Parathyroid Hormone In Osteoblastic Cells Research Articles

Connexin 43 hemichannels and intracellular signaling in bone cells.

Cell function and survival are controlled by intracellular signals, and modulated by surrounding cells and the extracellular environment. Connexin channels participate in these processes by mediating cell-to-cell communication. In bone cells, gap junction channels were detected in the early 1970s, and are present among bone resorbing osteoclasts, bone forming osteoblasts, and osteocytes - mature osteoblasts embedded in the mineralized matrix. These channels are composed mainly by Cx43, although the expression of other connexins (45, 46, and 37) has also been reported. It is now believed that undocked Cx43 hemichannels (connexons) formed in unopposed cell membranes facing the extracellular environment participate in the interaction of bone cells with the extracellular environment, and in their communication with neighboring cells. Thus, we and others demonstrated the presence of active hemichannels in osteoblastic and osteocytic cells. These hemichannels open in response to pharmacological and mechanical stimulation. In particular, preservation of the viability of osteoblasts and osteocytes by the anti-osteoporotic drugs bisphosphonates depends on Cx43 expression in vitro and in vivo, and is mediated by undocked hemichannels. Cx43 hemichannels are also required for the release of prostaglandins and ATP by osteocytes, and for cell survival induced by mechanical stimulation in vitro. Moreover, they are required for the anti-apoptotic effect of parathyroid hormone in osteoblastic cells. This review summarizes the current knowledge on the presence and function of undocked connexons, and the role of hemichannel regulation for the maintenance of bone cell viability and, potentially, bone health.

Connexin43 interacts with βarrestin: A pre-requisite for osteoblast survival induced by parathyroid hormone

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43-interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.

Capitalizing on Admixture in Genome-Wide Association Studies: A Two-Stage Testing Procedure and Application to Height in African-Americans

As genome-wide association studies expand beyond populations of European ancestry, the role of admixture will become increasingly important in the continued discovery and fine-mapping of variation influencing complex traits. Although admixture is commonly viewed as a confounding influence in association studies, approaches such as admixture mapping have demonstrated its ability to highlight disease susceptibility regions of the genome. In this study, we illustrate a powerful two-stage testing strategy designed to uncover trait-associated single nucleotide polymorphisms in the presence of ancestral allele frequency differentiation. In the first stage, we conduct an association scan by using predicted genotypic values based on regional admixture estimates. We then select a subset of promising markers for inclusion in a second-stage analysis, where association is tested between the observed genotype and the phenotype conditional on the predicted genotype. We prove that, under the null hypothesis, the test statistics used in each stage are orthogonal and asymptotically independent. Using simulated data designed to mimic African-American populations in the case of a quantitative trait, we show that our two-stage procedure maintains appropriate control of the family wise error rate and has higher power under realistic effect sizes than the one-stage testing procedure in which all markers are tested for association simultaneously with control of admixture. We apply the proposed procedure to a study of height in 201 African-Americans genotyped at 108 ancestry informative markers. The two-stage procedure identified two statistically significant markers rs1985080 (PTHB1/BBS9) and rs952718 (ABCA12). PTHB1/BBS9 is downregulated by parathyroid hormone in osteoblastic cells and is thought to be involved in parathyroid hormone action in bones and may play a role in height. ABCA12 is a member of the superfamily of ATP binding cassette transporters and its potential involvement in height is unclear.

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Potassium channel activator bl-1249 inhibits the c- fos response to parathyroid hormone in osteoblastic cells in a dose-dependent manner

Potassium channel activator bl-1249 inhibits the c- fos response to parathyroid hormone in osteoblastic cells in a dose-dependent manner

Evidences for a role of p38 MAP kinase in the stimulation of alkaline phosphatase and matrix mineralization induced by parathyroid hormone in osteoblastic cells

Increased bone formation by PTH mainly results from activation of osteoblasts, an effect largely mediated by the cAMP-PKA pathway. Other pathways, however, are likely to be involved in this process. In this study we investigated whether PTH can activate p38 MAPK and the role of this kinase in osteoblastic cells. Bovine PTH(1–34) and forskolin markedly increased alkaline phosphatase (ALP) activity and doubled osteocalcin (Oc) expression in early differentiating MC3T3-E1 cells. These effects were associated with increase in cellular cAMP and activation of the MAP kinases ERK and p38. Activation of these MAP kinases was detectable after 1 h incubation with 10 − 7 M PTH and lasted 1–2 h. Activation of p38 was mimicked by 10 μM forskolin and prevented by H89 suggesting a cAMP-PKA-dependent mechanism of p38 activation. Interestingly, PTH-induced ALP stimulation was dose-dependently inhibited by a specific p38 inhibitor with no change in the generation of cAMP and the production of osteocalcin. Similar inhibitory effect was obtained in cells stably expressing a dominant-negative p38 molecule. Finally, treatment of MC3T3-E1 cells with PTH for 3 weeks significantly enhanced matrix mineralization and this effect was markedly reduced by a selective p38 but not a specific MEK inhibitor. In conclusion, data presented in this study indicate that PTH can activate p38 in early differentiating osteoblastic cells. Activation of p38 is cAMP-PKA-dependent and mediates PTH-induced stimulation of ALP which plays a critical role for the calcification of the bone matrix.

