FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2092686483> ?p ?o ?g. }

• W2092686483 endingPage "26845" @default.
• W2092686483 startingPage "26839" @default.
• W2092686483 abstract "To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5–24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97–98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41–65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade. To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5–24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97–98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41–65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade. amphiregulin epidermal growth factor extracellular signal-regulated kinase heparin-binding EGF-like growth factor matrix metalloproteinase mitogen-activated protein kinase MAPK kinase receptor tyrosine kinase vascular endothelial growth factor Janus family of tyrosine kinases/signal transducers and activators of transcription phosphate-buffered saline 1,4-piperazinediethanesulfonic acid Among the many growth factors and cytokines that are important in skin physiology, several of the ErbB receptor ligands, including amphiregulin (AR),1heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α (TGF-α), are distinctive in that they are strongly overexpressed by keratinocytes in hyperproliferative skin disorders such as psoriasis (1Stoll S.W. Elder J.T. Exp. Dermatol. 1998; 7: 391-397Crossref PubMed Scopus (71) Google Scholar, 2Cook P.W. Pittelkow M.R. Keeble W.W. Graves Deal R. Coffey R.J., Jr. Shipley G.D. Cancer Res. 1992; 52: 3224-3227PubMed Google Scholar, 3Elder J.T. Fisher G.J. Lindquist P.B. Bennett G.L. Pittelkow M.R. Coffey R., Jr. Ellingsworth L. Derynck R. Voorhees J.J. Science. 1989; 243: 811-814Crossref PubMed Scopus (504) Google Scholar). In keratinocyte cultures, several ErbB ligands stimulate cell proliferation in an autocrine fashion (4Klein S.B. Fisher G.J. Jensen T.C. Mendelsohn J. Voorhees J.J. Elder J.T. J. Cell. Physiol. 1992; 151: 326-336Crossref PubMed Scopus (38) Google Scholar, 5Hashimoto K. Higashiyama S. Asada H. Hashimura E. Kobayashi T. Sudo K. Nakagawa T. Damm D. Yoshikawa K. Taniguchi N. J. Biol. Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar, 6Cook P.W. Mattox P.A. Keeble W.W. Pittelkow M.R. Plowman G.D. Shoyab M. Adelman J.P. Shipley G.D. Mol. Cell. Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (208) Google Scholar). These ligands interact in a complex fashion with receptors of the ErbB family (ErbB1–4), several of which are expressed by human keratinocytes and in skin (7Marques M.M. Martinez N. Rodriguez-Garcia I. Alonso A. Exp. Cell Res. 1999; 252: 432-438Crossref PubMed Scopus (34) Google Scholar, 8Stoll S.W. Kansra S. Peshick S. Fry D.W. Leopold W.R. Wiesen J.F. Sibilia M. Zhang T. Werb Z. Derynck R. Wagner E.F. Elder J.T. Neoplasia. 2001; 3: 339-350Crossref PubMed Scopus (71) Google Scholar, 9De Potter I.Y. Poumay Y. Squillace K.A. Pittelkow M.R. Exp. Cell Res. 2001; 271: 315-328Crossref PubMed Scopus (59) Google Scholar). Numerous studies indicate that ErbB receptor signaling is particularly critical for the regulation of wound healing responses. Addition of TGF-α or EGF to skin wounds accelerates wound healing (10Nanney L.B. J. Invest. Dermatol. 1990; 94: 624-629Abstract Full Text PDF PubMed Google Scholar, 11Brown R.L. Breeden M.P. Greenhalgh D.G. J. Surg. Res. 1994; 56: 562-570Abstract Full Text PDF PubMed Scopus (159) Google Scholar), and several important mediators of wound healing depend on ErbB signaling for their induction, including urokinase (12Jensen P.J. Rodeck U. J. Cell. Physiol. 1993; 155: 333-339Crossref PubMed Scopus (31) Google Scholar), hyperproliferative keratins K6 and K16 (13Jiang C.K. Magnaldo T. Ohtsuki M. Freedberg I.M. Bernerd F. Blumenberg M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6786-6790Crossref PubMed Scopus (170) Google Scholar), vascular endothelial growth factor (VEGF) (14Detmar M. Brown L.F. Claffey K.P. Yeo K.