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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02475

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

¹ Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109, USA
² Department of Pathobiology Graduate Program, University of Washington, N.E. Pacific Street, Seattle, WA 98195, USA
³ Program in Molecular and Cellular Biology, University of Washington, N.E. Pacific Street, Seattle, WA 98195, USA

View larger version (21K): [in a new window]   Fig. 1. (A) Suspension and re-adhesion of quiescent HKs activates leading keratinocytes for a wound model. HKs were grown at confluence to generate quiescence. Suspension with trypsin/EDTA activated the keratinocytes. Re-adhesion onto surfaces coated with laminin 5 was mediated by integrins 6ß4 and 3ß1. Re-adhesion was followed by cell spreading via integrin 3ß1. Spreading, but not adhesion, was blocked with Cytochalasin D. (B) cDNA microarray analysis of quiescent and suspended/re-adherent HKs. Levels of mRNA transcripts in quiescent and suspended/re-adherent HKs were compared by cDNA microarray analysis 1.5 hours post suspension/re-adhesion with spreading. Solid bars represent transcript levels of activated suspended/re-adherent HKs, whereas open bars represent transcript levels in quiescent cells. Transcript levels are reported as mean (±s.e.m.) fluorescent units (y-axis). Significant differences **P<0.05 and *P<0.10 in fluorescence levels were found between the groups indicated and the control. Microarray analysis was performed in five separate suspension/re-adhesion experiments.

View larger version (69K): [in a new window]   Fig. 2. (A) ATF3 is upregulated in LKs of in vitro scrape wounds. Monolayer cultures of quiescent HKs were fixed at (a) 0, (b) 3, (c) 6 and (d) 12 hours after scrape wounding. ATF3 protein was undetectable in quiescent unwounded cells at 0 hour. ATF3 protein expression levels are elevated and the protein is localized to the nucleus of LKs within 3 to 6 hours and returned to baseline levels within 12 hours. Following keratinocytes distant from the wound margin did not upregulate ATF3 protein expression. White arrows identify LKs. (B) ATF3 is upregulated in LKs that assemble focal adhesions at the wound edge. After wounding (2 hours), ATF3 was selectively expressed in nuclei (large white arrows) of LKs at the wound margin but not following cells. The LKs expressing ATF3 also assembled focal adhesions detected with anti-paxillin antibody (small white arrows). Bar, 25 µm.

View larger version (28K): [in a new window]   Fig. 3. (A) Levels of ATF3 mRNA increase on suspension but decline with or without re-adhesion. Semi-quantitative reverse-transcription PCR was used to evaluate levels of ATF3 mRNA in adherent quiescent HKs (Q), Quiescent HKs were suspended with trypsin EDTA or re-adhered to laminin 5 for the indicated times (2, 9, 24 and 48 hours). After 2 hours of suspension or re-adhesion, levels of ATF3 mRNA increased but subsequently declined over time. In controls, levels of GAPDH remained constant. (B) ATF3 protein is upregulated in suspended HKs but declines upon re-adhesion. Confluent quiescent HKs were suspended with trypsin-EDTA and re-adhered onto laminin 5 or held in suspension. Extracts were collected from the quiescent parent population (labeled Quiescent) and from suspended and re-adherent HKs 1-6 hours post suspension. Extracts were immunoblotted with an anti-ATF3 antibody. ATF3 protein was not detectable in quiescent cells, but was elevated in suspended cells throughout the assay (hours 1-6). Levels of ATF3 protein in adherent cells were maximal at 4 hours post activation, after which they declined. (C) Re-adhesion of MKs on laminin 5 suppresses ATF3 protein expression. Laminin 5 null MKs were grown to confluence, suspended and re-plated onto a non-adhesive BSA coated surface or onto a laminin 5-coated surface. Extracts were collected from the quiescent cell population, and from cells plated onto BSA or Laminin 5 at 2 hour intervals from 0-10 hours. Levels of ATF3 in the extracts were examined by immunoblotting. MKs plated onto BSA failed to adhere, upregulated ATF3 protein expression and maintained elevated ATF3 expression for the duration of the assay. MKs plated onto laminin 5 adhered, upregulated ATF3 initially but then suppressed ATF3 expression to baseline by 6 hours. The time course for the downregulation of ATF3 protein was confirmed in at least seven different blot experiments.

