MicroRNA-590-5p Stabilizes Runx2 by Targeting Smad7 During Osteoblast Differentiation.

Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co-factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR-590-5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR-590-5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR-590-5p and its mRNA and protein expression decreased after miR-590-5p mimic transfection in human osteoblast-like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR-590-5p. However, Runx2 protein, but not mRNA expression, increased after miR-590-5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3'-untranslated region of Smad7 was directly targeted by miR-590-5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2-mediated Runx2 degradation. Hence, based on our results, we suggest that miR-590-5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. J. Cell. Physiol. 232: 371-380, 2017. © 2016 Wiley Periodicals, Inc.

Runx2, a target gene for activating transcription factor-3 in human breast cancer cells.

Activating transcription factor (ATF-3) is a stress response gene and is induced by transforming growth factor beta 1 (TGF-ß1) in breast cancer cells. In this study, we dissected the functional role of ATF-3 gene in vitro by knocking down its expression stably in human bone metastatic breast cancer cells (MDA-MB231). Knockdown of ATF-3 expression in these cells decreased cell number, altered cell cycle phase transition, and decreased mRNA expression of cell cycle genes. Knockdown of ATF-3 expression in MDA-MB231 cells also decreased cell migration, and the expression levels of invasive and metastatic genes such as MMP-13 and Runx2 were found to be decreased in these cells. Most importantly, ATF-3 was associated with Runx2 promoter in MDA-MB231 cells and knockdown of ATF-3 expression decreased its association with Runx2 promoter. Hence, our results suggested that ATF-3 plays a role in proliferation and invasion of bone metastatic breast cancer cells in vitro and we identified for the first time that Runx2 is a target gene of ATF-3 in MDA-MB231 cell line.

MiR-4638-3p regulates transforming growth factor-ß1-induced activating transcription factor-3 and cell proliferation, invasion, and apoptosis in human breast cancer cells.

TGF-ß1 (transforming growth factor-beta1), a secreted polypeptide cytokine, stimulates ATF-3 (activating transcription factor-3) expression in a sustained and prolonged manner in human breast cancer cells (MDA-MB231), but not in normal human mammary epithelial cells (MCF-10A). Cyclin A (cell proliferation gene), Runx2 (metastasis gene), and MMP-13 (matrix metalloproteinase-13; invasive gene) were identified as ATF-3 target genes in these cells. Because ATF-3 has very few druggable sites, its direct targeting is difficult. Recent evidence has indicated that microRNAs (miRNAs) are key players in the post-transcriptional modulation of gene expression under several conditions. Bioinformatic analysis suggested a list of putative miRNAs that target ATF-3. Therefore, we hypothesized that TGF-ß1 downregulates the miRNAs that target ATF-3, resulting in the activation of genes that participate in breast cancer progression and skeletal metastasis. Our findings indicate that TGF-ß1 downregulated the expression of miR-4638-3p in MDA-MB231 cells. At the molecular level, forced expression of miR-4638-3p reduced the expression of ATF-3 and its downstream targets, Runx2 and MMP-13, in these cells. At the cellular level, overexpression of miR-4638-3p reduced proliferation, invasion, and migration, and induced G0/G1 cell cycle arrest and apoptosis in MDA-MB231 cells. Overall, this study highlights the possibility of utilizing miR-4638-3p as a therapeutic molecule to curb skeletal metastasis of breast cancer cells.

Parathyroid hormone-stimulation of Runx2 during osteoblast differentiation via the regulation of lnc-SUPT3H-1:16 (RUNX2-AS1:32) and miR-6797-5p.

