MITF mediates cAMP-induced protein kinase C-beta expression in human melanocytes.
The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-beta (protein kinase C-beta), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-beta were induced by 24 h. To determine whether MITF modulates PKC-beta expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-beta protein was reduced by 63+/-3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-beta promoter-CAT (chloramphenicol acetyltransferase) reporter construct (PKC-beta/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-beta/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-beta, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis.
Park
Hee-Young
HY
Department of Dermatology, Boston University School of Medicine, 609 Albany Street J-205, Boston, MA 02118-2394, USA.
Wu
Christina
C
Yonemoto
Laurie
L
Murphy-Smith
Melissa
M
Wu
Heng
H
Stachur
Christina M
CM
Gilchrest
Barbara A
BA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects*; D006801:Humans; D008544:Melanocytes / Q000187:drug effects* / Q000201:enzymology*; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
D051739:Microphthalmia-Associated Transcription Factor; D020033:Protein Isoforms; D012333:RNA, Messenger; D000242:Cyclic AMP; D014442:Monophenol Monooxygenase; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D015056:1-Methyl-3-isobutylxanthine