Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes.
Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin alpha6beta4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via beta1 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 beta4 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 alpha6beta4 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 alpha6beta4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and alpha6beta4 sustain P-p38 with probable pathological effects on transcription and migration.
Harper
Erin G
EG
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109, USA.
Alvares
Stacy M
SM
Carter
William G
WG
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D039161:Integrin alpha6beta4 / Q000378:metabolism; D015603:Keratinocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000293:injuries*; D014945:Wound Healing / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D007093:Imidazoles; D039161:Integrin alpha6beta4; D011725:Pyridines; D012333:RNA, Messenger; D048051:p38 Mitogen-Activated Protein Kinases; D000097794:Kalinin; C093642:SB 203580