The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice
Matthew B. Greenblatt, Jae-Hyuck Shim, Weiguo Zou, Despina Sitara, Michelle Schweitzer, Dorothy Hu, Sutada Lotinun, Yasuyo Sano, Roland Baron, Jin Mo Park, Simon Arthur, Min Xie, Michael D. Schneider, Bo Zhai, Steven Gygi, Roger Davis, Laurie H. Glimcher
Matthew B. Greenblatt, Jae-Hyuck Shim, Weiguo Zou, Despina Sitara, Michelle Schweitzer, Dorothy Hu, Sutada Lotinun, Yasuyo Sano, Roland Baron, Jin Mo Park, Simon Arthur, Min Xie, Michael D. Schneider, Bo Zhai, Steven Gygi, Roger Davis, Laurie H. Glimcher
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Research Article Bone biology

The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Abstract

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member–encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-β–activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1–MKK3/6–p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38β and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38β agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

Authors

Matthew B. Greenblatt, Jae-Hyuck Shim, Weiguo Zou, Despina Sitara, Michelle Schweitzer, Dorothy Hu, Sutada Lotinun, Yasuyo Sano, Roland Baron, Jin Mo Park, Simon Arthur, Min Xie, Michael D. Schneider, Bo Zhai, Steven Gygi, Roger Davis, Laurie H. Glimcher

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Figure 6

Defective osteoblast differentiation in MKK3/6 and p38α/β-deficient cells.

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CCD in Tak1osx mice. (A) Sections were taken from the sagittal sutur...
(A) BMSCs were isolated from WT and Mkk3–/–Mkk6+/– mice and cultured under differentiation conditions; then ALP activity and mineralization were analyzed by colorimetric assay/Fast Blue staining and Von Kossa staining, respectively (left). Alternatively, total RNA was extracted for RT-PCR analysis (right). Values are mean + SD. Original magnification, ×25. (B) Primary CalvOb were isolated from p38afl/fl pups, infected by vector or cre lentivirus, and cultured under differentiation conditions. ALP activity was analyzed by colorimetric assay and Fast Blue staining (left). Alternatively, total RNAs were extracted for quantitative PCR analysis (right). Inducible deletion of p38α was analyzed by immunoblotting with anti-p38α antibody. Values are mean + SD. (C) Primary CalvOb were isolated from p38b+/+ and p38b–/– pups and cultured under differentiation conditions; then ALP activity was analyzed by colorimetric assay and Fast Blue staining (upper). Alternatively, total RNAs were extracted for quantitative PCR analysis (lower). Values are mean + SD. (D) p38b+/+ and p38b–/– CalvOb were infected by lentiviruses expressing control (con), p38α (Sh1), or mock (Sh2) shRNA and cultured under differentiation conditions; then ALP activity was analyzed by colorimetric assay. Values are mean + SD. (E) Primary WT CalvOb were cultured under differentiation conditions, and total RNAs were extracted at day 0, 10, and 20 for quantitative PCR analysis. (F) Primary WT CalvOb were cultured under differentiation conditions. Cells were lysed, immunoprecipitated with anti-phospho p38 antibody and protein A agarose, and immunoblotted with the indicated antibodies.