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Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts.

Zhang R, Edwards JR, Ko SY, Dong S, Liu H, Oyajobi BO, Papasian C, Deng HW, Zhao M - PLoS ONE (2011)

Bottom Line: These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts.Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells.Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biostatistics and Bioinformatics, Tulane University, New Orleans, Louisiana, United States of America.

ABSTRACT
Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

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PTH-CREB pathway promotes osteoblast differentiation.(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.
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pone-0020780-g002: PTH-CREB pathway promotes osteoblast differentiation.(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.

Mentions: It has been postulated that the anabolic effect of PTH on bone is mediated through osteoblasts. In this series of experiments, we characterized the role of the PTH-CREB signaling pathway in osteoblast differentiation. First, we determined the effects of PTH on alkaline phosphatase (ALP) activity of C2C12 cells by incubating cells with PTH for 24 hours. Measurement of ALP activity in cell lysates showed that treatment with PTH at 0–200 nM stimulated ALP activity in a pattern that approached dose-dependence, with a maximum effect (2-fold increase) at PTH concentration 100 nM (Fig. 2A). Real time PCR showed that the mRNA levels of osteoblast maturation genes, Runx2 and type I collagen 1a1 (Col1a1), were significantly enhanced by treatment with PTH at doses of 10–100 nM (Fig. 2B).


Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts.

Zhang R, Edwards JR, Ko SY, Dong S, Liu H, Oyajobi BO, Papasian C, Deng HW, Zhao M - PLoS ONE (2011)

PTH-CREB pathway promotes osteoblast differentiation.(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.
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getmorefigures.php?uid=PMC3111437&req=5

pone-0020780-g002: PTH-CREB pathway promotes osteoblast differentiation.(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.
Mentions: It has been postulated that the anabolic effect of PTH on bone is mediated through osteoblasts. In this series of experiments, we characterized the role of the PTH-CREB signaling pathway in osteoblast differentiation. First, we determined the effects of PTH on alkaline phosphatase (ALP) activity of C2C12 cells by incubating cells with PTH for 24 hours. Measurement of ALP activity in cell lysates showed that treatment with PTH at 0–200 nM stimulated ALP activity in a pattern that approached dose-dependence, with a maximum effect (2-fold increase) at PTH concentration 100 nM (Fig. 2A). Real time PCR showed that the mRNA levels of osteoblast maturation genes, Runx2 and type I collagen 1a1 (Col1a1), were significantly enhanced by treatment with PTH at doses of 10–100 nM (Fig. 2B).

Bottom Line: These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts.Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells.Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biostatistics and Bioinformatics, Tulane University, New Orleans, Louisiana, United States of America.

ABSTRACT
Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

Show MeSH
Related in: MedlinePlus