-T. Kocher O. Jackman R.W. Berse B. Dvorak H.F. J. Exp. Med. 1994; 180: 1141-1146Crossref PubMed Scopus (647) Google Scholar), and collagenase (15Pilcher B.K. Dumin J. Schwartz M.J. Mast B.A. Schultz G.S. Parks W.C. Welgus H.G. J. Biol. Chem. 1999; 274: 10372-10381Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We have previously shown that expression of the ErbB ligands HB-EGF and/or AR is rapidly increased in human and mouse skin organ culture, an ex vivo system that displays many similarities to the process of cutaneous wound healing in vivo (8Stoll S.W. Kansra S. Peshick S. Fry D.W. Leopold W.R. Wiesen J.F. Sibilia M. Zhang T. Werb Z. Derynck R. Wagner E.F. Elder J.T. Neoplasia. 2001; 3: 339-350Crossref PubMed Scopus (71) Google Scholar, 16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar). Expression of these ligands was markedly reduced after antibody-mediated or pharmacologic inhibition of ErbB-receptor tyrosine kinase (RTK) activity, suggestive of an autocrine stimulatory mechanism. Furthermore, selective inhibition of ErbB-RTK abrogates keratinocyte outgrowth from human skin explants (16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar). In contrast to the well established function of ErbB receptors during wound repair, little is known about the downstream signaling events that become activated during this process. The extracellular signal-regulated kinases ERK1 and ERK2 become activated in response to growth factor-related signals, whereas the c-Jun NH2-terminal kinases (JNK) and p38 kinases become activated in response to environmental stress and inflammatory cytokines (17Chang L. Karin M. Suomela S. Kariniemi A.L. Snellman E. Saarialho-Kere U. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4396) Google Scholar). Because ERK1 and ERK2 are generally associated with growth factor signaling and because our previous studies demonstrated a large increase in ErbB ligand expression during wound healing (16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar), we hypothesized that ERK acts as a downstream mediator of ErbB signaling to the nucleus during wound repair. To test this hypothesis, we measured the effects of pharmacologic inhibitors of MEK activation, ErbB-RTK activity, and matrix metalloproteinase (MMP) function on a variety of biochemical responses in human skin organ culture. Our results demonstrate that ERK activation is an important component of the signaling mechanism leading to gene expression in wounded skin and that ErbB signaling makes a major contribution to ERK activation during this process. Our data also indicate that proteolytic release of one or more ErbB ligands by matrix metalloproteinases is a major mechanism responsible for ErbB and subsequent MAPK activation in wounded skin. The MAP kinase kinase (MEK) inhibitor PD98059 and the metalloproteinase inhibitor BB2516 were from Calbiochem. The ErbB-RTKI PD158780 was provided by Drs. David Fry and Wilbur Leopold (Pfizer). Human recombinant EGF was from Sigma or from BD PharMingen Collaborative Biomedical Products (Franklin Lakes, NJ). Antibodies directed against non-phosphorylated and phosphorylated forms of ERK were from Cell Signaling Technology (Beverly, MA). Mouse monoclonal antibodies against ErbB family members were obtained from Transduction Laboratories (Franklin Lakes, NJ) and Labvision (Fremont, CA). The mouse monoclonal anti-phosphotyrosine antibody 4G10 and horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG were from Upstate Biotechnology (Lake Placid, NY). Radioisotopes were from PerkinElmer Life Sciences. All other chemicals were purchased from Sigma. Human skin keratomes at a depth of 0.2–0.4 mm were harvested from the buttocks of healthy volunteers as previously described (18Voorhees J.J. Duell E.A. Bass L.J. Powell J.A. Harrell E.R. Arch. Dermatol. 