Cyclic AMP differentially regulates cell proliferation of normal human keratinocytes through ERK activation depending on the expression pattern of B-Raf.
Intracellular cyclic AMP (cAMP) increased by extracellular stimuli induces various biological effects, such as cell proliferation, differentiation, and migration. Previous reports regarding the effect of cAMP on keratinocyte proliferation are contradictory and indicate that the effect apparently depends on cellular density. Recent studies have revealed that cAMP signaling regulates cell proliferation by modulating mitogen-activated protein kinase (MAPK) activity. The precise mechanism by which cAMP affects keratinocyte proliferation and/or the crosstalk between the cAMP and MAPK signaling pathways, however, remain to be determined. Using normal human keratinocytes (NHK), we investigated the effect of cAMP on keratinocyte proliferation and its molecular mechanism in terms of cellular density. In confluent NHK, cyclic AMP decreased extracellular regulated kinase (ERK) phosphorylation and cell proliferation in a Ras-independent and Rap1-dependent manner. The decreased cell proliferation by cAMP was blocked by the MEK-1 inhibitor, PD98059. In contrast, in subconfluent NHK, cAMP increased ERK phosphorylation and cell proliferation. Western blot analysis revealed that NHK expressed B-Raf and Rap-1. Although both 95 kDa and 62 kDa B-Raf isoforms were expressed in subconfluent NHK, only 62 kDa B-Raf was detected in confluent NHK. Transfection of 95 kDa B-Raf into confluent NHK resulted in a cAMP-dependent increase in ERK phosphorylation and cell proliferation. These findings indicate that differential expression of B-Raf isoforms is critical for cAMP-dependent regulation of NHK proliferation that depends on phosphorylation of ERK.
Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaoka higashi, Asahikawa, Hokkaido, Japan. ht@asahikawa-med.ac.jp
Hidetoshi
H
Takahashi
Masaru
M
Honma
Yuki
Y
Miyauchi
Satoshi
S
Nakamura
Akemi
A
Ishida-Yamamoto
Hajime
H
Iizuka
D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D015398:Signal Transduction; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D005576:Colforsin; D002772:Cholera Toxin; D000242:Cyclic AMP; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020827:rap1 GTP-Binding Proteins; D004837:Epinephrine