Faculty Bibliography

The expression of the vitamin D 24-hydroxylase is highly regulated in target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D), where it may modulate the action of 1,25(OH)2D. In UMR106 osteoblastic cells, 1,25(OH)2D and PTH synergistically induce 24-hydroxylase expression. The purpose of these studies was to characterize the interaction between 1,25(OH)2D and PTH with regard to the messenger RNA (mRNA) levels of the cytochrome P450 component of the 24-hydroxylase (CYP24). PTH alone had no effect on CYP24 mRNA levels, and 1,25(OH)2D alone produced only a modest increase. However, 1,25(OH)2D and PTH together synergistically increased CYP24 mRNA levels 3-fold compared with 1,25(OH)2D alone. PTH also increased the sensitivity of UMR cells to 1,25(OH)2D from 10(-8) to 10(-10) M. PTH worked through the cAMP signaling pathway as evidenced by the lack of effect of PTH (3-34) and by the full activity of 8-bromo-cAMP. PTH in the presence of 1,25(OH)2D increased CYP24 gene transcription as shown by nuclear run-on studies and by activation of a CYP24 promoter-reporter construct after transfection. PTH also increased vitamin D receptor number in UMR cells, but this occurred at times later than the increase in transcription. These studies demonstrate that PTH in the presence of 1,25(OH)2D works through the cAMP-dependent signaling pathway to increase transcription of the CYP24 gene, to increase CYP24 protein levels, and to increase 24-hydroxylase activity

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 VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid 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 D3-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

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 leukemia-related factor(s) for this activity. Overexpression of c-Fos, c-Jun, osteoblast-specific factor-2, and core binding factor-beta 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

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

The major CRE in the c-fos gene is necessary for its activation in response to parathyroid hormone (PTH) treatment in UMR 106-01 rat osteosarcoma cells as determined through transient transfection of mouse c-fos 5'-deletion constructs. This CRE binds protein(s) from these cells which include CREB. We now provide further evidence indicative of a role for phosphoCREB and the major CRE in PTH activation of c-fos. To directly assess the importance of the c-fos major CRE, we have mutated this element in the largest of our c-fos promoter constructs (-356/os-CAT). This construct was transiently transfected into UMR 106-01 ceils and treated with PTH (1fr1 M). The mutation reduced basal expression to 10% of wild type. Most significantly, PTH inducibility was substantially decreased from 2.7 to 1.5 fold stimulation. Gel mobility shift confirmed that this mutation prevents CREB binding to this CRE. CREB involvement in c-fos promoter activity is directly addressed by cotransfection of the dominant inhibitor KCREB with the c-fos deletion constructs. KCREB expression substantially (average 46%) inhibits induction of all PTH-activatable promoter constructs. Of primary importance, PTH treatment causes phosphorylation of CREB protein in our cells with a time course and PTH dose dependency that parallels previously measured protein kinase A Induction. These data support our hypothesis that PTH-induced phosphoCREB binds the major CRE in the c-fos 5' regulatory region then activates transcription of this gene in UMR 106-01 cells.

We have previously identified a specific receptor for collagenase on UMR 106-01 rat osteosarcoma cells which is responsible for removal, internalization and degradation of PTH-induced extracellular collagenase. The present work shows that this is a member of aie low-density lipoprotein (LDL) receptor superfamily. The intracellular receptor associated protein (RAP) prevents ligand binding by three members of this family, the lipoprotein receptor related protein (LRP), gp330 and the VLDL receptor. RAP efficiently competed for binding of collagenase to UMR cells indicating that the collagenase receptor is another member of the LDL receptor superfamily. We have eliminated the possibility that the collagenase receptor is the LRP since equivalent collagenase binding was observed on LRP-null mouse embryo fibroblasts as on wild-type cells. Ligand blots of membranes from kidney, liver and UMR cells using 125I-RAP or collagenase show that the UMR membranes contain a similar receptor to LRP (liver) and gp330 (kidney) which is about 600kDa. Western blots using antiserum to rat gp330 suggest that the UMR collagenase receptor is not gp330. We have shown that normal rat osteoblasts and rat embryo fibroblasts also bind collagenase and endocytose and degrade the ligand. Our results suggest that the collagenase receptor is a member of the LDL receptor superfamily and is possibly a new member of mis family.

Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, c-fos, 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 cAMP-dependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene

Age-, postmenopause-, and disease-related conditions that result in low bone mass represent important public health issues. Maintenance of bone mass is a balance between bone resorption and formation and is influenced by diet, body composition, activity level, and the interactions between and among a large number of hormones, growth factors, and cytokines. Recent research has emphasized establishing a more complete understanding of the hormonal regulation of bone and developing anabolic agents with therapeutic potential for the treatment of low bone mass. The NIDDK at the NIH recently sponsored a Workshop, entitled Anabolic Hormones in Bone: Basic Research and Therapeutic Potential, that attempted to define the current state of the art knowledge of hormones, growth factors, and cytokines that affect bone mass, with particular emphasis on those that could potentially have a role as anabolic agents in bone. This review presents a condensed proceedings of that workshop along with a summary of the optimal requisites for the development of anabolic agents with therapeutic potential in bone

Interstitial collagenase plays an important role in both the normal and pathological remodeling of collagenous extracellular matrices, including skeletal tissues. The enzyme is a member of the family of matrix metalloproteinases. Only one rodent interstitial collagenase has been found but there are two human enzymes, human collagenase-1 and -3, the latter being the homologue of the rat enzyme. In developing rat and mouse bone, collagenase is expressed by hypertrophic chondrocytes, osteoblasts, and osteocytes, a situation that is replicated in a fracture callus. Cultured osteoblasts derived from neonatal rat calvariae show greater amounts of collagenase transcripts late in differentiation. These levels can be regulated by parathyroid hormone (PTH), retinoic acid, and insulin-like growth factors, as well as the degree of matrix mineralization. Much of the work on collagenase in bone has been derived from studies on the rat osteosarcoma cell line, UMR 106-01. All bone-resorbing agents stimulate these cells to produce collagenase mRNA and protein, with PTH being the most potent stimulator. Determination of secreted levels of collagenase has been difficult because UMR cells, normal rat osteoblasts, and rat fibroblasts possess a scavenger receptor that removes the enzyme from the extracellular space, internalizes and degrades it, thus imposing another level of control. PTH can also regulate the abundance of the receptor as well as the expression and synthesis of the enzyme. Regulation of the collagenase gene by PTH appears to involve the cAMP pathway as well as a primary response gene, possibly Fos, which then contributes to induction of the collagenase gene. The rat collagenase gene contains an activator protein-1 sequence that is necessary for basal expression, but other promoter regions may also participate in PTH regulation. Thus, there are many levels of regulation of collagenase in bone perhaps constraining what would otherwise be a rampant enzyme

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene