Involvement of FKHRL1 in melanoma cell survival and death.

Melanoma is a highly aggressive tumour characterized by a strong resistance to apoptotic stimuli that give rise to a selective advantage for tumour progression and metastasis formation. Therefore, it is of paramount importance to better understand the mechanisms involved in this resistance to apoptosis. In this report, we focused our attention on FKHRL1, a member of the forkhead family of transcription factors, which controls expression of genes involved in cell cycle progression and apoptosis. In melanoma cells, we show that IGF1, which exerts pro-survival properties, induces the phosphorylation and nuclear exclusion of FKHRL1 in a PI3K/AKT-dependent pathway. Moreover, we observe that over-expression of a non-phosphorylable mutant of FKHRL1 (FKHRL1-TM), constitutively localized to the nucleus, promotes apoptotic cell death of melanoma cells. Finally, we find that FKHRL1-TM decreases the expression of survivin, a member of the inhibitor of apoptosis protein and that survivin re-expression partially rescues the deleterious effects of FKHRL1. Taken together, these findings reveal, in melanoma cells, that endogenous FKHRL1 is a downstream target of the PI3K/AKT pathway and suggest that the phosphorylation of this transcription factor may be involved in the pro-survival effects of growth factors such as IGF1. On the other hand, forced nuclear localization of FKHRL1 decreases melanoma cell growth and may serve as a therapeutic strategy against melanoma.

PI3K mediates protection against TRAIL-induced apoptosis in primary human melanocytes.

Melanocytes are cells of the epidermis that synthesize melanin, which is responsible for skin pigmentation. Transformation of melanocytes leads to melanoma, a highly aggressive neoplasm, which displays resistance to apoptosis. In this report, we demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), which was thought to kill only transformed cells, promotes very efficiently apoptosis of primary human melanocytes, leading to activation of caspases 8, 9 and 3, and the cleavage of vital proteins. Further, we show that stem cell factor (SCF), a physiologic melanocyte growth factor that activates both the phosphatidyl-inositol-3 kinase (PI3K) and the extracellular regulated kinase (ERK) pathways, strongly protects melanocytes from TRAIL and staurosporine killing. Interestingly, inhibition of PI3K or its downstream target AKT completely blocks the antiapoptotic effect of SCF, while inhibition of ERK has only a moderate effect. Our data indicate that protection evoked by SCF/PI3K/AKT cascade is not mediated by an increase in the intracellular level of FLIP. Further, only a sustained PI3K activity can protect melanocytes from apoptosis, thereby indicating that the PI3K/AKT pathway plays a pivotal role in melanocyte survival. The results gathered in this report bring new information on the molecular mechanisms involved in primary melanocyte apoptosis and survival that would help to better understand the process by which melanomas acquire their resistance to apoptosis.

Rab27a: A key to melanosome transport in human melanocytes.

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

Regulation of the microphthalmia-associated transcription factor gene by the Waardenburg syndrome type 4 gene, SOX10.

The absence of melanocytes from the cochlea and epidermis is responsible of deafness and hypopigmentation, two symptoms shared by the four Waardenburg syndrome (WS) subtypes. Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation. Mutations, which impair MITF function or expression, result in an abnormal melanocyte development leading to the WS2. WS1 and WS3 are caused by mutation in the gene encoding the transcription factor Pax3, which regulates MITF expression. Recently, mutations in SOX10, a gene encoding a SRY-related transcription factor, have been reported in patients with WS4. However, the molecular basis of the defective melanocyte development in these patients remained to be elucidated. In the present report, we demonstrate that Sox10 is a strong activator of the MITF promoter, and we identify a Sox10 binding site between -264 and -266 of the MITF promoter. Finally, we show that three SOX10 mutations found in WS4 abolish the transcriptional activity of the resulting Sox10 proteins toward the MITF promoter. Taken together, our observations bring new and meaningful information concerning the molecular process that leads to a defective melanocyte development in WS4 patients with SOX10 mutations.

TFE3, a transcription factor homologous to microphthalmia, is a potential transcriptional activator of tyrosinase and TyrpI genes.

Microphthalmia gene encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor involved in the development of the melanocyte lineage and plays a key role in the transcriptional regulation of the melanogenic enzymes, tyrosinase and TyrpI. Recently, we have shown that Microphthalmia mediates the melanogenic effects elicited by alphaMSH that up-regulates the expression of tyrosinase through the activation of the cAMP pathway. Therefore, Microphthalmia appears as a principal gene in melanocyte development and functioning. Among the transcription factors of the bHLH-Zip family, TFE3 and TFEB show a remarkably elevated homology with Microphthalmia. These observations prompted us to investigate the role of TFE3 and TFEB in the regulation of tyrosinase and TyrpI gene transcription. We show in this report that overexpression of TFE3 stimulates the tyrosinase and TyrpI promoter activities, while TFEB acts only on the TyrpI promoter. TFE3 and TFEB elicit their effects mainly through the binding to Mbox (AGTCATGTGCT) and Ebox motifs (CATGTG) of tyrosinase and TyrpI promoters. In B16 melanoma cells, the high basal expression of TFE3 is down-regulated by forskolin and by alphaMSH. Interestingly, endogenous TFE3 cannot bind as homodimers to the Mbox, and we did not detect TFE3/Mi heterodimers. According to these data, TFE3 is clearly endowed with the capacity to regulate tyrosinase and TyrpI gene expression. However, TFE3 binding to the melanogenic gene promoters is hindered, thereby preventing its potential melanogenic action. In specific physiological or pathological conditions, the recovery of its binding function would make TFE3 an important element in melanogenesis regulation.

Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.

Melanocyte differentiation characterized by an increased melanogenesis, is stimulated by alpha-melanocyte-stimulating hormone through activation of the cAMP pathway. During this process, the expression of tyrosinase, the enzyme that controls melanin synthesis is upregulated. We previously showed that cAMP regulates transcription of the tyrosinase gene through a CATGTG motif that binds microphthalmia a transcription factor involved in melanocyte survival. Further, microphthalmia stimulates the transcriptional activity of the tyrosinase promoter and cAMP increases the binding of microphthalmia to the CATGTG motif. These observations led us to hypothesize that microphthalmia mediates the effect of cAMP on the expression of tyrosinase. The present study was designed to elucidate the mechanism by which cAMP regulates microphthalmia function and to prove our former hypothesis, suggesting that microphthalmia is a key component in cAMP-induced melanogenesis. First, we showed that cAMP upregulates the transcription of microphthalmia gene through a classical cAMP response element that is functional only in melanocytes. Then, using a dominant-negative mutant of microphthalmia, we demonstrated that microphthalmia is required for the cAMP effect on tyrosinase promoter. These findings disclose the mechanism by which cAMP stimulates tyrosinase expression and melanogenesis and emphasize the critical role of microphthalmia as signal transducer in cAMP-induced melanogenesis and pigment cell differentiation.

In B16 melanoma cells, the inhibition of melanogenesis by TPA results from PKC activation and diminution of microphthalmia binding to the M-box of the tyrosinase promoter.

In B16 melanoma cells, cAMP-induced melanogenesis is inhibited by the tumor promoting phorbol ester, TPA. However, the role of PKC activation or depletion in the inhibition of melanogenesis by TPA remains controversial. In this report, using specific PKC inhibitors, we demonstrated that PKC inhibition does not impair cAMP-induced melanin synthesis and tyrosinase expression. Further, the inhibition of melanogenesis by TPA results from a decrease of the tyrosinase promoter transcriptional activity and this effect is mimicked by over-expression of a constitutively active form of PKC alpha. These findings clearly demonstrate that PKC activation accounts for the inhibition of melanin synthesis by TPA. Additional experiments were undertaken to elucidate the mechanism by which TPA inhibits the tyrosinase gene transcription. Deletions and mutation in the tyrosinase promoter showed that TPA acts on a M-box which is involved in tissue-specific expression and regulation by cAMP of the tyrosinase gene. We showed that TPA decreases the binding of microphthalmia, a basic helix-loop-helix transcription factor, to the M-box. Since microphthalmia, strongly stimulates the transcriptional activity of the promoter we propose that TPA, through PKC activation, decreases microphthalmia binding to the M-box of the tyrosinase promoter, thereby leading to a reduced tyrosinase expression and melanogenesis inhibition.

Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: pivotal role of M boxes (GTCATGTGCT) and of microphthalmia.

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.

Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product.

In melanocytes and in melanoma cells, upregulation of melanogenesis, by cAMP elevating agents, results from a stimulation of tyrosinase activity that has been ascribed to an increase in tyrosinase protein and messenger amount. However, the mechanism by which cAMP elevating agents increase tyrosinase mRNA remains to be elucidated. In this study, using a luciferase reporter plasmid containing the 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene, we showed that cAMP elevating agents lead to a strong stimulation (20-fold) of transcriptional activity of the tyrosinase promoter. Deletions and mutations in the mouse tyrosinase promoter showed that the M-box 70-bp upstream from the TATA-box and the E-box located downstream the TATA-box, near to the initiator site, are involved in the regulation of the tyrosinase promoter activity by cAMP. Additionally, we showed that microphthalmia, a b-HLH transcription factor associated with pigmentation disorders in mouse, binds to these regulatory elements and modulates the transcriptional activity of the tyrosinase promoter. Since cAMP stimulates the binding of microphthalmia to the M-box and to the E-box; it is tempting to propose that microphthalmia, through its interaction with cis-acting elements surrounding the TATA-box, plays a key role in the regulation of the mouse tyrosinase gene expression by cAMP.