Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

• Published in: Journal of dermatological science Vol. 25; no. 2; pp. 139 - 149
• Main Authors: Nakamura, Satoshi, Takahashi, Hidetoshi, Kinouchi, Motoshi, Manabe, Akira, Ishida-Yamamoto, Akemi, Hashimoto, Yoshio, Iizuka, Hajime
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ireland Ltd 01.02.2001
• Subjects: Apoptosis; c-Jun N-terminal kinase; Cell Death - drug effects; Cell Death - physiology; Cell Death - radiation effects; Cell Division - drug effects; Cell Division - physiology; Cell Division - radiation effects; Cell Line, Transformed; DNA Fragmentation; Epidermal Growth Factor - pharmacology; Extracellular signal-regulated protein kinase; Humans; JNK Mitogen-Activated Protein Kinases; Keratinocytes - cytology; Keratinocytes - enzymology; Keratinocytes - radiation effects; Keratinocytes - virology; Mitogen-Activated Protein Kinases - metabolism; Mitogen-Activated Protein Kinases - physiology; p38 kinase; p38 Mitogen-Activated Protein Kinases; Phosphorylation - drug effects; Phosphorylation - radiation effects; Simian virus 40 - physiology; Ultraviolet Rays; c-Jun N-terminal kinase; SVHK, SV40-transformed human keratinocyte; Extracellular signal-regulated protein kinase; EGF, epidermal growth factor; JNK, c-JUN N-terminal kinase; UV, ultraviolet; HB-EGF, heparin binding epidermal growth factor-like growth factor; MEK, MAP/ERK kinase; DMEM, Dulbecco's modified Eagles medium; p38 kinase; MAPKK/MKK, MAP kinase kinase; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; TGF, transforming growth factor; MAP kinase, mitogen-activated protein kinase; ROS, reactive oxygen species; Apoptosis
• ISSN: 0923-1811; 1873-569X
• DOI: 10.1016/S0923-1811(00)00123-7

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm 2) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min–2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.