Whether ␣6␤4 integrin regulates migration remains controversial. ␤4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express ␤4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type ␤4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with ␣6␤4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the ␣6␤4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.Migration of cells is an essential element of morphogenesis, remodeling following tissue damage and metastasis. For example, following wounding of the skin, epidermal cells migrate over a wound, a complex process involving changes in cytoskeleton organization, alterations in cell-cell and cell-matrix interactions, and modulation in gene and protein expression (1, 2).In particular, keratinocytes lose stable matrix adhesive structures called hemidesmosomes, migrate over exposed dermal collagen, and deposit a provisional matrix rich in laminin (LM) 3 -332 (old nomenclature: laminin-5), a heterotrimer consisting of ␣3, ␤3, and ␥2 subunits (1-5). This provisional matrix regulates the motility of keratinocytes (2-4). LM-332 is also believed to support tumor cell invasion (6 -12).Although cells in wounds and cancer cells migrate on a LM-332 substrate, LM-332 stabilizes epidermal cell adhesion in intact skin (3, 4,(12)(13)(14)(15)(16). These contradictory functions exhibited by LM-332, adhesion and motility, have been investigated by a number of laboratories, and results to date indicate that proteolytic processing of LM-332 has a profound impact on its functions. Specifically, in the extracellular matrix, the ␣3 and ␥2 subunits are processed from 190 to 160 kDa and from 140 to 100 kDa, respectively (4, 17-22). LM-332 containing an unprocessed ␣3 chain supports keratinocyte motility (20). Upon proteolytic cleavage of the ␣3 LM subunit within its G domain, LM-332 triggers hemidesmosome assembly, leading to decreased cell motility (3, 20 -24). However, further proteolysis of l...

The motility of keratinocytes is an essential component of wound closure and the development of epidermal tumors. In vitro, the specific motile behavior of keratinocytes is dictated by the assembly of laminin-332 tracks, a process that is dependent upon ␣6␤4 integrin signaling to Rac1 and the actin-severing protein cofilin. Here we have analyzed how cofilin phosphorylation is regulated by phosphatases (slingshot (SSH) or chronophin (CIN)) downstream of signaling by ␣6␤4 integrin/Rac1 in human keratinocytes. Keratinocytes express all members of the SSH family (SSH1, SSH2, and SSH3) and CIN. However, expression of phosphatase-dead versions of all three SSH proteins, but not dominant inactive CIN, results in phosphorylation/inactivation of cofilin, changes in actin cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes. SSH activity is regulated by 14-3-3 protein binding, and intriguingly, 14-3-3/␣6␤4 integrin protein interaction is required for keratinocyte migration. We wondered whether 14-3-3 proteins function as regulators of Rac1-mediated keratinocyte migration patterns. In support of this hypothesis, inhibition of Rac1 results in an increase in 14-3-3 protein association with SSH. Thus, we propose a novel mechanism in which ␣6␤4 integrin signaling via Rac1, 14-3-3 proteins, and SSH family members regulates cofilin activation, cell polarity, and matrix assembly, leading to specific epidermal cell migration behavior.

In endothelial cells (ECs) ␤1 integrin function-blocking antibodies inhibit ␣v␤3 integrin-mediated adhesion to a recombinant ␣4-laminin fragment (r␣4LN fragment). ␤1 integrin sequestration of talin is not the mechanism by which ␤1 integrin modulates ␣v␤3 integrin ligand binding. Rather, treatment of the ECs with ␤1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases ␤3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of ␤1 integrin function-blocking antibodies on ␤3 integrin serine phosphorylation and EC-r␣4LN fragment binding. ␤3 integrin contains a serine residue at position 752. To confirm the importance of this residue in ␣v␤3 integrinr␣4LN fragment binding, we mutated it to alanine (␤3S752A) or aspartic acid (␤3S752D). Chinese hamster ovary (CHO) cells expressing wild type or ␤3S752A integrin attach robustly to ligand. CHO cells expressing ␤3S752D integrin do not. Because the ␤3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on ␤3 integrin. Instead, we have tested an hypothesis that PKA regulates ␤3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with ␤1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits ␣v␤3-mediated cell adhesion to the r␣4LN fragment when both PKA and ␤1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the ␤3 integrin cytoplasmic tail. These results indicate a novel mechanism by which ␤1 integrin negatively modulates ␣v␤3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.Angiogenesis is a complex process that not only depends on growth factors and their receptors but is also influenced by integrin-extracellular matrix interactions. Integrins are heterodimeric cell surface receptors for matrix molecules (1). They play central roles in complex cellular processes such as adhesion, migration, proliferation, and differentiation via their interactions with the extracellular matrix and ability to regulate various signaling pathways (1, 2).Integrin heterodimers exist in inactive and active conformations. High resolution microscopic and crystal structure analyses of ␣v␤3 integrin have shed light on the structural requirements that are involved (3-6). These studies demonstrate that integrins exists in two major conformations, a closed conformation, which has a low affinity for ligand, and an open conformation, which has a high ligand affinity (3-6). In general, integrins default to a low affinity state in cells. However, this is not always the case, and ultimately the activity of an integrin is influenced by the cellular environment (3-6). Indeed, activation of an integrin may occur when a cell is stimulated by extracellular signals (outside-in signaling) and may also be regulated by cyto...

We have demonstrated that in endothelial cells β1 integrin antagonists inhibited αvβ3 integrin mediated adhesion. Here we have performed vitronectin binding assays on endothelial cells in suspension. Vitronectin binding was inhibited upon β1 integrin clustering, indicating that β1 integrin modulated αvβ3 integrin affinity. Over‐expression of talin, a known modulator of integrin affinity, did not abrogate the impact of β1 integrin clustering on αvβ3 integrin‐ligand binding. Rather, β1 integrin clustering resulted in a two‐fold increase in PKA activity and β3 integrin Ser phosphorylation. PKA inhibition rescued cell adhesion when β1 integrin‐ligand binding was blocked and inhibited β3 integrin Ser phosphorylation. β3 integrin contains a Ser residue at position 752. We mutated it to Ala (β3S752A) or Asp (β3S752D). Cells expressing wild‐type or β3S752A integrin attached robustly to ligand. Cells expressing β3S752D integrin did not. Since the β3 cytoplasmic tail lacks a PKA consensus site, we hypothesized that PKA regulates β3 integrin Ser phosphorylation indirectly through protein phosphatase 1 (PP1). Indeed, blocking PP1 activity inhibited cell adhesion and increased β3 integrin Ser phosphorylation. These results indicate a novel mechanism by which clustered β1 integrin negatively modulates αvβ3 integrin‐ligand binding via activation of PKA and inhibition of PP1 activity. Supported by NCI.