1972; 105: 695-701Crossref PubMed Scopus (98) Google Scholar). All procedures involving human subjects were approved by the Institutional Review Board of the University of Michigan. Normal human keratinocytes (passage 2–4) were cultured in serum-free MCDB 153 medium optimized for high density keratinocyte growth (19Wille J., Jr. Pittelkow M.R. Shipley G.D. Scott R.E. J. Cell. Physiol. 1984; 121: 31-44Crossref PubMed Scopus (350) Google Scholar). The medium (Medium 154 CF, Cascade Biologics, Portland, OR) was purchased in calcium-free form and supplemented with CaCl2 to a final concentration of 0.1 mm as previously described (8Stoll S.W. Kansra S. Peshick S. Fry D.W. Leopold W.R. Wiesen J.F. Sibilia M. Zhang T. Werb Z. Derynck R. Wagner E.F. Elder J.T. Neoplasia. 2001; 3: 339-350Crossref PubMed Scopus (71) Google Scholar). At ∼40–50% confluence, the cells were depleted of exogenous growth factors by incubation in basal medium for 48 h. The cells were then pretreated with various concentrations of PD158780 (0.0016–1 μm) or PD98059 (0.08–50 μm) with a change of medium. After 1 additional hour, cells were stimulated with 16.5 nm EGF for 10 min and then lysed and processed for Western blotting as described below. Human skin keratome fragments (6-mm circular punches or 1-cm squares) were incubated at room temperature in keratinocyte basal medium containing 0.1 mm Ca2+ (Medium 154 CF, Cascade Biologics) in the presence or absence of the MEK inhibitor PD98059, the ErbB-RTK inhibitor PD158780 (20Rewcastle G.W. Murray D.K. Elliott W.L. Fry D.W. Howard C.T. Nelson J.M. Roberts B.J. Vincent P.W. Showalter H.D. Winters R.T. Denny W.A. J. Med. Chem. 1998; 41: 742-751Crossref PubMed Scopus (108) Google Scholar), or the metalloproteinase inhibitor BB2516 (21Glaser K.B. Pease L., Li, J. Morgan D.W. Biochem. Pharmacol. 1999; 57: 291-302Crossref PubMed Scopus (21) Google Scholar). In some experiments, 6-mm punch fragments were divided into eighths in order to increase the total area of wounded skin tissue. Organ cultures were allowed to stand for 30 min at room temperature to assure equilibration of the inhibitor and then warmed to 37 °C to initiate signaling. We have shown that organ cultures are transcriptionally inert when maintained at room temperature or below (16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar). Organ cultures were maintained at 37 °C for various times prior to harvest (0–24 h). In some experiments, EGF (100 ng/ml, 16.5 nm) was added to selected samples just prior to transferring the cultures to 37 °C. Organ cultures were processed for RNA or protein isolation and analyzed by Northern or Western blotting as described below. Total RNA was isolated from human skin keratomes (∼1 cm2 in size) as previously described (16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar). Equal microgram amounts (20–30 μg) were separated electrophoretically on 1% formaldehyde-agarose gels, transferred to nylon membranes (Zeta-probe, Bio-Rad), and hybridized against 32P-labeled c-Jun, c-Fos, HB-EGF, AR, VEGF, and 36B4 cDNA inserts as previously described (16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar, 22Elder J.T. Tavakkol A. Klein S.B. Zeigler M.E. Wicha M. Voorhees J.J. J. Invest. Dermatol. 1990; 94: 19-25Abstract Full Text PDF PubMed Google Scholar). Hybridization signals were quantitated by PhosphorImager (Amersham Biosciences) and normalized against the control gene 36B4 (23Laborda J. Nucleic Acids Res. 1991; 19: 3998Crossref PubMed Scopus (434) Google Scholar). Data are expressed as percentage of untreated controls. Cultured human keratinocytes were washed twice in 1× PBS, scraped into 1 ml of 1× Laemmli sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 50 mm dithiothreitol), and heated for 10 min at 100 °C (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). Human skin keratomes were cut into pieces using a 6-mm diameter biopsy punch. Two such pieces were placed in 500 μl of preheated 1× sample buffer and maintained at 100 °C for 10 min in a heating block, then vortexed for 10 s. The lysates were then cleared by centrifugation at 12,000 