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. 2016 Mar;231(3):587-96.
doi: 10.1002/jcp.25102.

V体育ios版 - Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors

Chunxi Ge  1 , William P Cawthorn  2 , Yan Li  1 , Guisheng Zhao  1 , Ormond A Macdougald  2 , Renny T Franceschi  1   3
Affiliations

Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors

Chunxi Ge et al. J Cell Physiol. 2016 Mar.

Abstract

In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity VSports手机版. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages. .

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Conflict of interest statement (VSports在线直播)

All authors state they have no conflicts of interest.

"V体育平台登录" Figures

Fig. 1
Fig. 1
Regulation of MAP kinase, Runx2 and PPARγ phosphorylation during osteoblast and adipocyte differentiation. ST2 mesenchymal cells were cultured in growth medium (GM), adipogenic medium (adipo) or osteogenic medium (osteo). Cells were stained with Oil Red O after 1 week or Alizarin Red after 3 weeks (A) or extracted for total protein after 1 week (B). RNA was isolated at the times indicated (C–I). (B) Immunoblot analysis of MAP kinase activation and Runx2/PPARγ phosphorylation. Samples were probed with antibodies to pERK1/2, total ERK, PPARγ-S112-P, total PPARγ, Runx2-S319-P and total Runx2 as indicated. (C–E) Osteoblast marker mRNAs. Runx2 (C), Bglap2 (D), Ibsp (E). (F–I) Adipocyte marker mRNAs. Pparg (F), Cebpb (G), Adipoq (H), and Fabp4 (I).
Fig. 2
Fig. 2
MAP kinase-dependent phosphorylation stimulates RUNX2 transcriptional activity while suppressing PPARγ. (A, B) RUNX2. COS7 cells were transfected with 6OSE2-luc reporter plasmid and expression vectors for wild type (WT) or phosphorylation mutant RUNX2 (S301A,S319A, SA; S301E,S319E, SE) and constitutively active (SP) or dominant-negative MEK1 (DN). (A) normalized luciferase activity; (B) immunoblot of RUNX2-S319-P and total RUNX2. (C, D) PPARγ. Parallel transfections were conducted with ARE-luc reporter plasmid and expression vectors encoding wild type (WT) or phosphorylation site mutant PPARγ (S112A, SA; S112E, SE) and constitutively active (SP) or dominant-negative MEK 1 (DN). Cells were treated with vehicle (−) or troglitazone (TZD) as indicated. (C) normalized luciferase activity; (D) immunoblot of PPARγ-S112-P and total PPARγ. *Statistical comparisons are indicated by bars; P < 0.001, n = 3.
Fig. 3
Fig. 3
Opposing actions of MAP kinase on osteoblast and adipocyte differentiation. ST2 cells were transduced with control adenovirus expression vector (LacZ) or vector encoding constitutively active (Meksp) or dominant-negative (Mekdn) MEK1 and grown in osteogenic (A–E) or adipogenic medium (F–K). (A–E) Osteoblast differentiation. (A) Alizarin Red staining, (B) Immunoblot detection of pERK, total ERK, RUNX2-S319-P and total RUNX2. (C–E) Osteoblast marker mRNAs; Runx2 (C), Bglap2 (D), Ibsp (E). (F–K) Adipocyte differentiation. (F) Oil Red O staining, (G) Immunoblot detection of pERK, total ERK, PPARγ-S112-P and total PPARγ. (H–K) Adipocyte marker mRNAs. PPARγ (H), Cebpa (I), Adipoq (J), and Fabp4 (K). *Statistical comparisons are indicated by bars; P < 0.001, n = 3.
Fig. 4
Fig. 4
Regulation of osteoblast and adipocyte differentiation by wild type and phosphorylation site mutant RUNX2. MEFs from Runx2−/− mice were transduced with empty vector (EV) or retrovirus expressing wild type (WT), S301A,S319A (SA) or S301E,S319E RUNX2 (SE). After selection, stable cell pools were grown in osteogenic (B, D–F) or adipogenic medium (C, G–J). (A) Western blot of total RUNX2 protein. (B) Alizarin Red staining. (C) Oil Red O staining. (D–F) osteoblast marker mRNAs. (G–J) adipocyte marker mRNAs. Statistical comparisons; a, Runx2-WT versus empty vector P < 0.001; b, Runx2-SA versus Runx2-WT P < 0.001; c, Runx2-SE versus Runx2-WT. P < 0.001, n = 3.
Fig. 5
Fig. 5
Regulation of osteoblast and adipocyte differentiation by wild type and phosphorylation site mutant PPARγ. MEFs from Pparg−/− cells were transduced with empty vector (EV) or retrovirus expressing wild type (WT), S112A (SA) or S112E mutant PPARγ (SE). Stable cell pools were grown in osteogenic (B, D–F) or adipogenic medium (C, G–J). (A) Western blot of total PPARγ protein. (B) Alizarin Red staining. (C) Oil Red O staining. (D–F) osteoblast marker mRNAs. (G–J) adipocyte marker mRNAs. Statistical comparisons; a, PPARγ-WT versus empty vector P <0.001; b, PPARγ-SA versus Runx2-WT P <0.001; c, PPARγ-SE versus PPARγ-WT. P <0.001, n=3.
Fig. 6
Fig. 6
Antagonism between RUNX2 and PPARγ is affected by phosphorylation state. (A and B) PPARγ inhibition of RUNX2 transcriptional activity. COS7 cells were transfected with a RUNX2 reporter plasmid (p6OSE2-luc), RUNX2-SE vector and the indicate amounts of wild type, SA or SE PPARγ vectors. A, luciferase activity; B, PPARγ and RUNX2 protein levels. (C and D) RUNX2 inhibition of PPARγ transcriptional activity. Cells were transfected with a PPARγ reporter plasmid (pARE-luc), PPARγ-SA and RXR vectors and the indicated amounts of wild type, SA or SE RUNX2. (C) luciferase activity, (D) RUNX2 and PPARγ protein. Statistical comparisons; a, PPARγ-SA versus PPARγ-WT (Panel A) or Runx2-SA versus Runx2-WT (Panel B); b, PPARγ-SE versus PPARγ-WT (Panel A) or Runx2-SE versus Runx2-WT (Panel B). P < 0.001, n = 3.
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