Epidermal growth factor receptor is a critical mediator of ultraviolet B irradiation-induced signal transduction in immortalized human keratinocyte HaCaT cells

Abstract

Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin.

Figure 1

PD169540 inhibits both EGF and UVB activation of EGFR in human keratinocytes. HaCaT cells were pretreated with 200 nmol/L PD169540 (+PD) or with vehicle DMSO (−PD) for 16 hours before treatment with EGF (10 ng/ml) or UVB irradiation (40 mJ/cm²). Whole cell lysates were prepared from cells treated with 10 ng/ml of EGF for 10 minutes or 15 minutes after UVB irradiation (40 mJ/cm²). Untreated cells were used as control. EGFRs were immunoprecipitated from the whole cell lysates and subjected to SDS-PAGE and Western blot using anti-phosphotyrosine antibody. A duplicate gel was probed with anti-EGFR antibody. Results are representative of three experiments.

Figure 2

EGFR mediates UVB activation of ERK, JNK, and p38 in human keratinocytes. HaCaT cells were pretreated with PD169540 (+PD, 200 nmol/L) or with DMSO (−PD) for 16 hours, UVB-irradiated (40 mJ/cm²), and whole cell lysates were prepared 15 minutes later. A: UVB activation of p44 and p42 ERK was analyzed by Western blot using total and phospho-ERK antibodies and quantified by STORM PhosphorImager. Data expressed as fold change relative to levels in nonirradiated, untreated control cells (CTRL). N = 4, *P < 0.05 when compared with UV-irradiated −PD samples (Student’s t-test, same below). B: UVB activation of p54 and p46 JNK were analyzed by Western blot using total and phospho-JNK antibodies and quantified by STORM PhosphorImager. Data are presented as fold change relative to levels in nonirradiated, untreated control cells (CTRL). N = 5, *P < 0.05. C: UVB activation of p38 was analyzed by Western blot using total and phospho-p38 antibodies and quantified by STORM PhosphorImager. Data are presented as fold change relative to levels in sham-irradiated, untreated control cells (CTRL). N = 5, *P < 0.05.

Figure 3

EGFR mediates UVB induction of c-Fos and c-Jun mRNA in human keratinocytes. Human HaCaT cells were treated with PD169540 (+PD, 200 nmol/L) or DMSO vehicle (−PD) for 16 hours before UVB irradiation (50 mJ/cm²) and total RNA was isolated. Cells were harvested at the indicated time points after UVB irradiation. c-Fos (A) and c-Jun (B) mRNA analysis was performed by real-time RT-PCR. Data are expressed as fold change relative to levels in untreated, sham-irradiated controls (0 time). A representative graph from three independent experiments is shown.

Figure 4

EGFR mediates UVB-induced phosphorylation of AKT, PKC, and PKA substrates in human keratinocytes. Human HaCaT cells were treated with PD169540 (+PD, 200 nmol/L) or DMSO vehicle (−PD) for 16 hours before UVB irradiation (50 mJ/cm²). Cells were harvested at the indicated time points, and whole cell lysates were subjected to Western blot probed with phospho-AKT substrate antibody (A), phospho-PKC substrate antibody (B), and phospho-PKA substrate antibody (C). Apparent molecular weights (MW) are indicated to the left of each band. Data are representative Western blots from three independent experiments.

Figure 5

EGFR-dependent ERK JNK activation in B82 cells. B82 cells lacking EGFR and B82K⁺ cells expressing EGFR were UVB (50 mJ/cm²)- or sham-irradiated, and whole cell lysates were prepared 15 minutes after treatment. A: p42 and p44 ERK were analyzed by Western blot using phospho-ERK antibody and quantified by STORM PhosphorImager. Data are presented as fold change relative to sham-irradiated cells. N = 3, *P < 0.05. B: p54 and p46 JNK were analyzed by Western blot using phospho-JNK antibody and quantified by STORM PhosphorImager. Data are presented as fold change relative to levels in sham-irradiated cells. N = 3, *P < 0.05.

Figure 6

UVB induction of c-Fos and c-Jun protein requires EGFR. B82 lacking EGFR and B82K⁺ EGFR-expressing cells were UVB-irradiated (50 mJ/cm²) or sham-irradiated, and whole cell lysate was prepared at the indicated time points after UVB irradiation. c-Fos (A) and c-Jun (B) protein levels were quantified by SDS-PAGE/Western blot at the indicated times after UVB. Data are presented as fold change relative to levels in sham-irradiated cells. N = 3, *P < 0.05.