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...

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.Matrix metalloproteinases play an essential role in physiological processes of tissue remodeling, including embryonic development, bone remodeling, ovulation, uterine involution, and wound healing (1, 2), and in pathological states such as rheumatoid and osteoarthritis and tumor invasion and metastasis (3-5). Recent studies have identified a novel matrix metalloproteinase from human breast carcinoma cells, collagenase-3 (matrix metalloproteinase-13) as an important metalloproteinase (6). Studies demonstrating a diminished response to PTHinduced bone resorption in collagenase-resistant mice implicate a role for collagenase-3 in the bone remodeling process (7). Collagenase-3 is a neutral metalloproteinase that can degrade types I, II and III fibrillar collagens and has been implicated in several disease states requiring the remodeling of extracellular matrices. Collagenase-3 has been detected in vivo in degenerative bone diseases including osteoarthritis and rheumatoid arthritis (4, 8, 9) as well as in several metastatic tumors including breast carcinomas (6), chondrosarcomas (10), and head and neck carcinomas (11). In addition, collagenase-3 has been detected during human fetal ossification (12, 13) and during murine fetal bone development (14), where it is likely to play an important role in bone development.Humans express three collagenases, fibroblast collagenase (collagenase-1 ...

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast.

Prostaglandins are essential for the initiation of parturition in mice. The peak in uterine prostaglandin F(2)(alpha) levels occurs at d 19.0 of gestation, just before the onset of labor. Our studies set out to determine the important regulatory step(s) involved in this increase of prostaglandin F(2)(alpha). We show that cytosolic phospholipase A(2) mRNA, protein, and activity do not significantly vary during mouse gestation. Rather, our studies demonstrate that cyclooxygenase-1 mRNA is abruptly induced at d 15.5 of gestation, but cyclooxygenase-1 protein levels only gradually increase throughout gestation. In contrast, cyclooxygenase-2 protein remains constant during gestation. We find that prostaglandin F synthase protein increases significantly during gestation reaching peak levels between d 15.5 and d 17.5 of gestation. We also find that the level of prostaglandin dehydrogenase, responsible for degradation of prostaglandins, decreases during late gestation. Taken together these results suggest that the regulation of prostaglandin F(2)(alpha) is a complex process involving the coordinate induction of synthetic enzymes along with a decrease in degradative enzymes involved in prostaglandin metabolism.

Prostaglandins are essential for the initiation of parturition in mice. The peak in uterine prostaglandin F(2)(alpha) levels occurs at d 19.0 of gestation, just before the onset of labor. Our studies set out to determine the important regulatory step(s) involved in this increase of prostaglandin F(2)(alpha). We show that cytosolic phospholipase A(2) mRNA, protein, and activity do not significantly vary during mouse gestation. Rather, our studies demonstrate that cyclooxygenase-1 mRNA is abruptly induced at d 15.5 of gestation, but cyclooxygenase-1 protein levels only gradually increase throughout gestation. In contrast, cyclooxygenase-2 protein remains constant during gestation. We find that prostaglandin F synthase protein increases significantly during gestation reaching peak levels between d 15.5 and d 17.5 of gestation. We also find that the level of prostaglandin dehydrogenase, responsible for degradation of prostaglandins, decreases during late gestation. Taken together these results suggest that the regulation of prostaglandin F(2)(alpha) is a complex process involving the coordinate induction of synthetic enzymes along with a decrease in degradative enzymes involved in prostaglandin metabolism.