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Parathyroid hormone induces collagenase-3 gene transcription in rat osteoblastic cells. Here, we characterized the basal, parathyroid hormone regulatory regions of the rat collagenase-3 gene and the proteins involved in this regulation. The minimal parathyroid hormone-responsive region was observed to be between base pairs ؊38 and ؊148. Deleted and mutated constructs showed that the activator protein-1 and the runt domain binding sites are both required for basal expression and parathyroid hormone activation of this gene. The runt domain site is identical to an osteoblast-specific element-2 or acute myelogenous leukemia binding sequence in the mouse and rat osteocalcin genes, respectively. Overexpression of an acute myelogenous leukemia-1 repressor protein inhibited parathyroid hormone activation of the promoter, indicating a requirement of acute myelogenous leukemiarelated factor(s) for this activity. Overexpression of c-Fos, c-Jun, osteoblast-specific factor-2, and core binding factor-␤ increased the response to parathyroid hormone of the wild type (؊148) promoter but not with mutation of either or both the activator protein-1 and runt domain binding sites. In summary, we conclude that there is a cooperative interaction of acute myelogenous leukemia/ polyomavirus enhancer-binding protein-2-related factor(s) binding to the runt domain binding site with members of the activator protein-1 transcription factor family binding to the activator protein-1 site in the rat collagenase-3 gene in response to parathyroid hormone in osteoblastic cells. Parathyroid hormone (PTH)1 is an essential regulator of calcium homeostasis (1). In addition to kidney, its major target tissue is bone, the body's main calcium store. While PTH increases serum calcium partly by activating osteoclasts, these cells do not display PTH receptors. Instead, PTH exerts a direct effect on osteoblasts, causing them to cease synthesis of type I collagen (2, 3), the major organic component of bone. Most relevant to the current study, we and others have demonstrated that, in vitro, PTH can stimulate the osteoblastic synthesis of interstitial collagenase, the enzyme that specifically degrades fibrillar collagens (4, 5). Although collagenase synthesis and secretion by osteoblasts has been well documented, the signaling mechanism through which PTH stimulates its expression in this cell type is not fully understood. We have employed the UMR 106-01 (UMR) rat osteosarcoma cell line to investigate PTH regulation of collagenase-3 gene expression in osteoblasts. This cell line displays classical osteoblastic markers including PTH receptors, type I collagen, and high alkaline phosphatase expression. Most importantly to the present study, UMR cells decrease collagen synthesis and begin production of interstitial collagenase in response to PTH treatment. Previously, we reported that UMR cell collagenase induction by PTH is due to an increase in transcription and is a secondary response since it requires de novo protein synthesis (6). In the present work, we have di...

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Previously we showed that the activator protein-1 site and the runt domain binding site in the collagenase-3 promoter act cooperatively in response to parathyroid hormone (PTH) in the rat osteoblastic osteosarcoma cell line, UMR 106-01. Our results of the expression pattern of core binding factor ␣1 (Cbfa1), which binds to the runt domain site, indicated that there is no change in the levels of Cbfa1 protein or RNA under either control conditions or after PTH treatment. The importance of posttranslational modification of Cbfa1 in the signaling pathway for PTH-induced collagenase-3 promoter activity was analyzed. PTH stimulation of collagenase-3 promoter activity was completely abrogated by protein kinase A (PKA) inhibition. To determine the role of PKA activity with respect to Cbfa1 activation (in addition to its known activity of phosphorylating cAMP-response element-binding protein to enhance c-fos promoter activity), we utilized the heterologous Gal4 transcription system. PTH stimulated the transactivation of activation domain-3 in Cbfa1 through the PKA site. In vitro phosphorylation studies indicated that the PKA site in the wild type activation domain-3 is a substrate for phosphorylation by PKA. Thus, we demonstrate that PTH induces a PKA-dependent transactivation of Cbfa1, and this transactivation is required for collagenase-3 promoter activity in UMR cells.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR⅐RXR heterodimer is not well understood, but it may involve interactions of VDR⅐RXR with transcriptional coactivator or corepressor proteins. Here, a yeast twohybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62,000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1,25-dihydroxyvitamin D 3 -activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D-and other nuclear receptor-mediated transcriptional pathways.1 is mediated through an intracellular receptor termed the vitamin D receptor (VDR). VDR is a member of the superfamily of nuclear receptors for steroid hormones, and it acts as a ligand-induced transcription factor that binds to specific DNA response elements in the promoter region of vitamin D-responsive genes (1-3). Vitamin D response elements (VDREs) consist of either exact or imperfect direct repeats of the hexonucleotide sequence, GGGTGA, generally separated by a three-nucleotide spacer. High affinity binding of VDR to VDREs requires an additional nuclear factor that is most likely retinoid X receptor (RXR), the nuclear receptor for 9-cis-retinoic acid (4 -6). Thus, VDR and RXR heterodimerize to form a complex that binds with high affinity to VDREs, and it is the VDR⅐RXR heterodimer that may be the functional transcription factor in vitamin D-mediated gene expression.The mechanism that links the heterodimeric receptor complex bound at the DNA response element to the transcriptional complex is not well understood, but it is presumed to involve protein-protein interactions between the heterodimer and other transcriptional coactivator proteins. Recently, a number of putative coactivator and corepressor proteins have been described for several members of the nuclear receptor superfamily (7). A general property of these transcriptional cofactors is their ability to selectively interact with liganded nuclear receptors and modulate their transcriptional activity. Putative coactivators include s...