Parathyroid hormone (PTH) acts as a regulator of calcium homeostasis and bone remodeling. Runx2, an essential transcription factor in bone, is required for osteoblast differentiation. Noncoding RNAs such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play crucial roles in regulating gene expression in osteoblasts. In this study, we investigated the effects of PTH on osteoblast differentiation via Runx2, lncRNA, and miRNA expression in human bone marrow stromal cells (hBMSCs) and human osteoblastic cells (MG63). PTH-treatment of hBMSCs for 24 h, 7 days, and 14 days stimulated Runx2 mRNA expression. Using bioinformatics tools, we identified 17 lncRNAs originating from human Runx2 gene. Among these, lnc-SUPT3H-1:16 (RUNX2-AS1:32) expression was highly up-regulated by the 7 d PTH-treatment in hBMSCs. We also identified miR-6797-5p as the putative target of lnc-SUPT3H-1:16 and Runx2 using bioinformatics tools. PTH-treatment increased the expression of miR-6797-5p in hBMSCs, and overexpression of miR-6797-5p decreased osteoblast differentiation in MG63 cells, suggesting a role for lnc-SUPT3H-1:16 as sponge molecule. A luciferase gene reporter assay identified direct targeting of miR-6797-5p with lnc-SUPT3H-1:16 and 3'UTR Runx2 in MG63 cells. Thus, PTH stimulated the expression of lnc-SUPT3H-1:16, miR-6797-5p and Runx2, and due to the sponging mechanism of lnc- SUPT3H-1:16 towards miR-6797-5p, Runx2 was protected, resulting in the promotion of osteoblast differentiation.

Morphological and biochemical characterization of four clonal osteogenic sarcoma cell lines of rat origin.

The ultrastructural and biochemical properties of four clonal osteogenic sarcoma lines, UMR 104, 105, 106, and 108, have been compared with uncloned osteogenic sarcoma cells and normal osteoblast-rich cells derived from newborn rat calvaria. High alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone were used as biochemical markers of osteoblastic cells. Cloning enriched both of these parameters above those of the parent tumor and far higher than that seen in normal cells, suggesting enrichment of the osteoblast phenotype. Both of these properties have been retained through many passages in culture. Morphologically, the clonal lines have also retained the "blast"-like appearance of the uncloned osteogenic sarcoma cells and consist mainly of flat, relatively featureless cells. Many cells with mitotic figures were observed, indicating continuous cell division taking place in the malignant cells. Each clonal line gave rise to characteristic tumors when reinjected into rats. It is concluded that the clonal osteogenic sarcoma lines are highly differentiated tumor lines which have conserved the differentiated properties of the mature osteoblast, making them a suitable model for the study of the effects of hormones on the growth of a differentiated tumor, as well as for the study of hormonal regulation of the osteoblast.

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis.

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells.

PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone inhibits collagen synthesis at both ribonucleic acid and protein levels in rat osteogenic sarcoma cells.

Cells of the clonal rat osteogenic sarcoma cell line, UMR 106-01, were used to investigate the regulation of collagen synthesis by PTH in osteoblastic cells. Monolayer cultures of cells were labeled with [3H] proline in order to determine both collagen type and rates of production. Analysis of labeled extracellular polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that UMR 106-01 cells synthesized predominantly type I collagen, accounting for 45.48 +/- 2.09% of the radioactivity incorporated into total protein. After 24-h treatment with bovine PTH (1-34, 10(-8) M), collagen synthesis (i.e. collagenase-digestible protein) was decreased to 29.45 +/- 1.39% of total protein production. This decrease was first observed 12 h after addition of hormone and greatest inhibition was achieved at 24 h. The effect of PTH was dose dependent, with half-maximal inhibition of collagen synthesis occurring at 5 x 10(-10) M after 24-h treatment. In contrast, when steady state levels of mRNA for type I collagen chains were examined by Northern blot analysis, the concentration of PTH that reduced collagen synthesis by 35-45% (10(-8) M), caused a net decrease of approximately 80-96% in the number of procollagen transcripts; a small reduction in beta-actin mRNA levels was also observed. The effect of the hormone on procollagen message level was dose dependent, with significant inhibition observed at 10(-10) M PTH and, as with collagen synthesis, maximal after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells.

