Gαs signaling controls intramembranous ossification during cranial bone development by regulating both Hedgehog and Wnt/ß-catenin signaling.

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gαs that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia (FD) or progressive osseous heteroplasia (POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gαs regulates Hh signaling in a ligand-independent manner. We further show that Gαs controls intramembranous ossification by regulating both Hh and Wnt/ß-catenin signaling. In addition, Gαs activation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

Hepatic Hippo signaling inhibits protumoural microenvironment to suppress hepatocellular carcinoma.

OBJECTIVE: Hippo signalling is a recently identified major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival and hepatocellular carcinoma (HCC) formation. Hippo kinase (Mst1 and Mst2) inhibits HCC proliferation by suppressing Yap/Taz transcription activities. As human HCC is mainly driven by chronic liver inflammation, it is not clear whether Hippo signalling inhibits HCC by shaping its inflammatory microenvironment. DESIGN: We have established a genetic HCC model by deleting Mst1 and Mst2 in hepatocytes. Functions of inflammatory responses in this model were characterised by molecular, cellular and FACS analysis, immunohistochemistry and genetic deletion of monocyte chemoattractant protein-1 (Mcp1) or Yap. Human HCC databases and human HCC samples were analysed by immunohistochemistry. RESULTS: Genetic deletion of Mst1 and Mst2 in hepatocytes (DKO) led to HCC development, highly upregulated Mcp1 expression and massive infiltration of macrophages with mixed M1 and M2 phenotypes. Macrophage ablation or deletion of Mcp1 in DKO mice markedly reduced hepatic inflammation and HCC development. Moreover, Yap removal abolished induction of Mcp1 expression and restored normal liver growth in the Mst1/Mst2 DKO mice. Finally, we showed that MCP1 is a direct transcription target of YAP in hepatocytes and identified a strong gene expression correlation between YAP targets and MCP-1 in human HCCs. CONCLUSIONS: Hippo signalling in hepatocytes maintains normal liver growth by suppressing macrophage infiltration during protumoural microenvironment formation through the inhibition of Yap-dependent Mcp1 expression, providing new targets and strategies to treat HCCs.

Induced GnasR201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/ß-catenin signaling.

Fibrous dysplasia (FD; Online Mendelian Inheritance in Man no. 174800) is a crippling skeletal disease caused by activating mutations of the GNAS gene, which encodes the stimulatory G protein Gαs FD can lead to severe adverse conditions such as bone deformity, fracture, and severe pain, leading to functional impairment and wheelchair confinement. So far there is no cure, as the underlying molecular and cellular mechanisms remain largely unknown and the lack of appropriate animal models has severely hampered FD research. Here we have investigated the cellular and molecular mechanisms underlying FD and tested its potential treatment by establishing a mouse model in which the human FD mutation (R201H) has been conditionally knocked into the corresponding mouse Gnas locus. We found that the germ-line FD mutant was embryonic lethal, and Cre-induced Gnas FD mutant expression in early osteochondral progenitors, osteoblast cells, or bone marrow stromal cells (BMSCs) recapitulated FD features. In addition, mosaic expression of FD mutant Gαs in BMSCs induced bone marrow fibrosis both cell autonomously and non-cell autonomously. Furthermore, Wnt/ß-catenin signaling was up-regulated in FD mutant mouse bone and BMSCs undergoing osteogenic differentiation, as we have found in FD human tissue previously. Reduction of Wnt/ß-catenin signaling by removing one Lrp6 copy in an FD mutant line significantly rescued the phenotypes. We demonstrate that induced expression of the FD Gαs mutant from the mouse endogenous Gnas locus exhibits human FD phenotypes in vivo, and that inhibitors of Wnt/ß-catenin signaling may be repurposed for treating FD and other bone diseases caused by Gαs activation.

Interacting network of Hippo, Wnt/ß-catenin and Notch signaling represses liver tumor formation.

Acquiring a selective growth advantage by breaking the proliferation barrier established by gatekeeper genes is a centrally important event in tumor formation. Removal of the mammalian Hippo kinase Mst1 and Mst2 in hepatocytes leads to rapid hepatocellular carcinoma (HCC) formation, indicating that the Hippo signaling pathway is a critical gatekeeper that restrains abnormal growth in hepatocytes. By rigorous genetic approaches, we identified an interacting network of the Hippo, Wnt/ß-catenin and Notch signaling pathways that control organ size and HCC development. We found that in hepatocytes, the loss of Mst1/2 leads to the activation of Notch signaling, which forms a positive feedback loop with Yap/Taz (transcription factors controlled by Mst1/2). This positive feedback loop results in severe liver enlargement and rapid HCC formation. Blocking the Yap/Taz-Notch positive feedback loop by Notch inhibition in vivo significantly reduced the Yap/Taz activities, hepatocyte proliferation and tumor formation. Furthermore, we uncovered a surprising inhibitory role of Wnt/ß-catenin signaling to Yap/Taz activities, which are important in tumor initiation. Genetic removal of ß-catenin in the liver of the Mst1/2 mutants significantly accelerates tumoriogenesis. Therefore, Wnt/ß-catenin signaling, known for its oncogenic property, exerts an unexpected function in restricting Yap/Taz and Notch activities in HCC initiation. The molecular interplay between the three signaling pathways identified in our study provides new insights in developing novel therapeutic strategies to treat liver tumors. [BMB Reports 2017; 50(1): 1-2].

Hippo signaling interactions with Wnt/ß-catenin and Notch signaling repress liver tumorigenesis.

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/ß-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/ß-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/ß-catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20-like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.