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptorrelated protein. Ligand blot analysis shows that 125 Icollagenase-3 binds specifically to two proteins (ϳ170 kDa and ϳ600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wildtype mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization. Collagenase-3 (MMP-13)1 is a member of the matrix metalloproteinase family of enzymes, which participates in extracellular matrix remodeling (1). Members of this family have a number of structural and functional features in common. In addition to sharing a similar domain structure, all are synthesized in inactive form, function at neutral pH, and require intrinsic zinc and calcium ions for their activity. Collagenase-3 is a highly regulated enzyme that cleaves native fibrillar collagens of types I, II, and III. The 57-kDa proenzyme is converted to its active 52-kDa form by the plasmin activation cascade, as well as by cathepsin B, stromelysin, and plasma kallikrein (2, 3). Collagenase-3 activity is inhibited by a family of tissue inhibitors of metalloproteinases (4, 5). A range of hormones and agents can also regulate expression of collagenase-3 (6). Parathyroid hormone is one of the hormones participating in this process. Osteoblastic cells respond to parathyroid hormone by increasing collagenase-3 synthesis (6 -8), plasminogen activator activity (9), and tissue inhibitors of metalloproteinases expression (8). In addition, experiments with UMR 106-01 rat osteosarcoma cells showed that over 80% of exogenous rat collagenase-3 was removed from the medium after 8 h of incubation (10). This rapid removal of rat collagenase-3 from the medium suggested the existence of a specific receptor that represents another level of regu...

Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, cfos, in this cell type through a cAMP-dependent pathway. The present work investigated the nuclear mechanisms involved in PTH regulation of c-fos in the osteoblastic cell line, UMR 106-01. By transiently transfecting c-fos promoter 5 deletion constructs into UMR cells, we demonstrated that PTH induction of the c-fos promoter requires the major cAMP response element (CRE). Point mutations created in the major CRE within the largest construct inhibited both PTH-stimulated and basal expression. This element, therefore, performs concerted basal and PTH-responsive cis-acting functions. Gel retardation and Western blotting techniques revealed that CRE-binding protein (CREB) constitutively binds the major CRE but becomes phosphorylated at its cAMP-dependent protein kinase consensus recognition site following PTH treatment. CREB was functionally implicated in c-fos regulation by coexpressing a dominant CREB repressor, KCREB (killer CREB), with the c-fos promoter constructs. KCREB suppressed both basal and PTH-mediated c-fos induction. We conclude that PTH activates c-fos in osteoblasts through cAMPdependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene.We have shown previously that parathyroid hormone (PTH) 1 stimulates c-fos transcription in the osteoblastic UMR 106-01 cell line through a cAMP-mediated pathway (1). However, the events that follow cAMP induction are less clear. The present work was undertaken to describe the nuclear mechanisms involved in PTH-mediated c-fos induction in osteoblasts. Many PTH-responsive genes in osteoblasts are thought to be secondary responses due to their delayed nature and requirement for ongoing protein synthesis (2, 3). By definition, these genes require the expression of primary response genes, such as c-fos, for their induction.Several in vivo models have identified Fos as a player in bone biology. This factor was first linked to bone when it was discovered in a mouse osteosarcoma as the product of v-fos, the viral homolog of c-fos (4). Similarly, several groups have engineered mice that overexpress c-fos and display bone abnormalities including non-malignant bone neoplasms and collagenaseproducing bone tumors (5, 6). Conversely, Fos null mice exhibit osteopetrosis and disorganized bone growth (7). Transgenic mice, which express a fos-lacZ fusion gene, also identified bone as one of the major sites for c-fos expression (8). In agreement with the rodent models, evidence for Fos involvement in human bone disease has been provided by high c-fos expression in Pagetic bone (9) and human osteosarcomas (10).c-fos is regulated in a cell-specific manner through a variety of mechanisms. These signaling pathways most likely act through different combinations of highly conserved sites within the promoter region (11). The mechanism for c-fos induction in osteoblasts by the bone r...

The clonal cell line UMR 106, which was originally derived from a rat transplantable osteogenic sarcoma with an osteoblastic phenotype, was subcloned after the emergence of a calcitonin-responsive adenylate cyclase was noted in late passages. Detailed studies on the stimulation of adenylate cyclase and activation profile of the cyclic AMP-dependent protein kinase isoenzymes in response to parathyroid hormone (PTH) and salmon calcitonin (SCT) were conducted on two subclones (UMR 106-01 and UMR 106-06). Both subclones responded in an identical manner to PTH, which stimulated adenylate cyclase and activated both isoenzyme I and isoenzyme II of cyclic AMP-dependent protein kinase. In contrast, only UMR 106-06 cells responded to calcitonin. At 3 X 10(-8)M SCT, there was a sevenfold stimulation of adenylate cyclase, 84% activation of isoenzyme I, and 44% activation of isoenzyme II. The activation profiles of the isoenzymes to PTH and SCT in UMR 106-06 were similar. Furthermore, their response to SCT correlates with the presence of specific, saturable binding of 125I-labeled SCT. Binding parameters indicate apparent Kd = 0.8 nM and 6,000 receptors/cell. These data point to a significant phenotypic change having taken place in this clonal cell line with prolonged maintenance in culture, with the emergence of a calcitonin receptor linked to adenylate cyclase and protein kinase activation.