Abstract
Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.
Highlights
CD82 belongs to the tetraspanin superfamily, in which members are involved in biological events ranging from cell fusion, cell adhesion, and cell migration to cell proliferation, synapse formation, and neurite outgrowth [1,2,3,4,5]
Our results indicate that 1) CD82 attenuates integrin ␣6 signaling during a cellular morphogenic process; 2) the decreased surface expression of ␣6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of ␣6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin ␣6 upon CD82 expression correlates with the down-regulation of cell surface integrin ␣6
The Cellular Morphogenesis of Du145 cells on Matrigel Depends on Integrin ␣6 —Du145 is an epithelial cell line originally isolated from the brain metastatic lesion of a prostate cancer patient [52] and is commonly used as an in vitro experimental model in the cellular behavior studies of prostate cancer
Summary
CD82 belongs to the tetraspanin superfamily, in which members are involved in biological events ranging from cell fusion, cell adhesion, and cell migration to cell proliferation, synapse formation, and neurite outgrowth [1,2,3,4,5]. The CD82-integrin complex is prevalent, and the role of CD82 in integrinmediated cell migration is well documented, it still remains to be assessed 1) whether or not CD82 directly regulates the functional status of its associated integrins and 2) if CD82 affects integrin activity, what regulatory mechanism exists To address these questions, we analyzed the effect of CD82 on integrin-dependent cellular morphogenic process on threedimensional extracellular matrices (ECM). Formation of cellular cable networks on a three-dimensional substratum appears to be a common morphogenic process for many cells ranging from endothelial cells, fibroblast cells, smooth muscle cells, epithelial cells, to cancer cells [29, 33, 35, 37, 39] This assay, could be widely applied to most cells commonly used for molecular and cellular studies as a morphogenetic readout, the physiological relevance of this morphogenesis for many cells still remains unclear. The integrin-tetraspanin interactions have been described for more than a decade, our discovery illustrates an important mechanism by which tetraspanins regulate integrin function
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