Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes

Abstract

Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin α6β4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via β1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin β4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via α6β4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via α6β4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and α6β4 sustain P-p38 with probable pathological effects on transcription and migration. Journal of Cell Science 3472 of polarized lamellipodium in epithelial cells through activation of Rac1 (Choma et al., 2004). This sequence of adhesion and signaling changes allows HKs to make the transition from quiescence to activation in wounds. Similarly, ligation of α3β1 in A549 adenocarcinoma cells by laminin 10/11 or laminin 5 preferentially activates the Rac-MKK-JNK/p38 stress pathway that mediates epithelial migration (Gu et al., 2001;Ono and Han, 2000;Xia and Karin, 2004). In addition to adhesion, interaction of cells with integrin ligands also regulates mRNA transcription and protein translation through activation of p38 MAPK (Balda and Matter, 2003). Ligation of α6β4 activates the Rac-PAK-MKK-p38 pathway to promote expression of IL-6 in thymic epithelial cells (Mainiero et al., 2003). Thus, cell suspension and re-adhesion onto dermal collagen or laminin 5 via integrins activates JNK and/or p38 by phosphorylation to alter protein expression and/or cell motility (Clark et al., 2003;Ono and Han, 2000). Here, we sought to: (1) identify changes in mRNA transcription and protein translation in quiescent keratinocytes as they transition into wound LKs; (2) identify cell signals necessary for the transcriptional changes in LKs; (3) understand the role of laminin 5 adhesion in regulating the signaling and transcriptional changes. We found that sus...

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

“…Thus, wounding generates a signaling cascade that changes cell behavior. Upon wounding, there is a dramatic alteration of membrane properties at the wound edge including the up-regulation of the tetraspanins CD9 and the transient expression of ATF3, regulated by the phosphorylation of p38 map kinase [6]. Additionally, we have identified L6, a tetraspanin-like protein as an example of a membrane protein that is upregulated soon after wounding [6] (Fig.…”

“…Upon integrin-mediated cell adhesion, membrane microdomains rich in glycosphingolipids and glycoproteins are recruited to the sites of receptor clustering to initiate signaling cascades that alter the cell's behavior [6]. In immune cells, components of microdomains are recruited to sites of antigen presentation in the immunological synapse-an adhesion and signaling site of immune cells-and affect cell signaling [7].…”

Ultrastructural Localization of Integrin Subunits β4 and α3 Within the Migrating Epithelial Tongue of In Vivo Human Wounds

“…Despite this discrete b4 localization to the leading edge, the global concentration of b4 remains within the keratin-rich trailing edge of the migrating keratinocyte. We believe this trailing edge concentration and leading edge paucity of b4 accounts for the repeating cell to cell "beads on a string" (Harper et al 2005) staining effect when viewing b4 localization on the migrating tongue of wounds at the light level.…”

ATF3 and p15PAF are novel gatekeepers of genomic integrity upon UV stress

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