Madhuri Wadehra

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion.

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the β1 integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that ∼60% of β1integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and β1 integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with α6β1 but not α5β1 integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the α6β1 integrin while decreasing that of the α5β1 protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of α6β1 integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.

Expression of epithelial membrane protein-2 is associated with endometrial adenocarcinoma of unfavorable outcome

Epithelial membrane protein 2 (EMP2) is an estrus‐regulated tetraspan protein that is required for endometrial competence in blastocyst implantation. EMP2 controls surface levels of several classes of integrin and other cell‐interaction molecules, and their trafficking to glycolipid‐enriched lipid raft domains is important in receptor signaling. These features suggest that EMP2 may contribute to neoplastic traits of endometrial cancer. The objective of this study was to determine the prevalence of EMP2 expression in endometrial neoplasms and its clinical significance.

Epithelial membrane protein-2 is expressed in discrete anatomical regions of the eye

Epithelial membrane protein-2 (EMP2) is a member of the four transmembrane superfamily (TM4SF) and is thought to mediate trafficking of diverse proteins such as alpha6beta1 integrin and MHC class I to lipid raft microdomains. EMP2 has also recently been recognized as a putative tumor suppressor gene in certain model systems. Normally, EMP2 is expressed at discrete locations in the body including high levels in the eye, lung, heart, thyroid, and uterus. Here we examine in detail the subanatomic distribution of EMP2 in murine and human ocular tissue. We observe that EMP2 is localized to epithelial layers of the cornea, ciliary body, and retinal pigmented epithelium-choroid, the stromal layers of the sclera, and the nerve fiber layer of the retina and optic nerve. This distribution is distinct from other TM4SF proteins and may relate to a role in apical membrane recycling.

The tetraspan protein EMP2 increases surface expression of class I major histocompatibility complex proteins and susceptibility to CTL-mediated cell death

Dysregulation of class I major histocompatibility (MHC1) expression is an important mechanism of immunologic resistance for certain virus-infected or neoplastic cells. This study characterizes a new molecule affecting MHC1 expression and CTL cytotoxicity. Epithelial membrane protein 2 (EMP2) is a tetraspan protein recently identified for its role in suppressing B lymphoma tumorigenicity. The biochemistry of EMP2 suggests that it regulates the surface expression of certain membrane proteins, notably those destined for lipid raft microdomains. In this study, retroviral overexpression of EMP2 in target cells increased their susceptibility to CTL cytotoxicity. Conversely, down-expression of EMP2 using an EMP2-specific ribozyme rendered target cells CTL-resistant. EMP2 expression increased the surface levels of MHC1, CD54, and GM1 glycolipids. Biochemical fractionation indicated that these molecules reside with EMP2 in a lipid raft membrane compartment. Among MHC1 proteins, surface display of H-2D was particularly dependent on EMP2 expression, and blocking antibodies demonstrated that H-2D was critical for allogeneic CTL recognition. This study demonstrates an unexpected role for a tetraspan protein in CTL-mediated cell death and MHC1 surface trafficking.

FAK activation and the role of epithelial membrane protein 2 (EMP2) in collagen gel contraction.

PURPOSE Proliferative vitreoretinopathy (PVR) occurs in approximately 10% of patients after retinal detachment. PVR results from a multiphase process that leads to an aberrant wound-healing strategy with contractile cellular forces and tractional retinal detachment (TRD). Epithelial membrane protein (EMP) 2 controls cell surface expression and function of integrin isoforms associated with cellular contraction in many cell types. Since EMP2 is highly expressed in retinal pigment epithelium, this study investigates the role of EMP2 in collagen gel contraction. METHODS EMP2 expression was recombinantly modified in the ARPE-19 cell line. Cell surface integrin expression was assessed by flow cytometry. Collagen gel contraction was assessed by using an in vitro assay and the percentage of contraction was quantified. Proliferation and migration were measured by BrdU incorporation and a wound-healing assay, respectively. Cellular invasion was investigated with polycarbonate membranes coated with collagen. RESULTS EMP2 expression levels correlated positively with the ability to contract collagen gels. Compared with wild-type ARPE-19 cells, the cells with increased EMP2 expression exhibited enhanced contraction (P = 0.02), and decreased EMP2 expression concomitantly resulted in decreased contraction (P = 0.002). EMP2 overexpression resulted in reduced proliferation, migration, and integrin alpha1 and alpha2 integrin expression. EMP2 overexpression was associated with a 70% increase in FAK activation (P = 0.0003) and relative resistance of gel contraction to inhibitors of FAK/Src activation. CONCLUSIONS ARPE-19-mediated collagen gel contraction is a multistep process that requires integrin ligation and activation of the FAK/Src complex. EMP2 positively modulates collagen gel contraction by ARPE-19 cells through increased FAK activation.

