Activation of cAMP-dependent protein kinase is required for optimal alpha-melanocyte-stimulating hormone-induced pigmentation.
The cAMP-dependent pathway has been long presumed to play a critical role in mediating alpha-melanocyte-stimulating hormone (alpha-MSH)-induced pigmentation, but it has never been demonstrated that this pathway is obligatory. In order to determine whether the cAMP-dependent pathway is required for a alpha-MSH-induced pigmentation, we inhibited the activity of cAMP-dependent protein kinase (PKA), the main kinase mediating in this pathway, by introducing a physiologic cAMP-dependent protein kinase inhibitor (PKI) into S91 murine melanoma cells and then measuring pigment response after alpha-MSH stimulation. Cells were stably transfected either with the pMXX-PKI expression vector that encodes the active part of PKI (the amino terminal 1-31 amino acids) under a metallothionein-inducible promoter and the pSV2-Neo expression vector alone. As expected, treatment of transfected cells with 1 microM CdCl2 for 24 h induced the expression of PKI mRNA in cells transfected with both vectors, but not in cells transfected with the pSV2-Neo expression vector alone. Subsequent treatment of these transfected cells with alpha-MSH for 5-6 days in the continual presence of 1 microM CdCl2 resulted in inhibition of PKA activity by 30-40% in cells expressing PKI. Parallel measurements revealed that alpha-MSH-increased melanin content five- to six-fold in control cells transfected with pSV2-Neo alone, while there was only a two-fold increase in PKI-expressing cells, a 40-50% inhibition in alpha-MSH-induced total melanin content. alpha-MSH-induced tyrosinase activity and tyrosinase mRNA and protein levels measured in parallel were also inhibited by 40-50% in PKI-expressing cells compared to control cells transfected with pSV2-Neo alone. Together, these results demonstrate for the first time that activation of PKA through the cAMP-dependent pathway is required for optimal alpha-MSH-induced pigmentation.
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Department of Dermatology, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts, 02118, USA.
Park
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Olaizola-Horn
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Gilchrest
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D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008545:Melanoma; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D010858:Pigmentation / Q000187:drug effects; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured; D000521:alpha-MSH / Q000502:physiology*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C015482:protein kinase modulator; D000521:alpha-MSH; D014442:Monophenol Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases