Proliferation and expression of a collagen-associated gene in 11β-HSD1 knockout mouse dermal fibroblasts.

<p>(A) The relative expression of 11β-HSD1 in dermal fibroblasts derived from newborn mouse skin and 1-year-old mouse skin was assessed with rtPCR. GAPDH was used as an internal control (N = 3; **P<0.01, Student's t-test). (B) The expression of 11β-HSD1 in dermal fibroblasts derived from newborn mouse skin and 1-year-old mouse skin was assessed with Western blotting. (C) The relative expression of 11β-HSD1 in dermal fibroblasts derived from wild-type (WT), heterozygous knockout (HT), and homozygous knockout (KO) mouse skin was assessed with rtPCR. GAPDH was used as an internal control (N = 3). **P<0.01, one-way ANOVA followed by the Bonferroni-Dunn test for multiple comparisons. (D) Proliferation of dermal fibroblasts derived from WT, HT, and KO mice was assessed with an MTS assay on days 1, 2, and 3. Day 2 and 3 data were normalized to day 1 data for each group. Bars indicate the mean ± SD [N = 6; *P<0.05, **P<0.01, two-way ANOVA followed by the Bonferroni-Dunn test for multiple comparisons. There was also a significant interaction effect of genotype and time (F = 13.33, P<0.001)]. (E) Proliferation of dermal fibroblasts derived from WT mice treated with 11β-HSD1 selective inhibitor (10 μM) for 3 days was assessed by MTS assay. DMSO was applied as vehicle control. Inhibitor group data were normalized to vehicle group. Bars indicate the mean ± SD (N = 10; *P<0.05 Student's t-test). (F) Western blot analysis of phospho-Akt (Thr308 and Ser473), Akt, phospho-Erk, and Erk in dermal fibroblasts derived from WT and KO mice. Bars show the results of densitometric analysis relative to β-actin. Mean ± SD of each group are shown. (N = 4, *P<0.05, Student's t-test) (G) The MTS assay was used to assess proliferation of dermal fibroblasts derived from WT and KO mice treated with an Akt inhibitor (LY294002, 10 μM) or an Erk inhibitor (U0126, 10 μM). Bars indicate the mean ± SD (N = 6; *P<0.0001, two-way ANOVA followed by the Bonferroni-Dunn test for multiple comparisons.) (H) The relative expression of Col1A1 and MMP-13 in dermal fibroblasts derived from WT, HT, and KO mice. GAPDH was used as an internal control (N = 3; **P<0.01, one-way ANOVA followed by the Bonferroni-Dunn test for multiple comparisons).