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Genetic determination of the cellular basis of the ghrelin-dependent bone remodeling.

Ma C, Fukuda T, Ochi H, Sunamura S, Xu C, Xu R, Okawa A, Takeda S - Mol Metab (2015)

Bottom Line: We performed molecular, genetic and cell biological analyses of Ghsr- mice and Ghsr- mice with tissue specific Ghsr restoration.Ghsr- mice showed a low bone mass phenotype with poor bone formation.Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr- mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery and Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT

Objective: Bone mass is maintained through a balance of bone formation and resorption. This homeostatic balance is regulated by various systems involving humoral and local factors. The discovery that the anorexigenic hormone leptin regulates bone mass via neuronal pathways revealed that neurons and neuropeptides are intimately involved in bone homeostasis. Ghrelin is a stomach-derived orexigenic hormone that counteracts leptin's action. However, the physiological role of ghrelin in bone homeostasis remains unknown. In this study, through the global knockout of ghrelin receptor (Ghsr) followed by tissue-specific re-expression, we addressed the molecular basis of the action of ghrelin in bone remodeling in vivo.

Methods: We performed molecular, genetic and cell biological analyses of Ghsr- mice and Ghsr- mice with tissue specific Ghsr restoration. Furthermore, we evaluated the molecular mechanism of ghrelin by molecular and cell-based assays.

Results: Ghsr- mice showed a low bone mass phenotype with poor bone formation. Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr- mice. Cell-based assays revealed ghrelin induced the phosphorylation of CREB and the expression of Runx2, which in turn accelerated osteoblast differentiation.

Conclusions: Our data show that ghrelin regulates bone remodeling through Ghsr in osteoblasts by modulating the CREB and Runx2 pathways.

No MeSH data available.


Related in: MedlinePlus

Ghrelin regulates bone remodeling via the ERK and/or PKA–CREB and Runx2 pathways. (A, F, I, J and K) Protein analysis in ghrelin-treated osteoblastic cells. (B–E) Effect of ERK inhibitor (B), siERK (C, D), PKA inhibitor (E) or siCreb2 treatment (G, H) on the expression of ERK (Mapk3) (C) or Creb2 (G) and osteoblast differentiation (B, D, H). (L) The effect of ghrelin on osteocalcin gene promoter activity. (A–L) MC3T3-E1 cells were treated with 10–7 M ghrelin and/or 10 μM MEK inhibitor (U0126) and 10 μM PKA inhibitor (H89). Phosphorylated protein/total protein ratios are indicated (A). P-, phosphorylated; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; ERK, Extracellular signal-regulated kinase; CREB, cAMP response element-binding protein, n = 10. The error bars indicate the standard deviation (s.d.). *P < 0.05, **P < 0.01. (M) Schematic representation of ghrelin-dependent osteoblast differentiation.
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fig6: Ghrelin regulates bone remodeling via the ERK and/or PKA–CREB and Runx2 pathways. (A, F, I, J and K) Protein analysis in ghrelin-treated osteoblastic cells. (B–E) Effect of ERK inhibitor (B), siERK (C, D), PKA inhibitor (E) or siCreb2 treatment (G, H) on the expression of ERK (Mapk3) (C) or Creb2 (G) and osteoblast differentiation (B, D, H). (L) The effect of ghrelin on osteocalcin gene promoter activity. (A–L) MC3T3-E1 cells were treated with 10–7 M ghrelin and/or 10 μM MEK inhibitor (U0126) and 10 μM PKA inhibitor (H89). Phosphorylated protein/total protein ratios are indicated (A). P-, phosphorylated; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; ERK, Extracellular signal-regulated kinase; CREB, cAMP response element-binding protein, n = 10. The error bars indicate the standard deviation (s.d.). *P < 0.05, **P < 0.01. (M) Schematic representation of ghrelin-dependent osteoblast differentiation.

