Figure 3.

TAK1 promotes cell survival through the SCF/c-Kit/PKBα pathway. (a and b) Expression level of SCF (a) protein and (b) mRNA in human keratinocytes transduced with either control (K_CTRL) or TAK1 siRNA (K_TAK1). SCF protein levels were determined from culture medium of K_CTRL and K_TAK1 by ELISA. Platelet-derived growth factor (PDGF)-AA levels were also measured as a control. Real-time qPCR revealed the relative gene expression of SCF and PDGF-AA in K_TAK1 and K_CTRL. Ribosomal L27 was used for normalization. Values (mean±S.D.) were from five independent experiments. (c) Expression levels of SCF (upper panel) and TAK1 (lower panel) proteins in K_CTRL and K_TAK1 transfected with either an empty vector or an expression vector encoding a TAK1 cDNA with a silent third-codon mutation in the siRNA targeting region (upper panel). Two independent transfections are shown. SCF and TAK1 proteins were measured by ELISA and immunoblot, respectively. β-Tubulin was used as a loading and transfer control. (d) Immunoblot analysis of total and phosphorylated c-Kit and its downstream mediators of the PI3K/PKBα pathway in K_CTRL and K_TAK1 epidermis from OTCs constructed with underlying collagen. The level of p(Y721)c-Kit, the activated form of the SCF receptor, was measured in K_CTRL and K_TAK1 epidermis. Total c-Kit, PKBα and Coomassie-stained blots were used as loading and transfer controls. (e) Quantification of Ki67 and TUNEL-positive K_CTRL and K_TAK1 in OTCs supplemented with either vehicle (PBS) as a control or recombinant SCF (rec. SCF). Mean numbers of proliferating and apoptotic cells were numerated after detection by anti-Ki67 antibody or TUNEL, respectively. Values are means from five standardized microscopic fields per section, performed on seven sections from three independent OTCs. (f) Immunodetection of cleaved caspase-3 (apoptotic marker) and PCNA (proliferation marker) in the epithelia from K_CTRL- and K_TAK1-derived OTCs treated with either PBS (−), anti-PDGF antibody or recombinant SCF (+). β-Tubulin served as a transfer and loading control. Values below each band represent the mean fold differences (n=3) in expression level compared with K_CTRL, which was assigned the value of 1. (g and h) TAK1 regulates SCF expression via MKK7/JNK/c-Jun pathway. Expression level of SCF (g) mRNA and (h) protein in K_CTRL treated with either DMSO (vehicle control) or specific inhibitors of the indicated kinases in the presence of TNF-α (10 ng/ml). The various kinase inhibitors were NF-κB (I): BAY 11-7082; NF-κB (II): SN50; JNK: 1,9-pyrazoloanthrone; p38: (2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one); and ERK1/2: PD98059. Ribosomal L27 was used for normalization in qPCR. Protein levels were determined by ELISA. (i) Transactivation assay in keratinocytes co-transfected with a luciferase reporter gene driven by the human SCF promoter (pGL-hSCFpro-luciferase), cDNA encoding for indicated constitutively active (ca) kinases and pEF1-β-galactosidase as control for transfection efficiency. Luciferase activity was measured, and normalized reporter activity is shown as fold induction compared with reporter construct-transfected K_CTRL (control). The SCF promoter reporter construct with a mutated AP-1 site is denoted pGL-mSCFpro-luciferase. Values (mean±S.D.) are from three independent experiments. (j) Relative SCF mRNA expression in K_CTRL transfected with increasing amount of dominant-negative c-Jun (TAM67). Ribosomal L27 was used for normalization. (k) EMSA of the human SCF AP-1-binding site. Biotin-labeled SCF AP-1 binding sequence was incubated with nuclear extract isolated from K_CTRL. NSC denotes non-specific competitor, a scrambled AP-1-binding sequence. SC denotes non-labeled consensus AP-1 sequence (conAP-1). As positive control, conAP-1 was used. Mutated SCF AP-1 site is denoted as SCF mAP-1. (l) Human SCF is a direct AP-1 target gene. Chromatin immunoprecipitation was done in K_CTRL and K_TAK1 using pre-immune IgG (pre) or antibody against phospho-c-Jun (Ab). The promoter region with the AP-1-binding site was immunoprecipitated and specifically amplified in K_CTRL using Ab. No amplified signal was obtained in K_TAK1 or using pre-immune IgG (pre). A control region upstream of the AP-1-binding site served as a negative control. M: 100-bp DNA marker