Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila , the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2 , the two mouse homologs of the Drosophila Hpo , control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFα was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFα is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFα-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2 .

The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1), effectors of the Hippo pathway, have been identified as mediators for mechanical stimuli. However, the role of YAP/TAZ in haemodynamics-induced mechanotransduction and pathogenesis of atherosclerosis remains unclear. Here we show that endothelial YAP/TAZ activity is regulated by different patterns of blood flow, and YAP/TAZ inhibition suppresses inflammation and retards atherogenesis. Atheroprone-disturbed flow increases whereas atheroprotective unidirectional shear stress inhibits YAP/TAZ activity. Unidirectional shear stress activates integrin and promotes integrin-Gα interaction, leading to RhoA inhibition and YAP phosphorylation and suppression. YAP/TAZ inhibition suppresses JNK signalling and downregulates pro-inflammatory genes expression, thereby reducing monocyte attachment and infiltration. In vivo endothelial-specific YAP overexpression exacerbates, while CRISPR/Cas9-mediated Yap knockdown in endothelium retards, plaque formation in ApoE mice. We also show several existing anti-atherosclerotic agents such as statins inhibit YAP/TAZ transactivation. On the other hand, simvastatin fails to suppress constitutively active YAP/TAZ-induced pro-inflammatory gene expression in endothelial cells, indicating that YAP/TAZ inhibition could contribute to the anti-inflammatory effect of simvastatin. Furthermore, activation of integrin by oral administration of MnCl reduces plaque formation. Taken together, our results indicate that integrin-Gα-RhoA-YAP pathway holds promise as a novel drug target against atherosclerosis.

Ellipse Technologies.

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP -/-limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP -/-embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP -/-embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP -/-embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/β-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.

Both the Wnt/␤-catenin and Ihh signaling pathways play essential roles in crucial aspects of endochondral ossification: osteoblast differentiation, chondrocyte proliferation and hypertrophy. To understand the genetic interaction between these two signaling pathways, we have inactivated the ␤-catenin gene and upregulated Ihh signaling simultaneously in the same cells during endochondral skeletal development using ␤-catenin and patched 1 floxed alleles. We uncovered previously unexpected roles of Ihh signaling in synovial joint formation and the essential function of Wnt/␤-catenin signaling in regulating chondrocyte survival. More importantly, we found that Wnt and Ihh signaling interact with each other in distinct ways to control osteoblast differentiation, chondrocyte proliferation, hypertrophy, survival and synovial joint formation in the developing endochondral bone. ␤-catenin is required downstream of Ihh signaling and osterix expression for osteoblast differentiation. But in chondrocyte survival, ␤-catenin is required upstream of Ihh signaling to inhibit chondrocyte apoptosis. In addition, Ihh signaling can inhibit chondrocyte hypertrophy and synovial joint formation independently of ␤-catenin. However, there is a strong synergistic interaction between Wnt/␤-catenin and Ihh signaling in regulating synovial joint formation.

Osteoarthritis is one of the leading causes of pain and disability in the aged population due to articular cartilage damage. This warrants investigation of signaling mechanisms that could protect cartilage from degeneration and degradation. Here we show in a murine model of experimental osteoarthritis that YAP activation by transgenic overexpression or by deletion of its upstream inhibitory kinases Mst1/2 preserves articular cartilage integrity, whereas deletion of YAP in chondrocytes promotes cartilage disruption. Our work shows that YAP is both necessary and sufficient for the maintenance of cartilage homeostasis in osteoarthritis. Mechanistically, inflammatory cytokines, such as TNFα or IL-1β, trigger YAP/TAZ degradation through TAK1-mediated phosphorylation. Furthermore, YAP directly interacts with TAK1 and attenuates NF-κB signaling by inhibiting substrate accessibility of TAK1. Our study establishes a reciprocal antagonism between Hippo-YAP/TAZ and NF-κB signaling in regulating the induction of matrix-degrading enzyme expression and cartilage degradation during osteoarthritis pathogenesis.

Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration.

Hedgehog (Hh) signaling is required for osteoblast differentiation from mesenchymal progenitors during endochondral bone formation. However, the role of Hh signaling in differentiated osteoblasts during adult bone homeostasis remains to be elucidated. We found that in the postnatal bone, Hh signaling activity was progressively reduced as osteoblasts mature. Upregulating Hh signaling selectively in mature osteoblasts led to increased bone formation and excessive bone resorption. As a consequence, these mutant mice showed severe osteopenia. Conversely, inhibition of Hh signaling in mature osteoblasts resulted in increased bone mass and protection from bone loss in older mice. Cellular and molecular studies showed that Hh signaling indirectly induced osteoclast differentiation by upregulating osteoblast expression of PTHrP, which promoted RANKL expression via PKA and its target transcription factor CREB. Our results demonstrate that Hh signaling in mature osteoblasts regulates both bone formation and resorption and that inhibition of Hh signaling reduces bone loss in aged mice.