Deposition of Laminin 5 by Keratinocytes Regulates Integrin Adhesion and Signaling

doi:10.1074/jbc.M006379200

Deposition of Laminin 5 by Keratinocytes Regulates Integrin Adhesion and Signaling*

From the ^‡Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and the ^§Department of Pathobiology, University of Washington, Seattle, Washington 98195

Abstract

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin α₆β₄ activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin α₃β₁ on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via α₂β₁ is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of α₆β₄ increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH₂-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by α₃β₁ and phosphorylation of c-Jun NH₂-terminal kinase. Studies with keratinocytes lacking α₆β₄ or laminin 5 confirm that deposition of laminin 5 and ligation by α₆β₄ are required for PI3K-dependent spreading via α₃β₁. We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via α₆β₄and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin α₃β₁ on laminin 5.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA49259 and AR21557 (both to W. G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109. Tel.: 206-667-4478; Fax: 206-667-3331; E-mail: wcarter@fhcrc.org.
Published, JBC Papers in Press, August 3, 2000, DOI 10.1074/jbc.M006379200
Abbreviations:

BM

basement membrane

BSA

bovine serum albumin

FAK

focal adhesion kinase

GJIC

gap junctional intercellular communication

HFF

human foreskin fibroblasts

HFK

human foreskin keratinocyte

JEB-G

junctional epidermolysis bullosa-gravis

JEB-PA

junctional epidermolysis bullosa-pyloric atresia

JNK

Jun kinase

mAb

monoclonal antibody

PI3K

phosphoinositide 3-OH-kinase

PIP₂

phosphatidylinositide biphosphate

PIP₃

phosphatidylinositide triphosphate

PBS

phosphate-buffered saline

Ab

antibody

LG

laminin globular
- Received July 18, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.