rpm for 10 min in a microcentrifuge. Protein concentrations were estimated byA280, and equal microgram amounts were separated on 4–20% Tris-glycine gels. The proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Invitrogen) according to the manufacturer's instructions. Western blots were blocked by incubation in 1× PBS/0.1% Tween 20 (PBST) containing 5% nonfat dry milk for 30 min and incubated with anti-ERK (1:1000), anti-phospho ERK (1:1000), anti-ErbB1 (TL13, Transduction Laboratories, 0.5 μg/ml), anti-ErbB2 (Ab-17, Labvision, 0.5 μg/ml) or anti-phosphotyrosine antibodies (0.5 μg/ml) for 16 h at 4 °C. The blots were washed three times for 5 min in PBST (ERK antibodies) or two times for 30 min in 1× PBS (anti-phosphotyrosine and anti-ErbB antibodies) and then incubated with 1 μg/ml horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Upstate Biotechnology) for 1 h. The membranes were washed as described above except that blots incubated with anti-ErbB1, anti-ErbB2, and anti-phosphotyrosine antibodies were washed for an additional 5 min in PBST prior to detection of specific protein-antibody complexes by enhanced chemiluminescence (ECL Plus,Amersham Biosciences). Chemiluminescence was quantitated by densitometry of x-ray films using an Amersham Biosciences densitometer. Base-line signals were obtained from an area of equal size just above the phospho-ERK band. Whenever subtraction of the base-line signal yielded a negative number, the phospho-ERK signal intensity for that lane was assigned a zero value. Normal human keratinocytes were depleted of growth factors for 48 h and treated with EGF as described above. The cells were then lysed in buffer A (10 mm PIPES, pH 6.8, 250 mm sucrose, 3 mm MgCl2, 150 mm KCl, 5 mm EGTA, 100 mm sodium fluoride, 5 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 10% glycerol, 1% Triton X-100, and 1× protease inhibitor mixture, Roche Molecular Biochemicals) and immunoprecipitated as previously described (8Stoll S.W. Kansra S. Peshick S. Fry D.W. Leopold W.R. Wiesen J.F. Sibilia M. Zhang T. Werb Z. Derynck R. Wagner E.F. Elder J.T. Neoplasia. 2001; 3: 339-350Crossref PubMed Scopus (71) Google Scholar) using antibodies directed against ErbB1 or ErbB2 (Ab-13 or Ab-2, respectively, Labvision). Controls contained the same concentration of an isotype-matched control mouse IgG (Sigma). Equal aliquots of the immunoprecipitates were subjected to Western blotting and immunodetection as described above. As shown in Fig.1A, left panels, the MEK activation inhibitor PD98059 inhibited EGF-induced ERK phosphorylation in cultured human keratinocytes in a dose-dependent manner. Inhibition to basal levels was observed at 50 μm PD98059. EGF markedly stimulated tyrosine phosphorylation of a 170–190-kDa band in cultured keratinocytes (Fig. 1A, right panels). The pan-ErbB inhibitor PD158780 reduced tyrosine phosphorylation of this band in a dose-dependent fashion. At concentrations of 0.2 and 1 μm, PD158780 also inhibited ERK phosphorylation in a dose-dependent manner to below basal levels. Immunoprecipitation experiments (Fig. 1B) confirmed that EGF stimulates tyrosine phosphorylation of ErbB1 under these conditions and that EGF-stimulated tyrosine phosphorylation of ErbB1 can be blocked by PD158780. In contrast, ErbB2 did not undergo tyrosine phosphorylation after EGF stimulation (Fig. 1B, right panels). ErbB1 protein levels were consistently increased by PD158780 pretreatment, presumably because of inhibition of ongoing ErbB degradation. Using phospho-specific antibodies, we next determined the activation status of ERK in normal and organ-cultured human skin. Levels of ERK phosphorylation were quite variable in freshly harvested skin not subjected to organ culture (compare Figs.2 and 4). The time course of ERK phosphorylation was also variable despite constant levels of total ERK. This variability is illustrated in the two independent experiments shown in Fig. 2A. Replicate samples of the same keratome