How TGF‐β and PAX3 regulate suntanning

The processes by which ultraviolet(UV) radiation induces melanin synthe-sis to induce a suntan is complex,involving signaling between keratino-cytes and melanocytes. Although muchis known about the enzymes involved inmelanin production, there have beengaps in understanding how key tran-scriptional regulators of melanogenesisare regulated. A recent study by Cuiand colleagues elucidated how expres-sion of PAX3, a gene that encodes atranscription factor that is involved inmelanocyte development, is regulatedin response to UV irradiation (Yanget al., 2008).PAX3 is a member of the paired-boxfamily of transcription factor genes andis expressed during development inneuroepithelium, neural crest, andsomitic mesoderm (reviewed in Kubicet al., 2008). PAX3 mutations causepigmentation defects in both mice andhumans (cited in Yang et al., 2008).PAX3 is one of the transcription factorsthat regulates expression of Mitf, whichencodes a transcription factor that regu-lates major pigment enzyme genes aswell as melanocyte proliferation andsurvival (cited in Yang et al., 2008).PAX3 has been detected in many pri-mary melanomas, but not in surround-ing benign tissue, and knocking downPAX3 expression with antisense oligo-nucleotides induces apoptosis of PAX3-positive melanoma cells in culture (citedin Yang et al., 2008).TGF-b expression is down-regulatedin skin in response to UV irradiation(cited in Yang et al., 2008). TGF-b inhib-its melanocyte proliferation and melaninsynthesis (cited in Yang et al., 2008).This prompted Cui and colleagues toinvestigate whether TGF-b might nega-tively regulate PAX3 expression, andhow PAX3 regulates Mitf expression.Using primary human keratinocytes,they found that UV irradiationsuppressed TGF-b mRNA and pro-tein production. The inhibition of TGF-bexpression is in response to UV radia-tion-induced activation of p53, andp53-induced activation of AP-1, whichinhibits TGF-b expression. There appearto be p53-independent effects of UVirradiation on AP-1 activation, as well.TGF-b suppressed expression of PAX3mRNA and protein in melanocytes. Thesuppression of PAX3 expression is dueto direct transcriptional repression byTGF-b-activated Smad4, which binds toa transcriptional regulatory region 100 bp upstream of the PAX3 tran-scription initiation site.In addition to indirectly inhibitingTGF-b expression, p53 directly regu-lates expression of proopiomelanocor-tin⁄melanocyte-stimulating hormone(POMC⁄MSH) (cited in Yang et al.,2008). a-MSH is the ligand for the mela-nocortin-1 receptor (MC1R). Binding ofa-MSH to MC1R stimulates adenylatecyclase activity, and cAMP signalingstimulates Mitf transcription (cited inYang et al., 2008). By transfecting aseries of Mitf-luciferase reporters intoB16 mouse melanoma cells along withplasmids expressing PAX3 or SOX10,Cui and colleagues showed that PAX3and SOX10 act in a synergistic fashionto stimulate Mitf expression, but thatthese effects require the presence of aCREB binding site on the Mitf pro-moter. Interestingly, there are PAX3and SOX10 binding sites adjacent toeach other (between )268 and )244)on the Mitf promoter, and they are 100 bp upstream of the CRE ()147 to)139). It is possible that PAX3 andSOX10 promote high afﬁnity binding ofeach other to the Mitf promoter. Fur-ther investigation will be necessary todetermine whether this is the case, andwhy the CRE is required for the tran-scriptional effects of PAX3 and SOX10,for example, whether this involvesco-activation by CBP. A diagram whichsketches the ﬂow of signaling pathwaysby which UV irradiation leads to melaninproduction is shown in Figure 1.Noting that TGF-b inhibits melanocytegrowth, and that TGF-b inhibits PAX3expression, Cui and colleagues testedwhether PAX3 regulates melanocyteproliferation or survival. Using ShPAX3RNA interference in melanoma cells,they observed that knockdown of PAX3mRNA induced G1 cell cycle arrest.Conversely, transfection of a PAX3expression vector rescued B16 mousemelanoma cells from TGF-b-induced cellcycle arrest. Thus, consistent with aprevious observation that PAX3 anti-sense oligonucleotides induce apoptosisin primary melanoma cultures (cited inYang et al., 2008), these results demon-strate that PAX3 promotes melanomasurvival by preventing cell cycle arrest,and moreover, that over expression ofPAX3 is sufﬁcient to prevent cell cyclearrest.It should be noted that the involve-ment of PAX3 in preventing cell cyclearrest was demonstrated in melanomacells, not normal skin melanocytes.Whereas normal melanocytes aregrowth-inhibited by TGF-b, there aremany melanoma cell lines that are notgrowth-inhibited by TGF-b. Therefore, inmelanoma cells that are refractory toTGF-b, PAX3 expression may be consti-tutively expressed, thereby continuallymaintaining the cell in a proliferativemode. Presumably, in TGF-b-responsivemelanoma cells (such as the B16 lineused by Cui and colleagues), there areother mechanisms leading to constitu-tive PAX3-dependent cell cycle stimula-tion [for example, ampliﬁcation of agene that positively regulates PAX3expression, similar to ampliﬁed N-MYCin some neuroblastoma lines (cited inYang et al., 2008)]. Given that PAX3appears to play a pivotal role in prevent-ing melanoma cell cycle withdrawal,inhibiting PAX3 expression, or destabi-lizing PAX3 mRNA or protein, might beCoverage on: Yang et al. (2008) Inhibi-tion of PAX3 by TGF-b modulates mela-nocyte viability. Mol. Cell 32: 554–563.