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First published online 17 January 2006
doi: 10.1242/jcs.02744

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The role of protein kinase C activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation

¹ Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, 52900 Israel
² Department of Dermatology, Ehime University School of Medicine, Onsen-gun, Ehime 791-0295, Japan
³ Osaka University Graduate School of Medicine, 2-2 Yamadaoka Suita, Osaka 545-8585, Japan
⁴ Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

View larger version (35K): [in a new window]   Fig. 1. Insulin induces STAT3 activation and nuclear translocation in mouse primary keratinocytes. (A) Keratinocytes following 5 days in culture were stimulated with 10–7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-p-tyr-STAT3 (top panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). Relative optical density of four representative blots is presented in arbitrary units (mean ± s.d.). (B) Keratinocytes were plated on glass slides. Cultures (5 days old) were stimulated with insulin for 5 minutes, fixed in ethanol and analyzed by immunofluorescence, using anti-p-tyr-STAT3, followed by FITC-conjugated secondary antibody. Cells were viewed by confocal microscopy. (C) Keratinocytes were stimulated with 10–7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-IRß antibody (top panel) and with anti-IGF 1Rß antibody (middle panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). The experiment was repeated twice. Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 2. Protein kinase C (PKC) specifically associates with STAT3. Keratinocytes (5 day cultures) were infected with isoform-specific PKC , , and recombinant adenoviruses, or with adenovirus encoding ß-galactosidase (ß-Gal) as a control, for 1 hour. OE, overexpression. Following infection, cells were incubated for 24 hours, then extracted and immunoprecipitated (IP) with antibodies against PKC, PKC, PKC and PKC. Immunoprecipitates were subjected to western blot analysis using isoform-specific anti-PKCs or anti-STAT3 antibodies. Results presented are representative of at least three experiments.

View larger version (38K): [in a new window]   Fig. 3. Insulin-induced PKC activation regulates STAT3 association and phosphorylation. (A) Keratinocytes were untreated (–) or treated with insulin for 5 minutes (+). PKC immunoprecipitates were subjected to western blot analysis using antibodies against STAT3, anti-phosphotyrosine-705-STAT3 (p-tyr), anti-phosphoserine-727-STAT3 (p-ser) and anti-PKC. Relative optical densities of the blots are presented in arbitrary units. Relative optical density of four blots is presented in arbitrary units (mean ± s.d.). (B) Primary keratinocytes were either untreated or infected for 1 hour with isoform-specific PKC recombinant adenovirus, or with ß-galactosidase (ß-Gal) adenovirus as control. Control cells were either untreated (C) or stimulated with insulin for 5 minutes (Ins 5'). Cells were extracted and immunoprecipitated (IP) with isoform-specific PKC antibodies. The immunoprecipitates were subjected to western blot analysis using anti-PKCs, anti-STAT3 or anti-phosphoserine-727-STAT3 (STAT p-ser) antibodies. Experiments were repeated three times.

View larger version (35K): [in a new window]   Fig. 4. Insulin-induced PKC-STAT3 association and activation depends on PKC activity. (A) Keratinocytes were infected (OE) for 1 hour with recombinant ß-Gal (control), wild-type (WT) PKC and dominant-negative PKC (DN PKC) adenovirus constructs. Following infection, cells were incubated for 18 hours, and left untreated (–) or stimulated with insulin for 5 and 30 minutes. STAT3 immunoprecipitates were subjected to western blot analysis and probed with anti-PKC and anti-STAT3 antibodies. (B) Keratinocytes were either infected for 1 hour with ß-Gal or WT STAT3 adenovirus constructs, or double infected with DN PKC followed by WT STAT3 recombinant adenovirus infection. Following infection, cells were incubated for 24 hours and overexpressing cells were untreated (–) or stimulated with insulin for 5 and 30 minutes. PKC immunoprecipitates were subjected to western blot analysis and probed with anti-phosphoserine-727-STAT3 (STAT3-p-ser), anti-PKC and anti-STAT3 antibodies. (C) Keratinocytes were either uninfected (–) or infected (OE) for 1 hour with recombinant dominant-negative PKC (DN PKC) adenovirus. After 24 hours, cells were left untreated (–) or treated with insulin for 5 minutes. PKC immunoprecipitates were subjected to western blot analysis and probed with anti-phosphotyrosine-705-STAT3 (STAT3-p-tyr) and anti-PKC antibodies. Relative optical density of three blots are presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times.

View larger version (21K): [in a new window]   Fig. 5. Overexpression of WT STAT3 and DN STAT3 using recombinant adenoviruses. (A,B,C) Primary keratinocytes were infected for 1 hour using ß-galactosidase (ß-Gal), WT STAT3 and DN STAT3 recombinant adenoviruses containing a hemagglutinin (HA) tag, and experiments were conducted 24 hours after infection. (A) Total cell lysates were subjected to western blot analysis using anti-STAT3 antibody. (B) Overexpressing cells were either untreated (–) or treated with insulin for 5, 15 and 30 minutes. Lysates were immunoprecipitated with anti-HA antibody and immunoprecipitates analyzed by western blotting, using a anti-p-tyr-STAT3 antibody. (C) Overexpressing cells were treated with insulin for 5 and 30 minutes. PKC immunoprecipitates were analyzed by western blotting, using anti-HA and anti-STAT3 antibodies. Equal loading of gels was confirmed by reblotting with PKC antibody. Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were either infected (OE) for 1 hour with WT STAT3 adenovirus constructs or double infected with DN PKC followed by WT STAT3 recombinant adenovirus infection. 24 hours following infection, cells were untreated (–) or treated with insulin for 5 minutes. HA immunoprecipitates were subjected to western blot analysis and probed with anti-phosphotyrosine and anti-HA antibodies. Experiments were repeated at least three times.