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of collagenase production by rat osteosarcoma cells can occur in a subpopulation of cells.

Recent studies have indicated that neutral collagenase can be produced in bones of rats. In addition, it has been demonstrated by in vitro studies that the enzyme is likely secreted by osteoblasts. Cells of the osteoblastic tumor cell line UMR-106 can be stimulated to produce not only collagenase, but also collagenase inhibitor and plasminogen activator. However, it is conceivable that not all osteoblasts produce all of these proteins. In this study, in which UMR cells were maximally stimulated with PTH, only a subpopulation of cells was observed to produce enhanced levels of collagenase but all cells had the ability to synthesize plasminogen activator. Cells of the rat osteosarcoma line UMR-106-01 were stained for the presence of collagenase and tissue plasminogen activator using an immunohistochemical procedure. In many cases, the cells were exposed to monensin for the final 3 h of incubation as well as to the inducing agent PTH. Monensin prevented export of the enzymes, enabling them to be visualized within their cell or origin. Maximal stimulation of collagenase was demonstrated to occur 8 h after exposure to 10(-8) -10(-7) M PTH. Under these conditions, 14-17% of the cells appeared to synthesize elevated amounts of collagenase (as determined by intense staining). Without PTH stimulation, there was a low level of collagenase in all cells, but less than 1% of the cells stained heavily for the enzyme. In contrast, strong staining for plasminogen activator was observed in all cells with or without PTH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse.

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.

Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts.

The plasminogen activator (PA) activity of clonal rat osteogenic sarcoma cell (phenotypically osteoblast) and of osteoblast-rich rat calvarial cells is shown to be increased by treatment with the bone-resorbing hormones, PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, and epidermal growth factor. Dose-dependent increases were observed, after a lag period of 4 to 8 h. Stimulated and control PA activities were inhibited by actinomycin D and cycloheximide but not by cytosine arabinoside. Glucocorticoid hormones prevented the hormone stimulation, but other steroids did not. Calcitonin had no effect either on basal or on hormone-treated PA activity. Isobutyl-methylxanthine alone increased PA activity and enhanced responsiveness to PTH and to prostaglandin E2. These data point to a common pathway in the actions upon osteoblasts of several hormones with diverse initial cellular actions and raise the possibility that the PA/plasmin system may contribute to cellular mechanisms of bone turnover.

Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3',5'-monophosphate response element-binding protein at serine 133.

PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 133 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1-34) and -(1-31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3-34), -(13-34), and -(28-48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression.

Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells.

Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

Activity ratio measurements reflect intracellular activation of adenosine 3',5'-monophosphate-dependent protein kinase in osteoblasts.

Parathyroid hormone, prostaglandin E2, and prostacyclin activate cAMP-dependent protein kinase in osteoblast-rich normal rat calvarial cells and in clonal rat osteogenic sarcoma cells of osteoblastic phenotype. The present study was undertaken to determine the activation of the enzyme in relation to cellular cAMP concentrations at increasing doses of the three hormones and also to test that the activity ratio measurement of the enzyme (ratio of the activity in the absence of cAMP to the activity in the presence of excess cAMP) was a true reflection of intracellular activation of the enzyme. With each hormone, using either normal or malignant osteoblasts, activation of the enzyme took place at hormone concentrations lower than those required to produce detectable changes in cAMP concentrations in the incubations. Stimulation of activity was abolished by addition of the heat-stable inhibitor of cAMP-dependent protein kinase, indicating that activation was of cAMP-dependent protein kinase alone. To demonstrate that protein kinase activation occurred intracellularly and not during sample preparation, charcoal was added at the time of cell disruption to absorb free cAMP. Under these conditions, no change was observed in the concentration of bovine parathyroid hormone required to cause activation of cAMP-dependent protein kinase. Finally, addition of purified cAMP-dependent protein kinase type I or type II to treated cells at the time of lysis did not result in significant activation of added isoenzyme, except at hormone concentrations sufficient to increase the total cAMP concentration of incubations. It is concluded that activity ratio measurement reflects the intracellular state of activation of cAMP-dependent protein kinase in the osteoblast-like cells treated by hormones and, furthermore, that only a fraction of the maximally generated cAMP is necessary for full enzyme activation.

