G Protein betagamma subunits augment UVB-induced apoptosis by stimulating the release of soluble heparin-binding epidermal growth factor from human keratinocytes.
UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, including MAPKs. The betagamma subunit of the heterotrimeric GTP-binding protein (Gbetagamma) was found to mediate UV-induced p38 activation via epidermal growth factor receptor (EGFR). However, it is not known how Gbetagamma mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human keratinocytes with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like growth factor (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal keratinocytes and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of Gbeta(1)gamma(2) increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of Gbetagamma by the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2ct) produced the opposite effect. The activation of Src increased UVB-induced, Gbetagamma-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of Gbetagamma increased UVB-induced apoptosis, and the overexpression of GRK2ct decreased this apoptosis. We conclude that Gbetagamma mediates UVB-induced human keratinocyte apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.
Seo
Miran
M
Department of Biochemistry and Molecular Biology and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-779.
Lee
Mi-Jeong
MJ
Heo
Jin Hee
JH
Lee
Yun-Il
YI
Kim
Yeni
Y
Kim
So-Young
SY
Lee
Eun-So
ES
Juhnn
Yong-Sung
YS
D017209:Apoptosis; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000502:physiology*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D017077:Culture Media, Conditioned; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases