Harper Erin G, Alvares Stacy M, Carter William G
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109, USA.
J Cell Sci. 2005 Aug 1;118(Pt 15):3471-85. doi: 10.1242/jcs.02475.
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.
静止的表皮通过整合素α6β4与基底膜中的层粘连蛋白5相连。受伤会提高层粘连蛋白5的表达，产生通过β1整合素迁移的前导角质形成细胞（LKs）。研究评估了层粘连蛋白5作为细胞信号调节因子以及在LKs中的mRNA和蛋白质表达情况。基于静止人角质形成细胞（HKs）的悬浮和重新黏附建立了体外伤口模型。DNA微阵列鉴定出悬浮和重新黏附1.5小时后多种mRNA水平升高，包括激活转录因子3（ATF3）。在体外和体内，悬浮或受伤2小时后，ATF3蛋白水平在LKs细胞核中升高，但在后续细胞的细胞核中未升高，不过在损伤后12 - 18小时下降。值得注意的是，层粘连蛋白5或整合素β4的无效缺陷会长期抑制黏附，从而在体内使ATF3显著升高。这表明与层粘连蛋白5而非其他配体的黏附会抑制激活。悬浮时，微阵列中的ATF3和其他转录本会被磷酸化的p38丝裂原活化蛋白激酶（P - p38）升高，P - p38是一种调节mRNA和细胞运动的应激激酶。用SB203580抑制P - p38可防止ATF2磷酸化，ATF2是LKs中ATF3的转录因子。相对于悬浮细胞，通过α6β4重新黏附到层粘连蛋白5会使P - p38去磷酸化并抑制ATF3蛋白。因此，静止HKs的受伤会破坏层粘连蛋白5的黏附以激活p38，产生定义LKs的mRNA转录本。通过α6β4黏附到层粘连蛋白5沉积物上会抑制P - p38和包括ATF3在内的激活mRNA。层粘连蛋白5和α6β4的缺陷会使P - p38持续存在，可能对转录和迁移产生病理影响。