Activation of adenosine 3',5'-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E2, and prostacyclin.

Hormonal activation of cAMP-dependent protein kinase has been studied in cultured cells derived from a rat osteogenic sarcoma and in osteoblast-rich cells grown from newborn rat calvaria. Both cell strains contain adenylate cyclase activities which respond to parathyroid hormone (PTH) and a variety of prostanoids. PTH, prostaglandin E2 (PGE2), and prostacyclin (PGI2) were all capable of activating cAMP-dependent protein kinase(s) in suspensions of the two cell types. Activation was very rapid in all cases, being detectable at 10 sec and maximal between 30-60 sec. Using saturating concentrations of hormones, the protein kinase activity ratio remained elevated (between 0.6-0.9) for up to 35 min after the start of PGE2 stimulation, but declined toward basal activity ratio 5-10 min after stimulation with PTH or PGI2. Each of the hormones caused a dose-dependent increase in activation of cAMP-dependent protein kinase in both cell types. Half-maximal activation of the enzyme occurred at 2 X 10(-9) M bovine PTH for calvarial cells, at 10(-8) M bPTH for osteogenic sarcoma cells, and at 2-4 X 10(-8) M PGE2 and 1-3 X 10(-7) M PGI2 for both cell types. Maximal activation of protein kinase occurred before maximal cAMP accumulated, implying that only a fraction of cAMP is biologically significant. These two cell strains provide a useful means of analyzing postreceptor events in the hormonal regulation of bone cells.

Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells.

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.

Second messenger signaling in the regulation of collagenase production by osteogenic sarcoma cells.

Recent work indicates that PTH can stimulate osteoblastic cells to secrete neutral collagenase, an enzyme thought to be linked to bone matrix turnover. Since recent studies suggest that the calcium/protein kinase-C (PKC) message system is involved in signal transduction stimulated by PTH, we examined the role of these putative second messengers of PTH in the regulation of collagenase production by the osteoblastic tumor cell line UMR 106-01. Immunohistochemical staining of cells exposed to PTH (10(-7) M) revealed that about 20% of the entire population was positive for collagenase, compared to less than 3% staining positively in control untreated cells. Incubation with the cAMP analog 8-bromo-cAMP (8BrcAMP) increased the number of collagenase-staining cells in a dose-dependent manner (ED0.5 = 2.5 x 10(-4) M), but to a lower level than PTH, with the maximal effect producing about 15% positive cells. The calcium ionophore ionomycin (10(-7) M) was ineffective, whereas phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased collagenase-specific staining to about 5%, but only at high concentrations (10(-5) M). Incubation of UMR 106-01 cells with ionomycin and PMA did not change the effect of the latter. When the three agents were used in combination, an additive effect was observed, which fully reproduced that of PTH. Similarly, the amount of collagenase released into the medium by cells stimulated with maximal concentrations of 8BrcAMP (10(-3) M) was only 80% of that induced by maximal doses of PTH (10(-7) M). PMA (10(-5) M) was slightly stimulatory, and ionomycin was ineffective alone, but they were synergistic with submaximal doses of 8BrcAMP (10(-4) M). In agreement with the immunohistochemical results, the full hormonal effect was reproduced when the three second messenger analogs were used in combination. In conclusion, signal transduction from PTH receptor to collagenase production is mediated mainly by cAMP; the Ca2+/PKC system appears to have a contributory role necessary for the full expression of hormonal response. These results support the hypothesis of a dual pathway of target cell activation by PTH.

Induction of the vitamin D 24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 is regulated by parathyroid hormone in UMR106 osteoblastic cells.

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.

Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells.

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.