Mentions: We next studied the molecular mechanism that is responsible for ghrelin/Ghsr-dependent osteoblast differentiation. Because ghrelin is known to exert various physiological functions through MAPK and PKA pathways [23–25], we studied the activation of the MAPK and PKA pathways after ghrelin treatment. Ghrelin induced the phosphorylation of ERK in osteoblastic cells but did not affect the phosphorylation of p38 or JNK (Figure 6A), indicating that ghrelin affects osteoblast differentiation through ERK. Indeed, blocking the ERK pathway, either through siRNA against ERK or by U0126, a known inhibitor of ERK, abolished ghrelin-dependent osteoblast differentiation (Figure 6B–D). The addition of a PKA inhibitor attenuated ghrelin-dependent osteoblast differentiation, indicating that PKA signaling is also involved (Figure 6E). CREB is an important transcription factor for osteoblast proliferation and differentiation [26,27]; we observed that ghrelin treatment induced the phosphorylation of CREB (Figure 6F). Moreover, the activation of CREB was required for the optimal induction of osteoblast differentiation by ghrelin, as a siRNA targeting Creb2 encoding CREB suppressed the ghrelin-mediated increase in ALP activity (Figure 6G,H). Interestingly, ghrelin-dependent CREB phosphorylation was attenuated by treatment with an ERK inhibitor (Figure 6I) or a PKA inhibitor (Figure 6J), indicating that both the ERK and PKA pathways are involved in ghrelin-Ghsr-dependent bone remodeling. Furthermore, we found that ghrelin treatment induced the expression of Runx2, a master regulator of osteoblast differentiation, in osteoblastic cells (Figure 6K). As a result, ghrelin increased the transcriptional activity of the osteocalcin promoter, a well-known target for Runx2 (Figure 6L). Taken together, our results indicate that ghrelin induces osteoblast differentiation through the activation of the osteoblastic ERK–CREB pathway and the induction of Runx2.


Genetic determination of the cellular basis of the ghrelin-dependent bone remodeling.

Ma C, Fukuda T, Ochi H, Sunamura S, Xu C, Xu R, Okawa A, Takeda S - Mol Metab (2015)

