Activation of vascular endothelial growth factor receptor 2 in a cellular model of loricrin keratoderma

Abstract

Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been identified in two dominantly inherited skin diseases, Vohwinkel syndrome with ichthyosis and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. We generated stable HaCaT cell lines that express wild-type (WT) loricrin and a mutant form found in Vohwinkel syndrome with ichthyosis, using an ecdysone-inducible promoter system. The cells expressing the mutant loricrin grew more rapidly than those expressing WT loricrin after induction for 5 days. Confocal immunofluorescence microscopy revealed that phospho-Akt occurred in the nucleolus where the mutant loricrin was also located. The level of activity of Akt kinase was about nine times higher in cells with the mutant than in those with WT loricrin. ERK1/2, the epidermal growth factor receptor, vascular endothelial growth factor (VEGF) receptor 2 and Stat3 were all phosphorylated in cells with the mutant loricrin. The docking proteins, Gab1 and c-Cbl, were also tyrosine-phosphorylated in these cells. Furthermore, chromatin immunoprecipitation assays showed that Stat3 protein bound to the VEGF promoter in cells with the mutant. Thus, this study suggests that VEGF release and the subsequent activation of VEGF receptor 2 link loricrin gene mutations to rapid cell proliferation in a cellular model of loricrin keratoderma.

FIGURE 1.

Establishment of HaCaT cell lines that express WT loricrin and mutant loricrin. Clones with the highest level of expression and tightest regulation were selected and named WL-1, WL-13, VL-2, and VL-5. WL-1 and WL-13 cell lines expressed WT loricrin. VL-2 and VL-5 cell lines expressed mutant loricrin. A, representative immunoblots obtained with the anti-V5 and anti-mutant loricrin (Mut Lor) antibodies. Immunoblotting using the anti-V5 antibody revealed that WT loricrin (35 kDa) and mutant loricrin (42 kDa) were expressed to almost the same degree. Immunoblotting using the anti-mutant loricrin antibody revealed that mutant loricrin was expressed only in VL-2 and VL-5 cells. Similar data were obtained in four to six experiments. B, dose dependence of the expression of WT and mutant loricrin in WL-1 and VL-5 cells, respectively. We performed experiments with WL-1 and VL-5 cells using different doses of muristerone A. The amounts of WT loricrin and mutant loricrin were dependent on the dose of muristerone A. Similar data were obtained in four to six experiments. C, representative merge images of the double stainings with anti-BrdUrd (BrdU) antibody (green) and propidium iodide (PI) (red). Scale bars, 100 μm. D, percentage of BrdUrd-positive cells. Ten thousand cells were chosen for the count. BrdUrd-positive cells were counted by three blinded examiners. Histogram vertical axis depicts percentage of BrdUrd-positive cells. Data are represented as mean ± S.E. of five independent cultures in each group. Average percentage of BrdUrd-positive cells was 19.5 ± 2.4 in mock cells, 13.1 ± 1.1 in WL-1 cells, and 24.7 ± 1.8 in VL-5 cells. *, p < 0.05 versus mock and VL-5; **, p < 0.05 versus mock and WL-1, by one-way ANOVA with Tukey-Kramer multiple comparisons test. E, to examine whether there is a difference in proliferation among cell lines, we treated mock, WL-1, and VL-5 cells with 1 mm muristerone A and counted the cells daily using trypan blue staining. VL-5 cells proliferated more actively than the mock cells. WL-1 cells proliferated less actively than the mock cells. Statistical significance was determined using the one-way ANOVA with Dunnett's multiple comparisons test (n = 5). Differences were considered statistically significant at p < 0.05. *, p < 0.05 versus mock and VL-5; **, p < 0.05 versus mock and WL-1.

FIGURE 2.

Expression of mutant loricrin promotes phosphorylation of the EGFR and VEGFR 2. To determine whether either receptor is phosphorylated in mock, WL-1, WL-13, VL-2, and VL-5 cells, we conducted an immunoblot analysis with anti-phospho-EGFR (Tyr-992), anti-phospho-EGFR (Tyr-1068), and anti-phospho-VEGFR 2 (Tyr-1175) antibodies. Both EGFR and VEGFR 2 were phosphorylated in mock, WL-1, WL-13, VL-2, and VL-5 cells 2 h after the addition of muristerone A. EGFR and VEGFR 2 were still phosphorylated in VL-2 and VL-5 cells 20 h after the addition of muristerone A. Similar data were obtained in four to six experiments.

FIGURE 3.

Phosphorylation and activity of Akt after the transfection of VL-5 cells with Δp85. A, representative immunoblot obtained with the anti-phospho-Akt (Ser-473) antibody. Akt was phosphorylated in VL-5 cells 20 h after the addition of muristerone A. Neither mock cells nor WL-1 cells expressed Akt phosphorylated with serine 473 (n = 4). B, assay of Akt kinase activity in cells transiently transfected with SRα-Δp85 plasmid. The transient transfection of Δp85 resulted in a decrease in Akt activity. Open circles depict Akt activity in VL-5 cells not transfected with SRα-Δp85. Open squares represent Akt activity in VL-5 cells after the transfection. Similar data were obtained in five experiments.

FIGURE 4.

