Expression of alpha5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells.

The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is up-regulated at sites of penetrating injuries and stroke; however, CNS neurons down-regulate the fibronectin receptor alpha5beta1 integrin during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron-like cells (derived from NT2 precursor cells) have been used in preclinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells down-regulate alpha5beta1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing alpha5 integrin under the control of the murine stem cell virus 5' long terminal repeat. Sustained alpha5 integrin expression is compatible with the CNS-like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, alpha5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell-replacement therapies for CNS injury and disease.

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5.

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.