tissue displayed reproducible levels of ERK phosphorylation for any given time point (data not shown). Increasing the lateral surface area of wounding by cutting 6-mm keratome fragments into 8 smaller pieces increased ERK activation by only ∼50%, despite a 3.5-fold increase in the lateral surface area (data not shown).Figure 4Inhibition of metalloproteinase activity blocks and EGF restores MAP kinase activation in human skin organ culture. Organ cultures were incubated in the presence or absence of 20 μm BB2516. After 2 h, 100 ng/ml (16.5 nm) of EGF was added to some cultures for 10 min followed immediately by tissue lysis. Lysates were analyzed by Western blotting with phospho-ERK or total ERK antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Despite variable levels of ERK activity, the MEK activation inhibitor PD98059 consistently and markedly (by 97% or more) reduced ERK phosphorylation at 0.5, 2, and 4 h (Fig. 2B). The ErbB-RTK inhibitor PD158780 also produced a marked inhibition of ERK phosphorylation in organ culture, which was substantial (92%) even at 30 min. By 2 h of organ culture, inhibition by PD158780 was significantly less marked than that produced by PD98059 (p = 0.0017 by Tukey's HSD test), but it was still prominent (70.9%) (Fig. 2B). Fig.3A demonstrates that c-Fos and c-Jun mRNAs are strongly induced after as little as 30 min of organ culture. HB-EGF and AR mRNA were induced with a delay of 60–120 min relative to c-Jun and c-Fos, whereas VEGF messages were only detectable at 8 h. PD98059 markedly reduced the mRNA levels of all genes tested (Fig. 3A). Inhibition of HB-EGF gene expression by PD158780 and PD98059 was dose-dependent (Fig.3B). However, inhibition was incomplete, falling to only 35% of control levels in the presence of 50 μm PD98059 and to 45% of control in the presence of 10 μm PD158780. In contrast, inhibition of ERK phosphorylation was essentially complete at 50 μm PD98059 and at 1 μm PD158780 in keratinocyte cell cultures (Fig. 1) and in skin organ culture (Fig. 2). Incomplete inhibition of c-fos, c-jun, andVEGF gene expression by 50 μm PD98059 is also evident from inspection of Fig. 3A. The metalloproteinase inhibitor BB2516 markedly reduced ERK phosphorylation at 2 h of organ culture to 31 ± 6% of control values observed in untreated organ cultures (mean ± S.E., n = 8, Fig.2). In four of four experiments, addition of EGF to BB2516-pretreated organ cultures restored ERK phosphorylation to levels at least as high as those observed in untreated organ cultures (Fig. 4). Treatment with EGF alone had variable effects on ERK phosphorylation (194 ± 42% of control organ cultures without additives, mean ± S.E., n = 3). In three of three experiments, ERK phosphorylation in BB2516 + EGF-treated cultures was restored to at least the level observed in cultures treated with EGF alone. Quantitative comparison to the other inhibitors revealed that the effect of BB2516 was significantly less marked than that produced by PD98059 (Tukey's HSD test, p = 0.0003 at 0.5 h and p = 0.0006 at 2 h) (Fig. 2B). To determine whether the blockade of metalloproteinase and ERK activities in organ culture might lead to an inhibition of gene expression similar to that seen for ERK phosphorylation (Fig. 2B), we prepared RNA from organ cultures treated for various times with each of the three inhibitors. Northern blots were hybridized, stripped, and sequentially re-hybridized against c-Fos, c-Jun, HB-EGF, AR, and 36B4. A representative experiment is shown in Fig.5A. The results of multiple experiments are quantitated for c-Fos and c-Jun in Fig. 5Band for HB-EGF in Fig. 5C. Statistical analysis of these results revealed that PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of the rapid and transient increase in c-Fos and c-Jun mRNA at 30 min (Tukey's HSD test,p ⩽ 0.02, Fig. 5B). In contrast, there was no significant difference in the effectiveness of these three compounds as inhibitors of the more delayed HB-EGF mRNA response (Fig.5C). As was previously discussed for PD98059, PD158780 and BB2516 yielded only incomplete inhibition of gene responses, reaching a maximum of 55–70% inhibition (Fig. 5, B andC). Although the importance of ErbB signaling in wound healing is well recognized (12Jensen P.J. Rodeck U. J. Cell. Physiol. 