Yaohong Wang

A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis

Apoptosis is a key regulator for the normal turnover of the intestinal mucosa, and abnormalities associated with this function have been linked to inflammatory bowel disease and colorectal cancer. Despite this, little is known about the mechanism(s) mediating intestinal epithelial cell apoptosis. Villin is an actin regulatory protein that is expressed in every cell of the intestinal epithelium as well as in exocrine glands associated with the gastrointestinal tract. In this study we demonstrate for the first time that villin is an epithelial cell-specific anti-apoptotic protein. Absence of villin predisposes mice to dextran sodium sulfate-induced colitis by promoting apoptosis. To better understand the cellular and molecular mechanisms of the anti-apoptotic function of villin, we overexpressed villin in the Madin-Darby canine kidney Tet-Off epithelial cell line to demonstrate that expression of villin protects cells from apoptosis by maintaining mitochondrial integrity thus inhibiting the activation of caspase-9 and caspase-3. Furthermore, we report that the anti-apoptotic response of villin depends on activation of the pro-survival proteins, phosphatidylinositol 3-kinase and phosphorylated Akt. The results of our studies shed new light on the previously unrecognized function of villin in the regulation of apoptosis in the gastrointestinal epithelium.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth‐factor‐like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy‐resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with Ki values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.

Dimerization and Actin-bundling Properties of Villin and Its Role in the Assembly of Epithelial Cell Brush Borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration.

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.

Autotaxin and LPA1 and LPA5 receptors exert disparate functions in tumor cells versus the host tissue microenvironment in melanoma invasion and metastasis.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Tyrosine-phosphorylated villin regulates actin dynamics, cell morphology, and cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. In this study we report six new sites in the carboxyl-terminal region of the villin core. With this study we document all phosphorylatable tyrosine residues in villin and map them to functions of villin. In this study, we identify for the first time the functional relevance of the carboxyl-terminal domains of the villin core. Expression of the carboxyl-terminal phosphorylation site mutant, as well as the villin truncation mutant S1-S3, inhibited cell migration in HeLa and Madin-Darby canine kidney Tet-Off cells, confirming the role of the carboxyl-terminal phosphorylation sites in villin-induced cell migration. The carboxyl-terminal phosphorylation sites were found to be critical for the interaction of villin with its ligand phospholipase C-γ1 and for its localization to the developing lamellipodia in a motile cell. The results presented here elucidate the molecular basis for tyrosine-phosphorylated villin-induced changes in cell motility.

Actin reorganization as the molecular basis for the regulation of apoptosis in gastrointestinal epithelial cells

The gastrointestinal (GI) epithelium is a rapidly renewing tissue in which apoptosis represents part of the overall homeostatic process. Regulation of apoptosis in the GI epithelium is complex with a precise relationship between cell position and apoptosis. Apoptosis occurs spontaneously and in response to radiation and cytotoxic drugs at the base of the crypts. By contrast, the villus epithelial cells are extremely resistant to apoptosis. The molecular mechanism underlying this loss of function of villus epithelial cells to undergo apoptosis shortly after their exit from the crypt is unknown. In this study we demonstrate for the first time, that deletion of two homologous actin-binding proteins, villin and gelsolin renders villus epithelial cells extremely sensitive to apoptosis. Ultrastructural analysis of the villin-gelsolin−/− double-knockout mice shows an abnormal accumulation of damaged mitochondria demonstrating that villin and gelsolin function on an early step in the apoptotic signaling at the level of the mitochondria. A characterization of functional and ligand-binding mutants demonstrate that regulated changes in actin dynamics determined by the actin severing activities of villin and gelsolin are required to maintain cellular homeostasis. Our study provides a molecular basis for the regulation of apoptosis in the GI epithelium and identifies cell biological mechanisms that couple changes in actin dynamics to apoptotic cell death.

Both the anti- and pro-apoptotic functions of villin regulate cell turnover and intestinal homeostasis

In the small intestine, epithelial cells are derived from stem cells in the crypts, migrate up the villus as they differentiate and are ultimately shed from the villus tips. This process of proliferation and shedding is tightly regulated to maintain the intestinal architecture and tissue homeostasis. Apoptosis regulates both the number of stem cells in the crypts as well as the sloughing of cells from the villus tips. Previously, we have shown that villin, an epithelial cell-specific actin-binding protein functions as an anti-apoptotic protein in the gastrointestinal epithelium. The expression of villin is highest in the apoptosis-resistant villus cells and lowest in the apoptosis-sensitive crypts. In this study we report that villin is cleaved in the intestinal mucosa to generate a pro-apoptotic fragment that is spatially restricted to the villus tips. This cleaved villin fragment severs actin in an unregulated fashion to initiate the extrusion and subsequent apoptosis of effete cells from the villus tips. Using villin knockout mice, we validate the physiological role of villin in apoptosis and cell extrusion from the gastrointestinal epithelium. Our study also highlights the potential role of villin’s pro-apoptotic function in the pathogenesis of inflammatory bowel disease, ischemia-reperfusion injury, enteroinvasive bacterial and parasitic infections.

Tu1849 Defining and Targeting IRGM's Mechanism of Action in Crohn's Disease

Crohns disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGMs action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGMs role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.