Micropthalmia-associated Transcription Factor Research Articles

Roan, ticked and clear coat patterns in the canine are associated with three haplotypes near usherin on CFA38.

SummaryWhite coat patterning is a feature of many dog breeds and is known to be coded primarily by the gene micropthalmia‐associated transcription factor (MITF). This patterning in the coat can be modified by other factors to produce the attractive phenotypes termed ‘ticked’ and ‘roan’ that describe the presence of flecks of color that vary in distribution and intensity within otherwise ‘clear’ white markings. The appearance of the pigment in the white patterning caused by ticking and roaning intensifies in the weeks after birth. We applied genome‐wide association to compare English Cocker Spaniels of roan phenotype (N = 34) with parti‐color (non‐roan) English Cocker Spaniels (N = 9) and identified an associated locus on CFA 38, CFA38:11 057 040 (P raw = 8.9 × 10−10, P genome = 2.7 × 10−5). A local case–control association in English Springer Spaniels comparing 11 ticked and six clear dogs identified indicative association with a different haplotype, CFA38:11 122 467G>T (P raw = 1.7 × 10−5) and CFA38:11 124 294A>C (P raw = 1.7 × 10−5). We characterize three haplotypes in Spaniels according to their putative functional variant profiles at CFA38:11 111 286C>T (missense), CFA38:11 131 841–11 143 239DUP.insTTAA (using strongly linked marker CFA38:11 143 243C>T) and CFA38:11 156 425T>C (splice site). In Spaniels, the haplotypes work as an allelic series including alleles (t, recessive clear; T, dominant ticked/parti‐color; and T R, incomplete dominant roan) to control the appearance of pigmented spots or flecks in otherwise white areas of the canine coat. In Spaniels the associated haplotypes are t (CCT), T (TCC) and T R (TTT) for SNP markers on CFA38 at 11 111 286C>T, 11 143 243C>T and 11 156 425T>C respectively. It is likely that other alleles exist in this series and together the haplotypes result in a complex range of patterning that is only visible when dogs have white patterning resulting from the epistatic gene Micropthalmia‐associated transcription factor (the S‐locus).

The biology of human hair greying.

Hair greying (canities) is one of the earliest, most visible ageing-associated phenomena, whose modulation by genetic, psychoemotional, oxidative, senescence-associated, metabolic and nutritional factors has long attracted skin biologists, dermatologists, and industry. Greying is of profound psychological and commercial relevance in increasingly ageing populations. In addition, the onset and perpetuation of defective melanin production in the human anagen hair follicle pigmentary unit (HFPU) provides a superb model for interrogating the molecular mechanisms of ageing in a complex human mini-organ, and greying-associated defects in bulge melanocyte stem cells (MSCs) represent an intriguing system of neural crest-derived stem cell senescence. Here, we emphasize that human greying invariably begins with the gradual decline in melanogenesis, including reduced tyrosinase activity, defective melanosome transfer and apoptosis of HFPU melanocytes, and is thus a primary event of the anagen hair bulb, not the bulge. Eventually, the bulge MSC pool becomes depleted as well, at which stage greying becomes largely irreversible. There is still no universally accepted model of human hair greying, and the extent of genetic contributions to greying remains unclear. However, oxidative damage likely is a crucial driver of greying via its disruption of HFPU melanocyte survival, MSC maintenance, and of the enzymatic apparatus of melanogenesis itself. While neuroendocrine factors [e.g. alpha melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), ß-endorphin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)], and micropthalmia-associated transcription factor (MITF) are well-known regulators of human hair follicle melanocytes and melanogenesis, how exactly these and other factors [e.g. thyroid hormones, hepatocyte growth factor (HGF), P-cadherin, peripheral clock activity] modulate greying requires more detailed study. Other important open questions include how HFPU melanocytes age intrinsically, how psychoemotional stress impacts this process, and how current insights into the gerontobiology of the human HFPU can best be translated into retardation or reversal of greying.

MiR-148a-3p inhibits alpaca melanocyte pigmentation by targeting MITF

microRNAs (miRNAs) are well established as post-transcriptional regulators of gene expression, and function by binding regulatory regions, such as the 3′ untranslated region (UTR) of their target mRNA sequences. They have been implicated in the regulation of pigmentation, including the determination of alpaca hair-fiber color; however, the mechanisms underlying their effects are not yet well understood. Thus, the present study investigated whether the miRNA miR-148a-3p regulates the expression of the micropthalmia-associated transcription factor (MITF), since MITF is known to control the expression of key genes linked to coat color. The results of the conducted analyses showed that overexpressing miR-148a-3p in alpaca melanocytes caused the expression of MITF, pigmentation-associated protein tyrosinase (TYR), and both tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2, respectively) to be reduced, and thereby led to a decrease in the overall melanin content of the analyzed cells. Thus, the present study supports that miR-148a-3p is likely an important regulator of MITF expression.

Nutrient deprivation and lysosomal stress induce activation of TFEB in retinal pigment epithelial cells

BackgroundInduction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB.MethodsExpression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes.ResultsOur results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes.ConclusionsCollectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.

Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation

BackgroundHyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented.MethodsIn this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed.ResultsThe results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-β were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components.ConclusionIn summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.

Plumbagin Suppresses α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells by Inhibiting Tyrosinase Activity.

