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...

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.

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...

No abstract

We have previously shown that in the rat osteoblastic osteosarcoma cell line-UMR 106-01-PTH induces maximal collagenase mRNA levels at 4 hours. Since this response to PTH requires de novo protein synthesis, it may be mediated by the combined temporal expression of members of the activator protein-1 (AP-1) gene family. We have demonstrated that maximal mRNA levels of two of the members of this family, c-fos and c-jun, occur 30 min after stimulation by PTH. Phorbol myristate acetate (PMA) elicits a similar increase in c-fos and c-jun mRNAs, but is unable to stimulate transcription of collagenase in these cells. To investigate further the involvement of the AP-1 gene family, we examined PTH and PMA stimulation of jun-B, jun-D, fos B, and fra-1 mRNAs in UMR 106-01 cells. The mRNA for jun-D was abundant under control conditions and showed no variation in response to PTH (10(-8) M). The fos B transcripts were not detected under control conditions, whereas jun-B and fra-1 mRNAs were present at low basal levels. PTH caused an increase in fos B mRNA that reached a maximal 4- to 5-fold plateau between 45 and 60 min. An increase in jun-B mRNA in response to PTH was detectable at 30 min, but reached a maximal 6- to 7-fold increase at 2 hours. After PTH stimulation, the fra-1 transcript showed a 10- to 11-fold peak at 4 hours. PMA (2.6 x 10(-7) M) stimulated fos B mRNA to maximal abundance at 1 hour, similar to PTH. In contrast, PMA caused a maximal increase in jun-B mRNA at 30 min and fra-1 mRNA at 2 hours, which was earlier than the response to PTH. To determine whether an increase in jun-B at the same time as c-fos and c-jun would inhibit collagenase gene transcription, we cotransfected an expression vector for jun-B with a rat collagenase promoter-reporter gene construct. This resulted in a decrease in PTH-stimulation of promoter activity. Thus, it appears that the differential temporal stimulation of the AP-1 genes by PTH and PMA, particularly an increase in jun-B at the same time as c-fos and c-jun, explains the difference seen in their ability to induce transcription of collagenase.