Effect of PI3K inhibitors on Akt activity and distribution of phospho-Akt in VL-5 cells. A, to determine whether PI3K is really required for VL-5 cells to proliferate, we used two PI3K inhibitors at various concentrations and conducted an immunoblot analysis. Wortmannin and LY294002 were added immediately after the treatment with 1 μm muristerone A. We also conducted an Akt kinase activity assay. Akt activity is represented as the intensity of bands on the phospho-GSK-3α/β (Ser-21/9) immunoblot. When wortmannin was used at 0.2 μm, phospho-Akt (Thr-308) was not detected. When this concentration was increased to 1 μm, neither phospho-Akt (Ser-473) nor phospho-Akt (Thr-308) was detected. When LY294002 was used at 50 μm, neither phospho-Akt (Ser-473) nor phospho-Akt (Thr-308) was detected. B, density of immunostaining was measured in five experiments and quantified with NIH Image software. The level of Akt activity was nine times higher in VL-5 cells than the control cells (mock and WL-1 cells). C, representative double stainings with anti-phospho-Akt (Ser-473) antibody (green) and anti-V5 antibody (red) of WL-1 and VL-5 cells. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear detection. Lower column of composite images is magnified views of framed rectangles in upper column of composite images. A substantial portion of phospho-Akt (Ser-473) colocalized with aggregates of mutant loricrin (arrows) in VL-5 cells. Scale bars, 80 μm. Mut, mutant; Lor, loricrin.

FIGURE 5.

Mutant loricrin expression stimulates Gab1 and c-Cbl tyrosine phosphorylation. A, mock, WL-1, and VL-5 cells were stimulated with 1 mm muristerone A for 20 h. The cells were lysed and processed for Gab1 immunoprecipitation (IP) followed by anti-phosphotyrosine (PY), anti-p85, and anti-SHP-2 immunoblotting (left panel) (n = 4). The lysates were also subjected to anti-phospho-Gab1 (Tyr-307) and anti-phospho-Gab1 (Tyr-627) immunoblotting (right panel) (n = 4). B, mock, WL-1, and VL-5 cells were stimulated with 1 mm muristerone A for 20 h. The cells were lysed and processed for c-Cbl immunoprecipitation (IP) followed by anti-phosphotyrosine (PY), anti-Grb2, and anti-Shc immunoblotting (left panel) (n = 4). The lysates were also subjected to anti-phospho-c-Cbl (Tyr-731) and anti-phospho-c-Cbl (Tyr-774) immunoblotting (right panel) (n = 4).

FIGURE 6.

Mutant loricrin expression stimulates ERK1/2 phosphorylation. Mock, WL-1, WL-13, VL-2, and VL-5 cells were stimulated with 1 mm muristerone A for 20 h. The cells were lysed and processed for anti-phospho-ERK1/2 (A), anti-phospho-p38 MAPK (MAP kinase) (B), and anti-phospho-SAPK/JNK (C) immunoblotting.

FIGURE 7.

ChIP assays and immunoblotting using anti-phospho-Stat3 antibody. A, ChIP assay was performed in VL-5 cells stimulated with 1 mm muristerone A for 20 h using either an anti-Stat3-specific antibody, an irrelevant antibody (anti-Rho), or no antibody. PCR primers were designed to yield a 130-bp product, which includes the Stat3-binding site (−848) of the VEGF promoter. As a negative control, a PCR using primers for the mouse albumin gene was included in these experiments. Input lanes represent 0.02% of total chromatin used in ChIP assays. Similar data were obtained in five experiments. B, mock, WL-1, and VL-5 cells were stimulated with 1 mm muristerone A for 20 h. The cells were lysed and processed for anti-phospho-Stat3 (Tyr-705) (top) and anti-phospho-Stat3 (Ser-727) (middle) immunoblotting (n = 4).

FIGURE 8.

Mutant loricrin induces release of TGF-α. A, activity of TGF-α in the culture medium (n = 4). The amount of TGF-α in the medium was measured with a solid phase enzyme-amplified sensitivity immunoassay. B, immunoblot obtained with the anti-TGF-α antibody (1:500) representative of five experiments.

FIGURE 9.

Mutant loricrin induces release of VEGF. A, activity of VEGF in the culture medium (n = 4). The amount of VEGF in the medium was measured with a solid phase enzyme-amplified sensitivity immunoassay. B, immunoblot obtained with the anti-VEGF antibody (1:500) representative of five experiments.

FIGURE 10.

Effect of VEGF inhibitors (CBO-P11 and JE-11) plus EGFR tyrosine kinase inhibitor (PD 153035) on VL-5 cells. A, proliferation of VL-5 cells was stimulated with 1 mm muristerone A. 20 μm CBO-P11 plus 1 μm PD 153035 inhibited the muristerone A-induced proliferation of VL-5 cells. Statistical significance was determined using the one-way ANOVA with Dunnett's multiple comparisons test (n = 5). *, **, difference was considered statistically significant at p < 0.05. B, 10 μm CBO-P11 plus 1 μm PD 153035 inhibited the muristerone A-induced proliferation of VL-5 cells. Statistical significance was determined using the one-way ANOVA with Dunnett's multiple comparisons test (n = 5). **, difference was considered statistically significant at p < 0.05. C, immunoblot obtained with the diluted anti-phospho-VEGFR 2 (Tyr-1175) antibody (1:500) representative of four experiments. D, immunoblot obtained with the diluted anti-phospho-VEGFR 2 (Tyr-1175) antibody (1:500) representative of four experiments.