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...

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and DetergentResistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior.

Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of lategeneration rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.germ line | fertility | tudor domain protein