The Tetraspan Protein EMP2 Regulates Expression of Caveolin-1

Caveolin-1 is the primary component of caveolae and functions in a variety of intracellular activities, including membrane trafficking and signal transduction. EMP2 (epithelial membrane protein 2) is a tetraspan protein recently identified as a novel regulator of caveolin-1 expression. In this study, we analyzed the mechanism of EMP2-mediated caveolin-1 regulation. In NIH 3T3 cells and in the human retinal pigment epithelium cell line (ARPE-19), EMP2 regulates caveolin-1 transcription and more substantially its protein levels. EMP2-mediated down-regulation of caveolin-1 does not affect caveolin-1 translational efficiency, phosphorylation, or proteasome-mediated degradation. Analysis of caveolin-1 protein half-life indicates the EMP2-mediated loss of caveolin-1 occurs rapidly. Protease inhibition and laser confocal microscopy associates this fate with specific intracellular compartmentalization, including early lysosomal delivery. These findings elucidate a new mechanism of caveolin-1 regulation and define an additional role for EMP2 as a key regulator of cell membrane composition.

Pilot Study on “Pericytic Mimicry” and Potential Embryonic/Stem Cell Properties of Angiotropic Melanoma Cells Interacting with the Abluminal Vascular Surface

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the “pericytic mimicry” by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Steroid hormone regulation of EMP2 expression and localization in the endometrium

BackgroundThe tetraspan protein epithelial membrane protein-2 (EMP2), which mediates surface display of diverse proteins, is required for endometrial competence in blastocyst implantation, and is uniquely correlated with poor survival from endometrial adenocarcinoma tumors. Because EMP2 is differentially expressed in the various stages of the murine and human estrous cycle, we tested the hypothesis that the steroid hormones progesterone and estrogen influence EMP2 expression and localization.MethodsFrozen human proliferative and secretory endometrium were collected and analyzed for EMP2 expression using SDS-PAGE/Western blot analysis. The response of EMP2 to progesterone and estradiol was determined using a combination of real-time PCR, SDS-PAGE/Western blot analysis, and confocal immunofluorescence in the human endometrial carcinoma cell line RL95-2. To confirm the in vitro results, ovariectomized mice were treated with progesterone or estradiol, and EMP2 expression was analyzed using immunohistochemistry.ResultsWithin normal human endometrium, EMP2 expression is upregulated in the secretory phase relative to the proliferative phase. To understand the role of steroid hormones on EMP2 expression, we utilized RL95-2 cells, which express both estrogen and progesterone receptors. In RL95-2 cells, both estradiol and progesterone induced EMP2 mRNA expression, but only progesterone induced EMP2 protein expression. To compare steroid hormone regulation of EMP2 between humans and mice, we analyzed EMP2 expression in ovarectomized mice. Similar to results observed in humans, progesterone upregulated endometrial EMP2 expression and induced EMP2 translocation to the plasma membrane. Estradiol did not promote translocation to the cell surface, but moderately induced EMP2 expression in cytoplasmic compartments in vivo.ConclusionThese findings suggest that targeting of EMP2 to specific locations under the influence of these steroid hormones may be important for integrating the molecular responses required for implantation competence.

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Purpose: Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design: Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Results: Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. Conclusions: These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

Functional consequences of interactions between FAK and epithelial membrane protein 2 (EMP2).

PURPOSE Collagen gel contraction by ARPE-19 is controlled by epithelial membrane protein 2 (EMP2) through focal adhesion kinase (FAK) activation. The purpose of this study was to test the role of EMP2 in the cellular context of FAK activation. METHODS The ARPE-19 cell line was recombinantly modified to increase the expression of EMP2 and was used in this study. Quantification of FAK and Src phosphorylation was determined with Western blot analysis of whole cell lysates with the use of specific antibodies for different target sites of phosphorylation. Coimmunoprecipitation of whole cell lysates with an antibody against EMP2, followed by Western blot analysis and identification of FAK, was performed. Focal adhesions and their relationship to EMP2 were identified with immunofluorescence and confocal microscopy. F-actin distribution was identified using fluorescence microscopy, and alpha- smooth muscle actin (alpha-SMA) expression was quantified with Western blot analysis and specific antibodies. Adhesion to collagen type I was determined with a binding assay. RESULTS EMP2 overexpression led to increased FAK phosphorylation at all measured phosphorylation sites. Coimmunoprecipitation and confocal microscopy provided evidence for a physical association between EMP2 and FAK. Increased EMP2 was also associated with altered distribution of focal adhesions, changes in actin organization, increased alpha-SMA expression, and increased adherence to a collagen-coated surface. CONCLUSIONS The EMP2-FAK association represents a novel protein-protein interaction, not previously reported, that demonstrates significant functional cellular responses in the context of in vitro models of proliferative vitreoretinopathy (PVR).