Ghrelin regulates bone remodeling via the ERK and/or PKA–CREB and Runx2 pathways. (A, F, I, J and K) Protein analysis in ghrelin-treated osteoblastic cells. (B–E) Effect of ERK inhibitor (B), siERK (C, D), PKA inhibitor (E) or siCreb2 treatment (G, H) on the expression of ERK (Mapk3) (C) or Creb2 (G) and osteoblast differentiation (B, D, H). (L) The effect of ghrelin on osteocalcin gene promoter activity. (A–L) MC3T3-E1 cells were treated with 10–7 M ghrelin and/or 10 μM MEK inhibitor (U0126) and 10 μM PKA inhibitor (H89). Phosphorylated protein/total protein ratios are indicated (A). P-, phosphorylated; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; ERK, Extracellular signal-regulated kinase; CREB, cAMP response element-binding protein, n = 10. The error bars indicate the standard deviation (s.d.). *P < 0.05, **P < 0.01. (M) Schematic representation of ghrelin-dependent osteoblast differentiation.
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fig6: Ghrelin regulates bone remodeling via the ERK and/or PKA–CREB and Runx2 pathways. (A, F, I, J and K) Protein analysis in ghrelin-treated osteoblastic cells. (B–E) Effect of ERK inhibitor (B), siERK (C, D), PKA inhibitor (E) or siCreb2 treatment (G, H) on the expression of ERK (Mapk3) (C) or Creb2 (G) and osteoblast differentiation (B, D, H). (L) The effect of ghrelin on osteocalcin gene promoter activity. (A–L) MC3T3-E1 cells were treated with 10–7 M ghrelin and/or 10 μM MEK inhibitor (U0126) and 10 μM PKA inhibitor (H89). Phosphorylated protein/total protein ratios are indicated (A). P-, phosphorylated; SAPK/JNK, stress-activated protein kinase/Jun-amino-terminal kinase; ERK, Extracellular signal-regulated kinase; CREB, cAMP response element-binding protein, n = 10. The error bars indicate the standard deviation (s.d.). *P < 0.05, **P < 0.01. (M) Schematic representation of ghrelin-dependent osteoblast differentiation.
Mentions: We next studied the molecular mechanism that is responsible for ghrelin/Ghsr-dependent osteoblast differentiation. Because ghrelin is known to exert various physiological functions through MAPK and PKA pathways [23–25], we studied the activation of the MAPK and PKA pathways after ghrelin treatment. Ghrelin induced the phosphorylation of ERK in osteoblastic cells but did not affect the phosphorylation of p38 or JNK (Figure 6A), indicating that ghrelin affects osteoblast differentiation through ERK. Indeed, blocking the ERK pathway, either through siRNA against ERK or by U0126, a known inhibitor of ERK, abolished ghrelin-dependent osteoblast differentiation (Figure 6B–D). The addition of a PKA inhibitor attenuated ghrelin-dependent osteoblast differentiation, indicating that PKA signaling is also involved (Figure 6E). CREB is an important transcription factor for osteoblast proliferation and differentiation [26,27]; we observed that ghrelin treatment induced the phosphorylation of CREB (Figure 6F). Moreover, the activation of CREB was required for the optimal induction of osteoblast differentiation by ghrelin, as a siRNA targeting Creb2 encoding CREB suppressed the ghrelin-mediated increase in ALP activity (Figure 6G,H). Interestingly, ghrelin-dependent CREB phosphorylation was attenuated by treatment with an ERK inhibitor (Figure 6I) or a PKA inhibitor (Figure 6J), indicating that both the ERK and PKA pathways are involved in ghrelin-Ghsr-dependent bone remodeling. Furthermore, we found that ghrelin treatment induced the expression of Runx2, a master regulator of osteoblast differentiation, in osteoblastic cells (Figure 6K). As a result, ghrelin increased the transcriptional activity of the osteocalcin promoter, a well-known target for Runx2 (Figure 6L). Taken together, our results indicate that ghrelin induces osteoblast differentiation through the activation of the osteoblastic ERK–CREB pathway and the induction of Runx2.

Bottom Line: We performed molecular, genetic and cell biological analyses of Ghsr- mice and Ghsr- mice with tissue specific Ghsr restoration.Ghsr- mice showed a low bone mass phenotype with poor bone formation.Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr- mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery and Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT

Objective: Bone mass is maintained through a balance of bone formation and resorption. This homeostatic balance is regulated by various systems involving humoral and local factors. The discovery that the anorexigenic hormone leptin regulates bone mass via neuronal pathways revealed that neurons and neuropeptides are intimately involved in bone homeostasis. Ghrelin is a stomach-derived orexigenic hormone that counteracts leptin's action. However, the physiological role of ghrelin in bone homeostasis remains unknown. In this study, through the global knockout of ghrelin receptor (Ghsr) followed by tissue-specific re-expression, we addressed the molecular basis of the action of ghrelin in bone remodeling in vivo.

Methods: We performed molecular, genetic and cell biological analyses of Ghsr- mice and Ghsr- mice with tissue specific Ghsr restoration. Furthermore, we evaluated the molecular mechanism of ghrelin by molecular and cell-based assays.

Results: Ghsr- mice showed a low bone mass phenotype with poor bone formation. Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr- mice. Cell-based assays revealed ghrelin induced the phosphorylation of CREB and the expression of Runx2, which in turn accelerated osteoblast differentiation.

Conclusions: Our data show that ghrelin regulates bone remodeling through Ghsr in osteoblasts by modulating the CREB and Runx2 pathways.

No MeSH data available.


Related in: MedlinePlus