Yingzi Yang - US grants

G?s-Hedgehog signaling in intramembranous bone formation and expansion Summary The development of a functional skeletal system requires tight spatial and temporal control of osteoblast differentiation and maturation. How osteoblast cells are induced at the outset of bone development is a central question in understanding the organizational principles underpinning a functional skeletal system. Extraskeletal or heterotopic ossification (HO) occurs as a common complication of trauma or in rare genetic disorders and can be disabling and lethal. The precise cellular and molecular mechanisms underlying HO are not clear. Research in our lab has provided insights into the molecular and cellular regulation of bone development and recently we have identified a novel G?s-Hedgehog (Hh) signaling axis that critically regulates ectopic osteoblast differentiation in progressive osseous heteroplasia (POH). POH is a rare human genetic disease in which HO occurs predominantly through an intramembranous process and progresses from subcutaneous tissue into skeletal muscle and deep connective tissues. POH is caused by inactivating mutations in GNAS that encodes G?s that transduces signals from G protein coupled receptors (GPCRs). We have found that loss of G?s function in POH leads to ligand-independent activation of Hh signaling, which in turn induces osteoblast differentiation of mesenchyme cells in soft tissues, whereas activation of G?s signaling leads to Wnt/?-catenin signaling upregulation and reduced osteoblast differentiation in the human condition of fibrous dysplasia (FD). We have further observed in our preliminary studies that ectopic bone formation and expansion in POH bare cellular and molecular similarities to craniofacial bone development. Here we will build upon our unique perspectives and test our central hypothesis: Hh signaling activation by G?s inhibition induces osteoblast differentiation during intramembranous bone formation and recruits wild type cells into ectopic bone during progressive ossification in POH. In Specific Aim 1, we will investigate the role of G?s-regulated Hh signaling during formation and growth of intramembranous bone. In Specific Aim 2, we will extend findings in normal craniofacial bone growth to ectopic bone in POH. We will test our hypothesis that ectopic bone in POH expands by inducing a suture-like tissue where wild type osteogenic mesenchyme stem cells reside. Our proposed studies will provide an unprecedented level of insight in acquired HO and the regulation of osteoblast differentiation under both physiological and pathological conditions. Knowledge gained here from the mouse models of POH will be readily translatable to human diseases such as POH, acquired HO, FD, craniosynostosis and osteoporosis. We anticipate that our findings will have broad significance with respect to cell-fate specification and reprogramming processes during development, repair, and regeneration of many other organ systems where G?s-Hh and G?s-Wnt signaling plays a critical role and enhance our understanding of these signaling pathways in human diseases including cancer.

Alcoholic liver disease (ALD), a major cause of morbidity and mortality worldwide, includes a broad spectrum of disorders, ranging from simple steatosis to severe forms of liver injury such as steatohepatitis, alcoholic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma. Aside from the direct cytotoxic and the oxidative- stress?mediated effects that alcohol and its metabolites exert on hepatocytes, alcohol ingestion also activates both the innate and adaptive immune responses in the liver, and dysregulates several important signaling pathways in the liver, thereby contributing to the pathogenesis of ALD. Recent studies suggest that impaired liver regeneration and inflammation are two important mechanisms contributing to liver failure in patients with alcoholic hepatitis. However, the underlying mechanisms remain unclear. The Hippo (Hpo) signaling pathway has recently emerged as a critical one regulating hepatocyte proliferation, survival as well as inflammation. Central to the Hpo pathway is the control of Yap/Taz transcription factors by a kinase cascade starting from the Hpo kinase, which are Mst1 and Mst2 in mammals. As hepatocyte injury is a major driving force for ALD pathogenesis, the goal of this explorative R21 proposal is to determine whether alcohol attenuates liver regeneration and induces liver inflammation by dysregulating the Hpo signaling pathway in hepatocytes. Despite the critical functions of Hpo signaling in restricting hepatocyte proliferation and survival we and others have identified, the precise functions and molecular mechanisms whereby the Hpo signaling pathway participates in alcohol-induced liver injury, inflammation and regeneration are mostly unknown. Hence, there are many unanswered fundamental questions regarding Hpo signaling in ALD. The knowledge gained from the proposed studies will establish a solid new foundation for further mechanistic investigation of Hpo signaling in ALD and provide new targets and strategies to protect liver from alcohol induced injury. Our unpublished preliminary data show that in a short-term chronic-binge ALD (E1d-1B) model, Mst1, Mst2 and Yap protein levels were reduced. We have also found that, infiltrated macrophage numbers and expression of pro- inflammatory cytokines are increased in the hepatocyte-specific Mst1 and Mst2 double mutant (DKO) liver. We hypothesize that reduction in Yap expression leads to increased hepatocyte cell death and impaired hepatocyte regeneration; while Mst1 and Mst2 down-regulation in hepatocytes of the alcoholic liver contributes to chronic pro-injury liver inflammation. In Specific Aim 1, we will define the effects of alcohol consumption on the Hpo signaling pathway in hepatocytes. In Specific Aim 2, we will determine whether alcohol feeding inhibits liver regeneration by reducing Yap in hepatocytes. In Specific Aim 3, we will determine whether alcohol feeding causes liver inflammation by reducing Mst1 and Mst2 in hepatocytes.

