PTH counteracts Hippo signaling via Src-dependent YAP stabilization to enhance bone marrow stromal cell differentiation
Sara Monaci, Mengrui Wu, Hiroyuki Okada, Kedkanya Mesil, Byeong-Rak Keum, Maisa Monseff Rodrigues da Silva, Clifford J. Rosen, Francesca Gori, Roland Baron
Sara Monaci, Mengrui Wu, Hiroyuki Okada, Kedkanya Mesil, Byeong-Rak Keum, Maisa Monseff Rodrigues da Silva, Clifford J. Rosen, Francesca Gori, Roland Baron
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Research Article Bone biology Endocrinology

PTH counteracts Hippo signaling via Src-dependent YAP stabilization to enhance bone marrow stromal cell differentiation

Abstract

Parathyroid hormone (PTH) regulates serum calcium and phosphate through its actions in bone and kidney and is used to increase bone in osteoporosis treatment. In bone, PTH targets osteoblasts and osteocytes to regulate bone remodeling but also bone marrow stromal cells (BMSCs), regulating their differentiation in the osteoblast or the adipocyte lineage. PTH exerts its action through the PTH/PTH-related peptide (PTHrP) receptor, a G protein–coupled receptor (GPCR), activating adenylyl cyclase and phospholipase C (PLC). Although the effects of cAMP and PKA are well characterized, little is known about the effects of PLC activation or on the crosstalk between PTH signaling and other pathways. Here, bulk RNA-Seq of PTH-treated murine BMSC line (W-20) revealed significant changes in the Hippo pathway. In addition to increasing its transcription, PTH stabilized YAP protein, a key target of Hippo, by decreasing YAP/LArge Tumor Suppressor kinase 1 (LATS1) interaction, YAPS127 phosphorylation, and YAP ubiquitination, leading to YAP nuclear translocation and expression of YAP target genes. Similar events occurred in osteocyte cell lines. This occurred via an increase in Src kinase activity: We identified YAPY428 as a key tyrosine residue phosphorylated by Src in response to PTH. Preventing YAPY428 phosphorylation led to YAP instability, blocking both osteogenic and adipogenic differentiation of W-20 cells. These results demonstrate active crosstalk between the PTH/PTHrP and the Hippo signaling pathways and reveal that PTH signaling utilizes the PLC/Ca2+/Src tyrosine kinase signaling cascade to influence YAP stability, antagonizing Hippo signaling and favoring stromal cell differentiation. Thus, PTH signaling counteracts the effects of Hippo signaling in BMSCs to favor their differentiation.

Authors

Sara Monaci, Mengrui Wu, Hiroyuki Okada, Kedkanya Mesil, Byeong-Rak Keum, Maisa Monseff Rodrigues da Silva, Clifford J. Rosen, Francesca Gori, Roland Baron

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Figure 2

PTH promotes YAP stability in W-20 cells and in OCY454 and OmGFP66.

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PTH promotes YAP stability in W-20 cells and in OCY454 and OmGFP66. (A) ...
(A) Western analysis representative blots and quantification of ph-YAP(S127) and YAP with or without PTH treatment. Fold change is relative to Veh. Protein levels were normalized to the respective housekeeping proteins in the same sample. (B) Representative blots of co-IP of YAP with LATS1 and β-TrCP and ubiquitin levels with or without PTH treatment. (C) Western analysis representative blots and quantification of ph-YAP (S127) and YAP protein levels in the cytoplasmic and nuclear fractions with or without PTH treatment. Fold change is relative to Veh. Protein levels were normalized to the respective housekeeping proteins in the same fraction/sample. (D) Representative blots of co-IP of YAP with LATS1 in the cytoplasmic fraction and TEAD in the nuclear fraction. (E) Western analysis representative blots and quantification of ph-YAP(S127) and YAP and representative blots of co-IP of YAP with LATS1 and β-TrCP with or without PTH treatment in OCY454 cells. (F) Expression of selected YAP target genes with or without PTH treatment in OCY454 cells. (G) Western analysis representative blots and quantification of ph-YAP(S127) and YAP with or without PTH in OmGFP66 cells. Data are shown as the mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 by unpaired Student’s t test. The fold change is relative to the Veh. Panel F mean Ct values: Gene Ctgf Veh 24.7; PTH 22.78. Gene Cyr61 Veh 23.17; PTH 20.4. Gene Myc Veh 25.51; PTH 22.8.