1993; 155: 333-339Crossref PubMed Scopus (31) Google Scholar, 13Jiang C.K. Magnaldo T. Ohtsuki M. Freedberg I.M. Bernerd F. Blumenberg M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6786-6790Crossref PubMed Scopus (170) Google Scholar, 14Detmar M. Brown L.F. Claffey K.P. Yeo K.-T. Kocher O. Jackman R.W. Berse B. Dvorak H.F. J. Exp. Med. 1994; 180: 1141-1146Crossref PubMed Scopus (647) Google Scholar, 15Pilcher B.K. Dumin J. Schwartz M.J. Mast B.A. Schultz G.S. Parks W.C. Welgus H.G. J. Biol. Chem. 1999; 274: 10372-10381Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16Stoll S. Garner W. Elder J. J. Clin. Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (102) Google Scholar, 25Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar), the mechanisms by which ErbB signaling is activated and propagated to the nucleus remain poorly defined. In this study we have investigated whether the ERK pathway could act as a mediator of ErbB receptor signaling during wound repair. To confirm the potency of our inhibitors, we performed preliminary experiments in cultured keratinocytes rendered EGF responsive by growth factor depletion (26Coffey R.J., Jr. Derynck R. Wilcox J.N. Bringman T.S. Goustin A.S. Moses H.L. Pittelkow M.R. Nature. 1987; 328: 817-820Crossref PubMed Scopus (692) Google Scholar). EGF treatment clearly increased ERK phosphorylation under these conditions (Fig. 1A). Western blotting and immunoprecipitation experiments demonstrated that EGF markedly stimulates tyrosine phosphorylation of ErbB1 but not ErbB2 (Fig. 1B). This finding is consistent with our previous demonstration that ErbB1 is expressed on the keratinocyte cell surface, whereas ErbB2 is sequestered within intracellular vesicles (8Stoll S.W. Kansra S. Peshick S. Fry D.W. Leopold W.R. Wiesen J.F. Sibilia M. Zhang T. Werb Z. Derynck R. Wagner E.F. Elder J.T. Neoplasia. 2001; 3: 339-350Crossref PubMed Scopus (71) Google Scholar). PD158780 is a selective and reversible inhibitor of all known ErbB tyrosine kinases (20Rewcastle G.W. Murray D.K. Elliott W.L. Fry D.W. Howard C.T. Nelson J.M. Roberts B.J. Vincent P.W. Showalter H.D. Winters R.T. Denny W.A. J. Med. Chem. 1998; 41: 742-751Crossref PubMed Scopus (108) Google Scholar). This compound reduced ERK phosphorylation to below basal levels (Fig. 1A) and completely inhibited EGF-stimulated tyrosine phosphorylation of ErbB1 (Fig. 1B). Taken together, these findings indicate that virtually all the ERK phosphorylation present in growth factor-depleted keratinocytes is a consequence of basal ErbB1-RTK activity. PD98059 is a highly specific inhibitor of the activation of MEK1/2 (27Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar), which in turn phosphorylates and activates ERK1 and ERK2. Because no other major proximal activators of ERK1/2 other than MEK1/2 have been described (28Guan K.L. Cell. Signal. 1994; 6: 581-589Crossref PubMed Scopus (164) Google Scholar), PD98059 can be regarded as a specific inhibitor of ERK signaling. As expected, basal and EGF-induced ERK phosphorylation in human keratinocytes could be blocked by 50 μm PD98059 (Fig. 1A), confirming earlier studies indicating that EGF-induced activation of ERK is mediated by MEK in keratinocytes (29Zeigler M.E. Chi Y. Schmidt T. Varani J. J. Cell. Physiol. 1999; 180: 271-284Crossref PubMed Scopus (206) Google Scholar). The potency of PD98059 was consistent with that observed by others (27Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar,29Zeigler M.E. Chi Y. Schmidt T. Varani J. J. Cell. Physiol. 