View larger version (58K): [in a new window]   Fig. 4. ATF3 is upregulated in LKs in epidermal wounds in vivo. Cryostat sections were prepared from wounds in neonatal mouse epidermis 0 (a), 4 (b) and 18 hours (c) post wounding. The wounds were stained with an anti-ATF3 antibody. White arrows indicate the wound edge. (d) Composite image of a 4 hour wound showing expression of ATF3 in LKs adjacent to the wound edge but not in the following cells distant to the wound. Bar, 20 µm.

View larger version (64K): [in a new window]   Fig. 5. (A) Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2 and up-regulation of ATF3. Confluent HK monolayers were either untreated to serve as a control, or pretreated with SB203580 for 1 hour. Scrape wounds were generated and allowed to incubate for 2.5 hours in the presence or absence of inhibitor. ATF2 phosphorylation (P-ATF-2; a,b) and ATF3 (c,d) protein expression levels were examined by immunofluorescent staining. SB203580 blocked expression of P-ATF-2 and ATF-3. (B) Pretreatment of scrape wounds with the membrane-permeable form of the JNK peptide inhibitor (JNK Peptide Inhibitor 1, L stereoisomer; 50 µM, 30 minutes prior to wounding) (Bonny et al., 2001) prevents phosphorylation of jun but not expression of ATF3. Bar, 25 µm.

View larger version (67K): [in a new window]   Fig. 6 (A) P-p38 and P-ATF2 are elevated in LKs (white arrowheads) of scrape wounds. Scrape wounds were generated in confluent HK monolayers. Levels of phosphorylated p38 and ATF2 were examined 1 hour post wounding by immunofluorescence microscopy. (B). P-p38 is upregulated in LKs of wounds in vivo. Cryostat sections of human epidermis 0 hours (a), 1 hour (b) and 4 hours (c) post wounding were examined for p38 phosphorylation through immunofluorescence microscopy. In the control (d), a 0 hour wound section is stained with anti-laminin 5 mAb (C2-5) to identify the basement membrane. Wound margins are identified with white arrowheads. Direction of outgrowth is shown by white arrow. Bar, 20 µm.

View larger version (28K): [in a new window]   Fig. 7. (A) Scrape wounding increases phosphorylation of p38 and ATF2. HKs were grown to confluence and multiple scrape wounds were generated in the monolayer. Triton-soluble extracts were collected in non-wound HKs (Quiescent), or in wounded HKs at the time of wounding, (0 hour), and 30 minutes, 1 hour, 2 hours and 3 hours post wounding. Levels of p38 remained constant but P-p38 increased by 0.5 hours after wounding and P-ATF2 increased immediately after wounding. (B) p38 kinase activity is elevated in suspended or wounded HKs. Confluent quiescent HKs (Quiescent) were assayed for p38 kinase after suspension for 3 hours (Suspended) or scrape wounding for 6 hours (Wounded). In vitro assay of p38 kinase activity was performed as described (see Materials and Methods) using ATF2 as substrate and immunoblotting for P-ATF2. Aliquots of the cell extracts were also immunoblotted (Immunoblot) for P-p38 to confirm that levels of P-p38 correlated with p38 kinase activity.

