(B) Colocalization of vinculin and sites of HA (C) TMEM2 is the predominant hyaluronidase TMEM2 binds directly to integrins via interactions between the respective extracellular domains (ECDs)Colocalization of TMEM2-mediated HA degradation at FAs suggests that TMEM2 may physically associate with some FA component(s). Since TMEM2 is a transmembrane protein, we initially surmised that TMEM2 binds FA-associated cytoskeletal proteins via its cytoplasmic domain. However, we found that truncated TMEM2 lacking its cytoplasmic domain can still drive FA-targeted HA degradation7). Thus, we explored the possibility that TMEM2 interacts with integrins via extracellular interactions. Coimmunoprecipitation assays following the crosslinking of cell surface proteins revealed that α5β1 integrin is coimmunoprecipitated with TMEM2. Direct binding between TMEM2 and α5β1 was demonstrated by a pull-down assay with recombinant ECDs of TMEM2 and α5β1. The same assay also demonstrated the direct interaction between ECDs of TMEM2 and LFA-1 (αLβ2 integrin)7). These results demonstrate that TMEM2 binds multiple integrins via interactions between the respective ECDs, and that this interaction forms the basis for TMEM2 localization to FA sites.Our findings revealed a critical role for TMEM2 in the adhesion and migration of tumor cells on HA-rich ECM substratesFigure 2 depicts a model for the role of TMEM2 in integrin-mediated cell adhesion and migration, based on our data. The Progress after the granted period and future perspectiveWe have recently generated an inducible global Tmem2 knockout mice and demonstrated that TMEM2 plays an Figure 1. Tumor cells degrade substrate-bound HA at central tenet of this model is that TMEM2 removes matrix-associated HA in the vicinity of integrin-mediated cell–matrix adhesion, thereby promoting integrin–ligand engagement and FA formation and maturation. This model provides novel insight into the molecular mechanisms of the early phase of FA formation, an issue that has not been completely understood. Significantly, this function of TMEM2 in cell adhesion and migration has potential clinical relevance to cancer progression. It has been shown that HA is highly accumulated in the stromal tissue of high-grade adenocarcinomas, and that this HA accumulation is mediated mainly by stromal cells8). The results of our study suggest that tumor cells may use TMEM2 to degrade HA and remodel the stromal microenvironment in a way that is favorable for their expansive growth and invasion. In this context, it is of particular interest that increased TMEM2 expression correlates significantly with reduced overall patient survival in high-grade breast cancer9) and pancreatic cancer10). These clinical correlation data are consistent with the possibility that TMEM2 is a cancer progression-promoting molecule, and moreover, suggest that TMEM2 can be a new drug target for treatment of high-grade adenocarcinomas.FAs (A) Contact-dependent in situ degradation of substrate-immobilized HA by DU145, BT474, U2OS, and TRAMP-C2 cells. In situ HA degradation activity is detected as black areas in the fluorescent substratum.degradation. Cells cultured on the fluorescein-labeled HA substrate were immunostained with anti-vinculin antibody.that mediates contact-dependent in situ HA degradation. Hyaluronidases molecules expressed in U2OS cells (TMEM2, KIAA1199, HYAL1, and HYAL2) were individually knocked down by siRNA treatment, and in situ HA degradation assays were performed with siRNA-treated cells. siRNAs used in this study reduce respective mRNA levels by 90 to 95%7). Alexa Fluor 594-conjugated wheat germ agglutinin (WGA) was used to visualize cell morphology.The novel cell surface hyaluronidase TMEM2 in tumor cell invasionYu Yamaguchi92
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