1999; 180: 271-284Crossref PubMed Scopus (206) Google Scholar). We observed variable levels of basal ERK phosphorylation in skin samples not subjected to organ culture. We also observed a variable time course of ERK phosphorylation in untreated organ cultures (examples shown in Figs. 2 and 4). This variability was not due to sampling, because replicate fragments of the same keratome gave highly reproducible results at any given time point (data not shown). The source of this variability remains to be elucidated; however, it must lie at or upstream of MEK because the MEK activation inhibitor PD98059 produced a marked (97–98%) and very consistent inhibition of ERK phosphorylation at all time points tested (Fig. 2). The marked and consistent inhibition of ERK phosphorylation in organ-cultured skin by the ErbB inhibitor PD158780 (Fig. 2) clearly demonstrates that ErbB1-RTK activation is a major pathway leading to ERK activation in this system. The fact that inhibition of ERK activation was incomplete (Fig. 2B), even at doses of PD158780 sufficient to abrogate EGF-stimulated tyrosine phosphorylation of ErbB1 and phosphorylation of ERK in keratinocytes (Fig. 1), indicates that signal transduction pathways not present in cultured keratinocytes contribute to ErbB-independent ERK activation in wounded skin. These additional inputs to ERK activation remain to be determined. Dividing the keratome fragments into eighths increased the lateral wound surface area by 3.5-fold yet produced only a 50% increase in ERK activity. This observation suggests that the skin tissue is already substantially injured as a result of the keratome-harvesting procedure. When set to a depth of 0.2–0.4 mm, the keratome produces a substantial wound near the dermal-epidermal junction, frequently interrupting the epidermal basement membrane. Thus, overall epidermal injury should not be markedly increased by increasing the cut edges at the lateral margins of the specimen. The primarily epidermal nature of the keratome biopsy also limits the likelihood that cell types other than keratinocytes (e.g. fibroblasts, endothelial cells) make a substantial contribution to the process of ErbB1/ERK activation in this system. Thus, the observed events are likely to primarily involve keratinocytes. We previously reported the induction of c-Fos and c-Jun mRNA after 2 h of organ culture (22Elder J.T. Tavakkol A. Klein S.B. Zeigler M.E. Wicha M. Voorhees J.J. J. Invest. Dermatol. 1990; 94: 19-25Abstract Full Text PDF PubMed Google Scholar). In the present studies, we have demonstrated marked induction of these transcripts as early as 30 min (Fig. 3A). We have observed that c-Fos mRNA accumulates more rapidly than does c-Jun in skin organ culture (Fig.3A). This finding is consistent with the known requirement for increased c-Fos transcription and translation and the formation of c-Fos/c-Jun heterodimers in order to increase c-Jun transcription (30Shaulian E. Karin M. Kariniemi A.L. Snellman E." @default.
• W2092686483 created "2016-06-24" @default.
• W2092686483 creator A5010826903 @default.
• W2092686483 creator A5015004042 @default.
• W2092686483 creator A5023408670 @default.
• W2092686483 date "2002-07-01" @default.
• W2092686483 modified "2023-10-12" @default.
• W2092686483 title "Metalloproteinases Stimulate ErbB-dependent ERK Signaling in Human Skin Organ Culture" @default.
• W2092686483 cites W1505475872 @default.
• W2092686483 cites W1557197358 @default.
• W2092686483 cites W1595716544 @default.
• W2092686483 cites W1965560753 @default.
• W2092686483 cites W1967058955 @default.
• W2092686483 cites W1981602259 @default.
• W2092686483 cites W1989170373 @default.
• W2092686483 cites W1996405087 @default.
• W2092686483 cites W1996513552 @default.
• W2092686483 cites W2002212170 @default.
• W2092686483 cites W2007780460 @default.
• W2092686483 cites W2012104993 @default.
• W2092686483 cites W2015030147 @default.
• W2092686483 cites W2018793834 @default.
• W2092686483 cites W2019720769 @default.
• W2092686483 cites W2025357617 @default.
• W2092686483 cites W2026171842 @default.
• W2092686483 cites W2035936653 @default.
