Figure 4

PI-3K and Akt mediate insulin-induced keratinocyte migration. (A) HaCaT cells were either left untreated or treated with 10^-7 M insulin for the indicated time points, followed by immunoblot analysis of the cell lysates using an Ab that specifically recognizes phosphorylated Akt at Ser 473. This blot was reprobed with an Ab to Akt to ensure equal loading. Insulin increased Akt phosphorylation over time, with peak phosphorylation seen at 5 min. (B) HaCaT cells were either left untreated or treated with 10^-8 M to10^-5 M insulin for 5 min, and then analyzed as in (A). Insulin stimulated phosphorylation of Akt in a dose-dependent manner. (C) HaCaT cells grown to 60–70% confluence were infected with adenovirus expressing constitutively active Akt (Akt-CA), dominant-negative Akt (Akt-DN) or the null adenovirus (Null) by incubating cells with the adenovirus for 5 h. Forty-eight hours after infection, cells were treated with 10^-7 M insulin for 3 min or left untreated. Phosphorylation of Akt and total Akt were detected by immunoblot. The Akt-DN inhibited insulin-induced Akt phosphorylation. (D, E) HaCaT Cells grown to 50–60% confluence were infected with adenovirus expressing Akt-CA, Akt-DN, or null adenovirus, as mentioned above. Forty-eight hours after infection, scratch wounds were made, and cells were treated with10^-7 M insulin; migration distances were measured after 24 h of treatment. Again expression of the Akt-DN inhibited insulin-induced migration. (F) HaCaT cells were incubated with 50 μM of the PI3K inhibitor LY294002, 10^-7 M insulin, or LY294002 for 1 h, followed by treatment with 10^-7 M insulin. The migration distances were measured 24 h after treatment. Insulin induced keratinocyte migration was abolished by pre-treatment with the PI3K inhibitor LY294002. Results are representative of at least three independent experiments. *P < 0.05 vs control; ***P < 0.001 vs control.