TGF-␤s in bone and their importance as local skeletal growth regulators were first described in fetal rat calvariae, and cells derived from various bone tissues have provided much information about TGF-␤ activity and receptor expression during osteogenesis (reviewed in Ref. 1).Similar to cells found in many tissues, fetal rat bone cells express type I (53 kDa) and type II (73 kDa) glycoprotein receptors, as well as type III proteoglycans (Ͼ250 betaglycans) that could directly or indirectly influence TGF-␤ binding to the signaling types I and II sites (2-5). In this model, the association of TGF-␤ with each receptor varies with the basal state of bone cell differentiation, and relative mRNA abundance levels generally reflect the cell surface binding profiles found in untreated cells (5). Rapid changes in the proportion of TGF-␤ binding among receptors, induced by certain growth factors and hormones, are consistent with specific changes in its biological effects (5-8). For example, glucocorticoid decreases TGF-␤ binding to types I and II receptors, increases its binding to type III sites, and reduces its overall effectiveness (6). In contrast, bone morphogenetic protein (BMP)-2 reduces TGF-␤ binding to type II receptors and betaglycan, whereas it enhances binding to type I receptors. After BMP-2 treatment, the m...

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 E 2 (PGE 2 ) 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 estrogendependent 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 M PGE 2 increased IGF-I P1 activity by 5-fold. 17␤-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE 2 by up to 90% (ED 50 ϳ0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17␣-estradiol was 100 -300-fold less effective. By contrast, 17␤-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17␤-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 PGE 2 . 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 PGE 2 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.Although postmenopausal osteoporosis is one of the most prevalent age-related skeletal disorders, our understanding of the role of estrogen, which has a critical role in maintaining bone mass, remains incomplete. The principal laboratory animal model of postmenopausal osteoporosis is the ovariectomized (OVX) 1 rat. One consistent observation with this model is an increase in the rate of bone remodeling after OVX-induced estrogen depletion (1-5). Furthermore, consistent with a cause and effect relationship, estrogen administration re-establishes a reduced rate of bone remodeling in the OVX rat.Bone remodeling consists of two opposing events, i.e. bone resorption and formation (6). Skeletal integrity in adults relies on closely coupled remodeling where there is a balance between these catabolic and anabolic processes. Net bone loss in postmenopausal o...

Organization of the transforming growth factor-beta (TGF-beta) 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/PEBP2alpha) transcription factors. By gel mobility shift and immunological analyses, a principal osteoblast-derived nuclear factor that binds to these sites is CBFa1 (AML-3/PEBP2alphaA). 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-beta on osteoblast function.

Transforming growth factor (TGF-beta) binds several discrete membrane proteins. Of these, a type 1 receptor appears indispensable for signal transduction. Previous examination of TGF-beta receptor expression has been limited to changes in cell surface protein, and more recently, mRNA abundance. In order to learn more about TGF-beta function and receptor expression during osteogenesis, we have now cloned a 4 kilobase (kb) DNA fragment 5' proximal to the coding region of the rat TGF-beta type I receptor gene. Sequence analysis revealed multiple elements compatible with transcription initiation, including a properly positioned and oriented CCAAT box, six Sp1 binding sites (three defining GC boxes), and two strong AP2 binding sites within a 0.7 kb span directly upstream of the coding region. The 3' terminal 0.3 kb span comprises a GC-enriched (77%) so-called CpG island that, like other similarly organized promoters, lacks a TATA box. Primer extension and RNase protection studies with cRNAs from this area show multiple initiation sites within 220 bp 5' proximal to the initial methionine codon. Transient transfections using nested, deleted, and inverted promoter sequences demonstrated maximal reporter expression by a 1 kb fragment encompassing all of these elements. Truncation of the 1 kb fragment from the 5' and 3' ends indicated the need for several elements for peak promoter activity. These results, and transfections in fetal rat bone and dermal cells, suggest that this promoter contains elements that specify basal and conditional expression of the TGF-beta type I receptor in bone.