View larger version (14K): [in a new window]   Fig. 6. DN STAT3 blocks insulin-induced PKC activation. Keratinocytes were either uninfected (–), or infected with DN STAT3 adenovirus. Following infection (24 hours), cells were untreated, or stimulated with 10–7 M insulin (Ins) for the designated times (5 or 30 minutes). (A) PKC or (B) STAT3 were immunoprecipitated from protein extracts using specific anti-PKC and anti-STAT3 antibodies. PKC and STAT3 immunoprecipitates were analyzed for PKC activity using an in vitro kinase assay as described in Materials and Methods. Each bar represents the mean ± s.d. of three determinations in three separate experiments. Values are expressed as picomoles of ATP per dish per minute. Highly significant differences were found (P<0.0001) for both IP PKC and STAT3 compared with levels in the respective controls at 5 minutes.

View larger version (67K): [in a new window]   Fig. 7. Insulin-induced STAT3 nuclear translocation is abrogated by PKC inhibition. Primary keratinocytes were plated on glass slides and maintained for 5 days in low-Ca2+ MEM (0.05 mM) until they reached 80% confluence. (A) Cells were left untreated (upper panel) or pre-treated with 5 µM rottlerin (Rott) for 7 minutes (lower panel), followed by 10–7 M insulin for 5 and 30 minutes (Ins). Cells were fixed in methanol, washed and air-dried. Cultures were analyzed by immunofluorescence using anti-phospho-tyr-705-STAT3 (STAT3-p-tyr) antibody, followed by FITC-conjugated secondary antibody. Slides were viewed by confocal microscopy. (B) Cells were untreated (–) or treated with insulin (Ins) for 5 and 30 minutes. Following treatment, cells were fixed in methanol and analyzed by immunofluorescence using anti-PKC antibody followed by FITC conjugated secondary antibody and scanned by confocal microscope. Arrow in middle panel indicates translocation of PKC to the perinuclear membranes. Experiments were repeated at least three times. Bar, 20 µm.

View larger version (21K): [in a new window]   Fig. 8. Overexpression of serine mutant STAT3 using recombinant adenovirus. (A) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding ß-Gal and STAT3 serine mutant (SmS). After 18 hours, total cell lysates were subjected to western blot analysis utilizing anti-STAT3 (top panel), anti-STAT3-p-tyr (middle panel) and STAT3-p-ser (bottom panel) antibodies. (B,C) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding WT STAT3, DN STAT3 and STAT3 serine mutant (SmS). After 24 hours, cells were (B) left untreated or (C) stimulated with insulin for the designated time periods (0, 5, 15 or 30 minutes). HA immunoprecipitates were subjected to western blot analysis and probed with anti-STAT3-p-tyr (top panel), anti-STAT3-p-Ser (middle panel) and anti-STAT3 (bottom panel) antibodies. Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding WT STAT3 and STAT3 serine mutant (SmS). Cells were stimulated with insulin for 5 and 30 minutes. Lysates were immunoprecipitated with PKC antibody and analyzed by western blotting, using anti-HA. Equal loading of gels was confirmed by reblotting with PKC antibody. Relative optical density of blots is presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times.

View larger version (25K): [in a new window]   Fig. 9. Effects of overexpression of PKC and STAT3 on keratinocytes proliferation and cell death. Keratinocytes were infected for 1 hour with (A) recombinant adenoviruses ß-Gal, PKC, WT STAT3, DN STAT3 or double infected with DN PKC followed by WT STAT3 or with DN STAT3 followed by WT PKC infection. (B) with recombinant adenoviruses ß-Gal, PKC, DN PKC, WT STAT3, DN STAT3 and STAT3 serine mutant (SmS) and un-treated (–) or treated (+) with insulin. Twenty-four hours following infection, cell proliferation was analyzed by [3H]thymidine incorporation as described in Materials and Methods. Results are presented as cpm/µg protein. Each bar represents the mean of three determinations in a plate from the same culture. Experiments were repeated at least three times. (mean ± s.d.). Significant differences were observed between ß-gal control values and those for WT STAT3 (P<0.001) and PKC (P<0.0005). (C) Keratinocytes were infected with recombinant adenoviruses encoding ß-Gal, PKC, WT STAT3, DN STAT3 and STAT3 serine mutant (SmS). Following infection (24 hours) FACS cell-cycle analysis of propidium-iodide-stained keratinocytes was performed. Apoptotic cell death was defined as the sub-G1 population (Apo, percentage of sub-G1 population) and proliferation was identified as the population of S-phase cells (S, percentage of S-phase population).

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