During the last few years, controversial results concerning the regulation of tyrosinase, TRP1, and TRP2 mRNA levels have been published. For instance, Abdel-Malek et al. (2
) have shown that α-MSH-induced melanogenesis is accompanied by an increased amount of tyrosinase, TRP1, and TRP2 proteins without any changes in mRNA levels. On the other hand, Kuzumaki et al. (26
) demonstrated that cAMP-elevating agents increase both tyrosinase and TRP1 expression at mRNA levels. In the same way, TRP1 and TRP2 mRNA levels, in mice with different coat color mutations, were shown to tightly correlate with eumelanin synthesis (23
). In the course of investigating the regulation of TRP1 and TRP2 expression during cAMP-induced melanogenesis, we clearly showed in this report that cAMP increases tyrosinase, TRP1, and TRP2 at both protein and mRNA levels. Our results demonstrate a coordinated regulation of tyrosinase, TRP1, and TRP2 expression at mRNA levels during cAMP-induced melanogenesis. Next, we showed that cAMP-elevating agents such as forskolin and α-MSH or expression of the catalytic subunit of PKA stimulate the transcriptional activity of the TRP1 and TRP2 promoters. The role of PKA in the melanogenic pathway was confirmed by transfection with an expression plasmid encoding PKI, which dramatically reduces the effect of forskolin, α-MSH, and PKA. These results clearly demonstrate that α-MSH effects on melanogenesis are mediated through the activation of the cAMP pathway and PKA.
The regulation of tyrosinase, TRP1, and TRP2 promoters by PKA was observed in other human or mouse melanoma cell lines such as G361 or S91 (data not shown). Furthermore, we also observed a stimulation of the melanogenic promoter activities by PKA in normal human melanocytes, emphasizing the physiological relevance of the cAMP effects. It should be noted that human melanocytes express high basal levels of melanogenic proteins (13
), probably because culture conditions of normal melanocytes are unable to prevent a spontaneous differentiation of the cells. Thus, consequent to the high basal melanogenesis, the effect of PKA on tyrosinase, TRP1, and TRP2 promoters appears markedly less pronounced in human melanocytes than in mouse melanoma cells. In other cell types, such as NIH 3T3 mouse fibroblasts, cAMP does not change the activity of the melanogenic promoters (not shown). These results indicate that a cell-specific mechanism is involved in the cAMP response of the melanogenic promoters. We then attempted to localize and to identify the cis
-regulatory elements conferring on TRP1 and TRP2 promoters their cAMP responsiveness. We demonstrated that the M box and the E box upstream of the TATA box play a key role in the cAMP response of TRP1 and TRP2 promoters. Recently, we also reported that the cAMP response of the tyrosinase promoter involved a M box and an E box surrounding the TATA box (5
). Thus, the mechanisms of regulation of tyrosinase, TRP1, and TRP2 promoters by cAMP appear to rely on similar regulatory elements. However, it should be noted that in the tyrosinase promoter, the cAMP-sensitive E box located at the initiator site binds tightly to microphthalmia and is absolutely necessary for the cAMP response. In TRP1 and TRP2 promoters, the M boxes bind avidly to microphthalmia, but the E boxes, involved in the cAMP response, interact weakly with microphthalmia. Thus, microphthalmia appears to require the core motif CATGTG to provide a strong interaction with DNA, since this motif is found in all of the M boxes and in the tyrosinase E box, while TRP1 and TRP2 E boxes have CAAGTG and CAATTG, respectively, as core sequences. We cannot exclude the possibility that in intact cells and in the context of the intact promoter, microphthalmia tightly binds to TRP1 and TRP2 E boxes. However, mutations of TRP1 and TRP2 E boxes moderately affect cAMP sensitivity and the microphthalmia effect on the promoters, suggesting that in intact cells, microphthalmia does not interact strongly with these motifs. In the TRP2 promoter, a CRE-like sequence also participates in the cAMP response, indicating that transcription factors of the CREB family are involved in the stimulation of the TRP2 promoter activity by cAMP. However, this CRE motif has to cooperate with the M box to give full cAMP sensitivity to the TRP2 promoter. Noteworthy, the same cis
-acting elements (M and E boxes) were thought to mediate the tissue-specific expression (4
) and the cAMP responses of the tyrosinase, TRP1, and TRP2 genes. Thus, it should be considered that the cAMP pathway participates in the regulation of the tissue-specific expression of the melanocyte-specific genes.