Molecular Mechanism of Wnt/Planar Polarity Signaling Summary Directed cellular polarization as a key feature of organismal development is required for tissue and organ function and homeostasis. Planar Cell Polarity (PCP) is emerging as a fundamental mechanism regulating various morphogenetic processes including cartilage elongation in the limb, anterior-posterior (A-P) body axis elongation, neural tube closure, body hair orientation, orientation of inner ear sensory hair cells, left-right asymmetry and axon guidance in vertebrates. Mutations in PCP signaling components have been identified in human diseases such as brachydactyly type B1, Robinow syndrome, scoliosis, spinal bifida and epilepsy. Despite the fundamentally important roles of PCP, the mechanisms of PCP establishment by global instructive cues such as Wnts remain poorly understood and represent an exciting frontier in developmental and cell biology. The Wnt/Planar Cell Polarity (PCP) pathway is evolutionarily conserved and provides essential directional information during morphogenesis to orient cytoskeleton, cell division, cell migration, differential adhesion across cells, and to position cell extensions, such as cilia and axons. However, unlike the extensively studied Wnt/?-catenin pathway, Wnt signal transduction in the PCP pathway remains poorly understood. The PCP pathway is controlled by core PCP proteins including Van Gogh (Vang), Frizzled (Fzd) and Dishevelled (Dvl), which were originally identified in Drosophila. Wnt/PCP signaling in vertebrates is more complex and functionally diverse and vertebrate-specific features of PCP require rigorous genetic and biochemical studies of their own. The core PCP proteins are initially randomly distributed in the cell and gradually accumulate on one side of the cells instructed by global cues during PCP establishment. Wnt5a is a global cue required for establishing PCP in vertebrate long bone cartilage by inducing a novel receptor complex that contains Vang like 2 (Vangl2) and Ror2, a vertebrate specific PCP component. As a result, Vangl2 is phosphorylated in a Wnt5a dose-dependent manner and Vangl2 phosphorylation regulates its function. Our identification of Wnt induced PCP signalosome and Vangl2 phosphorylation as both an important readout and transducer of Wnt/PCP signaling opens a new door to find missing links in the Wnt/PCP signaling cascade. We propose to decipher novel Wnt5a signaling events that eventually lead to PCP establishment with rigorous genetic and biochemical approaches. In Specific Aim 1, we will define the functions and regulations of Vangl2 phosphorylation in vivo. In Specific Aim 2, we will define the molecular mechanism whereby Wnt5a signal is transduced through Vangl2 in the PCP pathway. In Specific Aim 3, we will identify additional regulatory components in Wnt/PCP signaling. Given the fundamental roles of Wnt/PCP signaling in many morphogenetic processes and identified WNT5A, VANGL and ROR2 mutations in human diseases, our studies of Wnt5a/PCP signaling in vertebrates will advance our understanding of Wnt signaling in human biology and pathology.

Summary Recent studies have expanded the concept that inflammation is a critical component of tumor progression. It is now clear that the tumor microenvironment largely orchestrated by inflammatory cells, is an indispensable participant in neoplastic process, fostering proliferation, survival and migration. In the last few decades, immunotherapy has become increasingly important in treating cancer. Therefore, there is an urgent need to better understand cancer-immune interactions, particular under specific contexts of cells, tissues and deficient molecular pathways involved. Human hepatocellular carcinoma (HCC), a primary malignancy of the liver and the second-leading cause of cancer mortality worldwide is an example of inflammation-induced cancer. Chronic viral hepatitis, metabolic liver diseases, and alcohol abuse cause chronic inflammation, which induces fibrosis, cirrhosis, and cancer. Macrophages function in the initiation and maintenance of inflammation and fibrosis and tumor associated macrophages (TAMs) play critical roles during cancer progression. However, the cellular and molecular mechanisms underlying reciprocal interaction between macrophages and pre-tumor/ tumor cells remain largely unknown. The Hippo signaling pathway has recently emerged as a major oncosuppressive pathway and play critical roles inhibiting hepatocyte proliferation, survival and HCC formation. Central to the Hippo pathway is the inhibition of Yap/Taz transcription factors by a kinase cascade starting from the Hippo kinase, which are Mst1 and Mst2 in mammals. As macrophage infiltration is dramatically increased in livers with Mst1 and Mst2 removed in hepatocytes, the goal of this proposal is to determine a previously unknown functional mechanism by which Hippo signaling in hepatocytes attenuates hepatocarcinogenesis by inhibiting macrophage infiltration and TAM differentiation. Our preliminary studies have led to the identification of two secreted effectors of Hippo signaling in hepatocytes, monocyte chemoattractant protein 1(Mcp1 or Ccl2) and Jagged 1 (Jag1), that each partially mediates Hippo effects in restricting inflammatory response and tumor growth. We hypothesize that a previously unknown function of Hippo signaling in hepatocytes is to regulate pro-tumor immune response by at least partially inhibiting Mcp1 and Jag1 expression. In Specific Aim 1, we will determine the molecular mechanism underlying TAM differentiation regulated by Hippo signaling in hepatocytes. In Specific Aim 2, we will determine the functions of macrophages in tumorigenesis in the hepatocyte specific Mst1/2 DKO, Mst1/2/Mcp1 TKO and Mst1/2/Jag1 TKO liver. In Specific Aim 3, we will determine the mechanisms whereby Hippo signaling in hepatocytes inhibits expression of Mcp1 and other factors. The knowledge gained from the proposed studies will establish a solid new foundation for further mechanistic investigation of hepatic Hippo signaling in inducing inflammation, tumor microenvironment remodeling and provide new targets and strategies to treat HCC.