View larger version (30K): [in a new window]   Fig. 8. (A) Adhesion, not cell spreading, dephosphorylates P-p38. Human keratinocytes null for laminin 5 were suspended and re-adhered to the indicated ligands (laminin 5, collagen) or immobilized mAbs: SP2, an irrelevant non-adhesive mAb; P4C11, a mAb against a 47 kDa non-integrin surface glycoprotein; CD44, a non-integrin cell surface antigen; mAbs against the indicated integrin subunits 2, 3,6, ß1. Cell adhesion and cell spreading induced by each ligand is indicated. Triton-soluble extracts were prepared from the adherent cells after washing then fractionated by SDS-PAGE and immunoblotted as indicated with antibodies against P-p38, p38 and focal adhesion kinase phosphorylated on Tyr397 (P-FAK). (B) Cytochalasin D inhibits adhesion and dephosphorylation of P-p38 on collagen and fibronectin but not laminin 5. Quiescent laminin 5 null HKs were suspended, treated with cytochalasin D (10 µM), then either left in suspension for 2 hours or incubated on laminin 5, collagen or fibronectin surfaces for 2 hours. Adhesion and spreading of the cells is indicated. Extracts of the indicated cells were blotted with anti-P-p38 and anti-p38 Abs. (C) Re-adhesion of suspended HKs on laminin 5 dephosphorylates P-p38 followed by transient re-phosphorylation. Confluent HKs (with null defects in laminin 5) were suspended using trypsin and either re-adhered to laminin 5 or collagen or suspended over BSA. Triton-soluble extracts were collected from the quiescent adherent HKs, suspended HKs and suspended/re-adherent cells at the indicated intervals (15 minutes, 30 minutes, etc). Expression of P-p38 was examined by immunoblotting. Blots for cells re-adhered on laminin 5 were re-probed with anti-p38 to control for levels of total p38 and are representative of all the blots.

View larger version (118K): [in a new window]   Fig. 9. Adhesion defects in laminin 5 or integrin ß4 chronically elevate ATF3. (A) Cryostat sections of skin from wild type neonatal mouse (a,d) and laminin 5 null neonatal mouse (b,c,e,f,) were stained with an anti-ATF3 (a,b,c) or anti-integrin ß4 antibody (d,e,f). ATF3 protein is not detectable in wild type mouse epidermis (arrows identify epidermal basal cells). ß4 was continuous and polarized to the basement membrane (d, arrow identifies ß4 staining in the basement membrane zone). In contrast, laminin 5 null mouse skin (3-/-) exhibited increased levels of nuclear ATF3 protein expression (b, c, arrow identifies ATF3 staining). Integrin ß4 was polarized to the basement membrane zone in laminin 5 null keratinocytes but was discontinuous (e,f) (arrows identify discontinuities in ß4 integrin). (B) Cryostat sections of skin from a normal control individual (a) and from an individual with null defects in the INTB4 gene encoding the integrin ß4 subunit (b) were stained with anti-ATF3 antibodies. ATF3 was elevated in most cell layers in the ß4 null epidermis. Bar, 20 µm (A); 20 µm (B).

View larger version (28K): [in a new window]   Fig. 10. Defects in laminin 5 deposition, prevent adhesion and dephosphorylation of P-p38. MKs from laminin 5 null mice (3-/-) and wild-type mice (3+/+) were suspended and replated onto either a BSA- or Laminin 5-coated surface. Triton-soluble extracts were collected at 2 hour intervals from 0 to 10 hours and immunoblotted with anti-P-p38 and anti-p38 antibodies. Quiescent 3+/+ and 3-/- MKs did not express phosphorylated p38. When plated onto a BSA-coated surface, 3+/+ MKs deposited laminin 5, adhered to the deposits on the BSA surface and dephosphorylated p38 within 4 hours. In contrast, 3-/- MKs were unable to deposit laminin 5 or adhere to the BSA or dephosphorylate p38 for the duration of the assay. Both 3+/+ MKs and 3-/- MKs rapidly adhered to exogenous laminin 5 and dephosphorylated P-p38, indicating exogenous laminin 5 was sufficient to rescue the ability of 3-/- MKs to adhere and dephosphorylate p38. Total levels of p38 did not change significantly in either the 3+/+ MK or 3-/- MK populations.

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