AP-1 and Cbfa/Runt Physically Interact and Regulate Parathyroid Hormone-dependent MMP13 Expression in Osteoblasts through a New Osteoblast-specific Element 2/AP-1 Composite Element

The expression of MMP13 (collagenase-3), a member of the matrix metalloproteinase family, is increased in vivo as well as in cultured osteosarcoma cell lines by parathyroid hormone (PTH), a major regulator of calcium homeostasis. Binding sites for AP-1 and Cbfa/Runt transcription factors in close proximity have been identified as cis-acting elements in the murine and rat mmp13 promoter required for PTH-induced expression. The cooperative function of these factors in response to PTH in osteoblastic cells suggests a direct interaction between AP-1 and Cbfa/Runt transcription factors. Here, we demonstrate interaction between c-Jun and c-Fos with Cbfa/Runt proteins. This interaction depends on the leucine zipper of c-Jun or c-Fos and the Runt domain of Cbfa/Runt proteins, respectively. Moreover, c-Fos interacts with the C-terminal part of Cbfa1 and Cbfa2, sharing a conserved transcriptional repression domain. In addition to the distal osteoblast-specific element 2 (OSE2) element in the murine and rat mmp13 promoter, we identified a new proximal OSE2 site overlapping with the TRE motif. Both interaction of Cbfa/Runt proteins with AP-1 and the presence of a functional proximal OSE2 site are required for enhanced transcriptional activity of the mmp13 promoter in transient transfected fibroblasts and in PTH-treated osteosarcoma cells.

Induction of rat interstitial collagenase (MMP-13) mRNA in a development-dependent manner by parathyroid hormone in osteoblastic cells.

The purpose of this study was to determine whether the production of interstitial collagenase mRNA in response to parathyroid hormone (PTH) changes with osteoblast phenotypic development. To accomplish this, cells derived from fetal rat calvaria were examined. The calvarial osteoblasts, which proliferate when placed in culture, can be made to differentiate after confluence. Studies were performed on cells while they were proliferating, at confluence, and during the differentiation process. The cells were treated with PTH for various times, and interstitial collagenase mRNA was quantified by RNase protection assay. We concluded that the ability of PTH to induce interstitial collagenase mRNA in these cells increased with osteoblast phenotypic development. We also determined that the response could be mimicked by combining the effect of 8-bromo-cAMP and 12-O-tetradecanoyl-phorbol-13-acetate, stimulators of the protein kinase A and protein kinase C pathways, respectively, both known to be activated by PTH. The binding of nuclear factors to two regions previously reported to be important for PTH induction of the gene in UMR 106-01 cells was also examined. These data indicated that the binding of nuclear factors to oligonucleotides encompassing the TRE (-51) or the PEA3 (-80) elements changed with development of the osteoblast phenotype. The latter was also shown to be PTH responsive.

Identification of a novel parathyroid hormone-responsive gene in human osteoblastic cells

Parathyroid hormone (PTH) is a potent stimulator of osteoblastic cell function in vitro and bone resorption and formation in vivo; however, the details of the molecular mechanism(s) responsible for PTH action and the regulation of gene expression in response to PTH remain unknown. In this study, we employed an mRNA differential display (DRD) approach to examine the initial events in gene expression in human osteoblast-like SaoS-2/B10 cells exposed to 10 −7 mol/L bPTH(1-34). This approach identified several differentially regulated mRNA species, including a novel paired-class homeobox protein, osteoblast-specific factor-2 (OSF-2), and a unique clone with no known sequence homology (clone G18). G18 is a previously unidentified human gene, expressed in a wide variety of human tissues, including heart, brain, placenta, skeletal muscle, and kidney, and is regulated by PTH in osteoblastic cells in vitro. This mRNA appears to be the product of a single gene, which is alternatively spliced to produce multiple transcript sizes observed in several tissues, except bone and bone-derived cells, in which a single predominant ∼1.8 kb transcript is observed. Our study has identified several genes that have expression altered significantly by treatment with bPTH(1-34), and which may provide insight into the immediate effects of PTH on osteoblast-like cells and ultimately on the mechanism of action and bioactivity of PTH.

Parathyroid Hormone Regulates the Rat Collagenase-3 Promoter in Osteoblastic Cells through the Cooperative Interaction of the Activator Protein-1 Site and the runt Domain Binding Sequence

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.

Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.

Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways.

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.