E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1 ؊/؊ keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins ␣ 5 , ␣ 6 , ␤ 1 , and ␤ 4 in E2F-1 ؊/؊ keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1 ؊/؊ keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-␥1. E2F-1 ؊/؊ keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G 1 and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1 ؊/؊ mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1 ؊/؊ mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of reepithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility

E2F1 is a transcription factor central for cell survival, proliferation, and repair following genomic insult. Depending on the cell type and conditions, E2F1 can induce apoptosis in transformed cells, behaving as a tumour suppressor, or impart growth advantages favouring tumour formation. The pleiotropic functions of E2F1 are a likely consequence of its ability to transcriptionally control a wide variety of target genes, and require tight regulation of its activity at multiple levels. Although sequestration of proteins to particular cellular compartments is a well-established regulatory mechanism, virtually nothing is known about its contribution to modulation of E2F1 target gene expression. We have examined the subcellular trafficking of E2F1 and, contrary to the widely held notion that this factor is constitutively nuclear, we now demonstrate that it is subjected to continuous nucleocytoplasmic shuttling. We have also defined two nuclear localization domains and a nuclear export region, which mediates CRM1-dependent transit out of the nucleus. The predominant subcellular location of E2F1 is likely determined by the balance between the activity of nuclear import and export domains, and can be modulated by differentiation stimuli in epidermal cells. Thus, we have identified a hitherto unrecognized mechanism to control E2F1 function through modulation of its subcellular localization.

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by downregulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca 2؉ -induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca 2؉ as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.The epidermis is formed by keratinocytes at different stages of differentiation (1, 2). Undifferentiated keratinocytes reside in the basal cell layer and are attached to a basement membrane that separates them from the dermis (3-5). Proliferation, adhesion, and motility of undifferentiated keratinocytes are regulated by integrins and associated proteins, which form focal adhesions (6). Upon receiving appropriate signals, basal keratinocytes initiate terminal differentiation, characterized by loss of proliferative capacity, decrease in integrin expression, detachment from the basement membrane, and migration upward to form postmitotic suprabasal keratinocyte layers. In culture, primary keratinocytes can be induced to differentiate by elevating the extracellular Ca 2ϩ concentration. Induction of differentiation by Ca 2ϩ is accompanied by loss of integrin expression and formation of epithelial sheets. Intercellular adhesion in differentiated keratinocytes occurs through tight junctions, cadherin-mediated formation of adherens junctions, and desmosomes (6 -9). Epidermal integrity and barrier function depend on these cell-cell junctions.Originally identified as a ␤ 1 integrin-binding protein, ILK 1 also interacts with paxillin, actopaxin, and affixin in focal adhesions. Accumulating evidence from a variety of cell types indicates that ILK can function as a kinase and as a scaffold protein that mediates interactions between integrins and the actin cytoskeleton through interaction with mult...

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.