• W2092686483 cites W2039202467 @default.
• W2092686483 cites W2042749187 @default.
• W2092686483 cites W2050265215 @default.
• W2092686483 cites W2054012934 @default.
• W2092686483 cites W2056263610 @default.
• W2092686483 cites W2067450274 @default.
• W2092686483 cites W2071972670 @default.
• W2092686483 cites W2077564758 @default.
• W2092686483 cites W2079127998 @default.
• W2092686483 cites W2086730539 @default.
• W2092686483 cites W2091053844 @default.
• W2092686483 cites W2100837269 @default.
• W2092686483 cites W2110228428 @default.
• W2092686483 cites W2113021215 @default.
• W2092686483 cites W2118146357 @default.
• W2092686483 cites W2126514623 @default.
• W2092686483 cites W2137033699 @default.
• W2092686483 cites W2138642580 @default.
• W2092686483 cites W2140475905 @default.
• W2092686483 cites W2149364554 @default.
• W2092686483 cites W2150732175 @default.
• W2092686483 cites W2158293853 @default.
• W2092686483 cites W2159141617 @default.
• W2092686483 cites W2273634713 @default.
• W2092686483 cites W2322523258 @default.
• W2092686483 cites W4232995978 @default.
• W2092686483 doi "https://doi.org/10.1074/jbc.m201108200" @default.
• W2092686483 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12016209" @default.
• W2092686483 hasPublicationYear "2002" @default.
• W2092686483 type Work @default.
• W2092686483 sameAs 2092686483 @default.
• W2092686483 citedByCount "18" @default.
• W2092686483 countsByYear W20926864832016 @default.
• W2092686483 countsByYear W20926864832020 @default.
• W2092686483 crossrefType "journal-article" @default.
• W2092686483 hasAuthorship W2092686483A5010826903 @default.
• W2092686483 hasAuthorship W2092686483A5015004042 @default.
• W2092686483 hasAuthorship W2092686483A5023408670 @default.
• W2092686483 hasBestOaLocation W20926864831 @default.
• W2092686483 hasConcept C109523444 @default.
• W2092686483 hasConcept C11960822 @default.
• W2092686483 hasConcept C185592680 @default.
• W2092686483 hasConcept C202751555 @default.
• W2092686483 hasConcept C2779251935 @default.
• W2092686483 hasConcept C28483552 @default.
• W2092686483 hasConcept C502942594 @default.
• W2092686483 hasConcept C55493867 @default.
• W2092686483 hasConcept C57074206 @default.
• W2092686483 hasConcept C62478195 @default.
• W2092686483 hasConcept C86803240 @default.
• W2092686483 hasConcept C95444343 @default.
• W2092686483 hasConceptScore W2092686483C109523444 @default.
• W2092686483 hasConceptScore W2092686483C11960822 @default.
• W2092686483 hasConceptScore W2092686483C185592680 @default.
• W2092686483 hasConceptScore W2092686483C202751555 @default.
• W2092686483 hasConceptScore W2092686483C2779251935 @default.
• W2092686483 hasConceptScore W2092686483C28483552 @default.
• W2092686483 hasConceptScore W2092686483C502942594 @default.
• W2092686483 hasConceptScore W2092686483C55493867 @default.
• W2092686483 hasConceptScore W2092686483C57074206 @default.
• W2092686483 hasConceptScore W2092686483C62478195 @default.
• W2092686483 hasConceptScore W2092686483C86803240 @default.
• W2092686483 hasConceptScore W2092686483C95444343 @default.
• W2092686483 hasIssue "30" @default.
• W2092686483 hasLocation W20926864831 @default.
• W2092686483 hasOpenAccess W2092686483 @default.
• W2092686483 hasPrimaryLocation W20926864831 @default.
• W2092686483 hasRelatedWork W1494554257 @default.
• W2092686483 hasRelatedWork W2081165140 @default.
• W2092686483 hasRelatedWork W2155030722 @default.
• W2092686483 hasRelatedWork W2292402945 @default.
• W2092686483 hasRelatedWork W2602725667 @default.

• next