Although microphthalmia and M boxes play a pivotal role in the regulation of tyrosinase, TRP1, and TRP2 promoter activities by cAMP, it appears that each promoter responds to cAMP through specific mechanisms because of the relative position of the regulatory elements or the intrinsic nature of the elements cooperating with the M box. This is particularly true for the TRP2 promoter, which contains a CRE-like element involved in cAMP sensitivity; in tyrosinase and TRP1 promoters, no CRE participates in the cAMP response. These observations reveal, besides a common regulatory mechanism, the existence of specific processes involved in the control of tyrosinase, TRP1, and TRP2 gene expression that could allow a differential expression pattern of the melanogenic enzymes under particular physiological and pathological conditions.
We have previously demonstrated that cAMP increases microphthalmia binding to the M box and to the E box. Since microphthalmia has a strong stimulating effect on the tyrosinase promoter, we have proposed that microphthalmia, through the binding to M and E boxes, mediates the effects of cAMP on tyrosinase gene expression (5
). In the present report, we clearly showed a strong stimulation of TRP1 and TRP2 promoter activities by microphthalmia. Consistently, microphthalmia or its human homolog MITF (microphthalmia-associated transcription factor) was shown to transactivate the TRP1 promoter (39
). However, the TRP2 promoter has been shown to be unresponsive to MITF (39
), while we clearly showed in this report that microphthalmia stimulates the TRP2 promoter activity. This discrepancy could be explained either by specific behaviors of human versus mouse microphthalmia or by the different cellular contexts.
Interestingly, the effect of microphthalmia on different TRP1 and TRP2 promoter constructs tightly correlates with their cAMP responsiveness. Further, we showed that cAMP increases the binding of microphthalmia to TRP1 and TRP2 M boxes. These data strongly suggest that the effect of cAMP on TRP1 and TRP2 gene expression is mediated through an increased interaction of microphthalmia with M boxes, thereby leading to a transactivation of TRP1 and TRP2 promoters. At least two hypothesis could explain the stimulation of microphthalmia binding on the M box after cAMP treatment. cAMP could increase either microphthalmia expression or affinity of microphthalmia for its target sequences. Previous immunofluorescence studies with antimicrophthalmia antibody did not show a stimulation of microphthalmia expression after 24 h with forskolin. Although this result does not support the first hypothesis, we have reassessed the effect of cAMP on microphthalmia expression. Metabolic labeling followed by immunoprecipitation with specific antibody to microphthalmia demonstrates that cAMP increases microphthalmia expression. Microphthalmia appears as two bands around 60 and 70 kDa. The lower mobility of the higher band results from its phosphorylation on serine 73 by mitogen-activated protein kinases (16
). The maximal expression of microphthalmia was obtained after 3 h of forskolin treatment, and consistent with our immunofluorescence studies, the level of microphthalmia returned near the basal level after 24 h with forskolin. The presence of a CRE in the microphthalmia promoter (10
) suggests that PKA regulates microphthalmia expression through the classical pathway involving transcription factors of the CREB family. However, this hypothesis remains to be demonstrated. Interestingly, Rungta et al. (33
) showed that α-MSH treatment significantly increases the tyrosinase mRNA levels within 16 h, and we previously observed an effect of forskolin on tyrosinase promoter after 6 h. Further, careful examination of the report of Abdel-Malek et al. (2
) shows that the TRP1 mRNA amount was increased after 6 h with α-MSH. Thus, if we compare the kinetics of microphthalmia induction with those of tyrosinase and TRP1, the upregulation of the melanogenic enzymes is observed clearly after the maximal expression of microphthalmia. These observations are consistent with our former hypothesis suggesting that microphthalmia plays a key role in the stimulation of melanogenic gene expression.
Considering the physiological aspect of our findings, it should be mentioned that in humans, melanogenesis is stimulated by UVB, which upregulates the production of α-MSH by epidermal keratinocytes. Further, subcutaneous injection of α-MSH has been shown to stimulate local pigmentation (28
). Thus, we can hypothesize that α-MSH, through the binding to its receptor coupled to the G protein αs
and adenylate cyclase, increases the cAMP content in melanocytes. Then cAMP, through the activation of PKA, leads to an augmentation of microphthalmia expression. Consequently, the amount of microphthalmia bound to M or E boxes increases resulting in a stimulation of the melanogenic promoter activities. Taken together, our results disclosed the cascade of molecular events involved in the regulation of the melanogenic genes that could be of paramount importance in the control of skin pigmentation.