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...

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...

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.

The -361 bp A/G polymorphism is common in all racial groups tested. The G allele was more active than the A allele in a transfection assay. The basis for this difference is not known. If the differences in activity of the promoter constructs were paralleled by differences in ALDH2 enzyme activity in the liver, this polymorphism could affect risk of alcoholism.

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