Ao Y, Park H Y, Olaizola-Horn S, Gilchrest B A
Department of Dermatology, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts, 02118, USA.
Exp Cell Res. 1998 Oct 10;244(1):117-24. doi: 10.1006/excr.1998.4086.
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.
长期以来，人们一直认为环磷酸腺苷（cAMP）依赖性途径在介导α-黑素细胞刺激素（α-MSH）诱导的色素沉着过程中起关键作用，但从未有人证明该途径是必不可少的。为了确定α-MSH诱导的色素沉着是否需要cAMP依赖性途径，我们通过将生理性cAMP依赖性蛋白激酶抑制剂（PKI）导入S91小鼠黑色素瘤细胞中，抑制该途径中主要的激酶——cAMP依赖性蛋白激酶（PKA）的活性，然后在α-MSH刺激后测量色素反应。细胞分别用pMXX-PKI表达载体和单独的pSV2-Neo表达载体进行稳定转染，其中pMXX-PKI表达载体在金属硫蛋白诱导型启动子下编码PKI的活性部分（氨基末端1-31个氨基酸）。正如预期的那样，用1μM氯化镉（CdCl2）处理转染细胞24小时，可诱导两种载体转染的细胞中PKI mRNA的表达，但单独用pSV2-Neo表达载体转染的细胞中则不会。随后，在持续存在1μM CdCl2的情况下，用α-MSH处理这些转染细胞5-6天，结果显示表达PKI的细胞中PKA活性被抑制了30-40%。平行测量结果表明，单独用pSV2-Neo转染的对照细胞中，α-MSH可使黑色素含量增加五至六倍，而在表达PKI的细胞中仅增加两倍，α-MSH诱导的总黑色素含量受到40-50%的抑制。与单独用pSV2-Neo转染的对照细胞相比，表达PKI的细胞中平行测量的α-MSH诱导的酪氨酸酶活性、酪氨酸酶mRNA和蛋白水平也受到40-50%的抑制。总之，这些结果首次证明，通过cAMP依赖性途径激活PKA是α-MSH诱导色素沉着达到最佳效果所必需的。
