PTH Counteracts Hippo Signaling via Src-dependent YAP Stabilization to Enhance Bone Marrow Stromal Cell Differentiation

Parathyroid hormone (PTH) regulates serum calcium and phosphate through its actions in bone and the 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 and/or the adipocyte lineages. PTH exerts its action through the PTH/PTH-related peptide (PTHrP) receptor (PTH1R), 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 cross-talk 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. PTH stabilized YAP, a key target of Hippo, by decreasing YAP/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 Y428 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. Overall design: To gain insight the cross-talk between parathyroid hormone (PTH) signaling and Hippo signaling pathways in bone marrow stromal cells (BMSCs) and bone cells, with a focus on understanding the role of PTH in regulating YAP (Yes-associated protein) stability and activity. The murine bone marrow stromal cell line W-20 was used as the primary model to study PTH-induced signaling and differentiation. Cells were treated with PTH to evaluate its effects on signaling pathways and cellular outcomes. The role of Src kinase in PTH-mediated YAP regulation was investigated, with a focus on YAP-Y428 phosphorylation.