We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin α6β4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the β4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of β4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with α6β4. By contrast, Src and Lck do not associate with α6β4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of β4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the β4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

Ligation of the α₆β₄ integrin induces tyrosine phosphorylation of the β₄ cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of β₄ that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr¹⁴⁴⁰, or secondarily Tyr¹⁴²², interacts with the SH2 domain of Shc, whereas Tyr¹⁵²⁶, or secondarily Tyr¹⁶⁴², interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by α₆β₄. In addition, mutation of β₄ Tyr¹⁵²⁶, which binds to the PTB domain of Shc, but not of Tyr¹⁴²² and Tyr¹⁴⁴⁰, which interact with its SH2 domain, abolishes the activation of ERK by α₆β₄. Phenylalanine substitution of the β₄ tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of α₆β₄ in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of β4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type β₄. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of β₄containing phenylalanine substitutions at Tyr¹⁴²² and Tyr¹⁴⁴⁰, at Tyr¹⁵²⁶ and Tyr¹⁶⁴², or at all four tyrosine phosphorylation sites. These results suggest that β₄ Tyr¹⁵²⁶ interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses α_Vβ₃- (but not α₅β₁-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041–1047). Here we investigated the role of the COX-2 metabolites PGE₂ and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE₂ accelerated α_Vβ₃-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist U46619 retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE₂ effects. α_Vβ₃-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked α_Vβ₃-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued α_Vβ₃-dependent spreading in the presence of NS398 or U46691, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. α₅β₁-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE₂, U46691, 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE₂ accelerates α_Vβ₃-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces α_Vβ₃-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins α_Vβ₃ by COX-2/PGE₂during tumor angiogenesis and inflammation.