Recent studies have shown that plumbagin has anti-inflammatory, anti-allergic, antibacterial, and anti-cancer activities; however, it has not yet been shown whether plumbagin suppresses alpha-melanocyte stimulating hormone (α-MSH)-induced melanin synthesis to prevent hyperpigmentation. In this study, we demonstrated that plumbagin significantly suppresses α-MSH-stimulated melanin synthesis in B16F10 mouse melanoma cells. To understand the inhibitory mechanism of plumbagin on melanin synthesis, we performed cellular or cell-free tyrosinase activity assays and analyzed melanogenesis-related gene expression. We demonstrated that plumbagin directly suppresses tyrosinase activity independent of the transcriptional machinery associated with melanogenesis, which includes micropthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1). We also investigated whether plumbagin was toxic to normal human keratinocytes (HaCaT) and lens epithelial cells (B3) that may be injured by using skin-care cosmetics. Surprisingly, lower plumbagin concentrations (0.5–1 μM) effectively inhibited melanin synthesis and tyrosinase activity but do not cause toxicity in keratinocytes, lens epithelial cells, and B16F10 mouse melanoma cells, suggesting that plumbagin is safe for dermal application. Taken together, these results suggest that the inhibitory effect of plumbagin to pigmentation may make it an acceptable and safe component for use in skin-care cosmetic formulations used for skin whitening.

Inhibition of oncogenic BRAF activity by indole-3-carbinol disrupts microphthalmia-associated transcription factor expression and arrests melanoma cell proliferation.

Indole-3-carbinol (I3C), an anti-cancer phytochemical derived from cruciferous vegetables, strongly inhibited proliferation and down-regulated protein levels of the melanocyte master regulator micropthalmia-associated transcription factor (MITF-M) in oncogenic BRAF-V600E expressing melanoma cells in culture as well as in vivo in tumor xenografted athymic nude mice. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing melanoma cells, I3C treatment inhibited phosphorylation of MEK and ERK/MAPK, the down stream effectors of BRAF. The I3C anti-proliferative arrest was concomitant with the down-regulation of MITF-M transcripts and promoter activity, loss of endogenous BRN-2 binding to the MITF-M promoter, and was strongly attenuated by expression of exogenous MITF-M. Importantly, in vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C selectively inhibited the enzymatic activity of the oncogenic BRAF-V600E but not of the wild type protein. In silico modeling predicted an I3C interaction site in the BRAF-V600E protomer distinct from where the clinically used BRAF-V600E inhibitor Vemurafenib binds to BRAF-V600E. Consistent with this prediction, combinations of I3C and Vemurafenib more potently inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells compared to the effects of each compound alone. Thus, our results demonstrate that oncogenic BRAF-V600E is a new cellular target of I3C that implicate this indolecarbinol compound as a potential candidate for novel single or combination therapies for melanoma. © 2016 Wiley Periodicals, Inc.

Biological characteristics of mouse skin melanocytes.

The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.

Oncogene addiction

Oncogene addiction

A Novel Isoform of Microphthalmia-Associated Transcription Factor Inhibits IL-8 Gene Expression in Human Cervical Stromal Cells

Cervical ripening during pregnancy is a profound change in cervix structure and function characterized by increases in the proinflammatory cytokine IL-8 and dissolution of the cervical extracellular matrix. Relatively little is known about the molecular mechanisms that underlie these events. Here, we report identification of a novel isoform of micropthalmia-associated transcription factor in human cervical stromal cells (MiTF-CX) that is down-regulated 12-fold during cervical ripening and that represses expression of IL-8. Ectopic expression of MiTF-CX in human cervical stromal cells resulted in substantial suppression of endogenous IL-8 mRNA and protein expression, whereas expression of dominant negative MiTF-CX mutants with impaired DNA binding resulted in dramatic increases in IL-8 production. Gel shift, reporter gene, and chromatin immunoprecipitation assays revealed one strong binding site (E-box (-397) CACATG(-391)) in the human IL-8 promoter that was crucial for mediating transcriptional repression by MiTF-CX. Moreover, we show that MiTF-CX expression in the cervix was itself positively autoregulated via two E-box motifs within a 2.1-kb promoter fragment. We therefore propose that maintenance of cervical competency during pregnancy is an active process maintained through suppression of IL-8 by the transcription factor MiTF-CX. During cervical ripening, loss of MiTF-CX would result in significant up-regulation of IL-8 mRNA and protein synthesis, thereby leading to recruitment and activation of leukocytes within the cervix and dissolution of the extracellular matrix.

Genome‐wide analysis of POU3F2/BRN2 promoter occupancy in human melanoma cells reveals Kitl as a novel regulated target gene

POU3F2 is a POU-Homeodomain transcription factor expressed in neurons and melanoma cells. In melanoma lesions, cells expressing high levels of POU3F2 show enhanced invasive and metastatic capacity that can in part be explained by repression of Micropthalmia-associated Transcription Factor (MITF) expression via POU3F2 binding to its promoter. To identify other POU3F2 target genes that may be involved in modulating the properties of melanoma cells, we performed ChIP-chip experiments in 501Mel melanoma cells. 2108 binding loci located in the regulatory regions of 1700 potential target genes were identified. Bioinformatic and experimental assays showed the presence of known POU3F2-binding motifs, but also many AT-rich sequences with only partial similarity to the known motifs at the occupied loci. Functional analysis indicates that POU3F2 regulates the stem cell factor (Kit ligand, Kitl) promoter via a cluster of four closely spaced binding sites located in the proximal promoter. Our results suggest that POU3F2 may regulate the properties of melanoma cells via autocrine KIT ligand signalling.

Abstract 5440: Study of the mechanism of action and pharmacodynamic response of JNJ-26481585, a potent histone deacetylase inhibitor in the setting of melanoma

Abstract Background: JNJ-26481585 is a potent histone deacetylase (HDAC) inhibitor. It is undergoing evaluation in phase I trials in solid tumours. The drug has shown plasma concentrations consistent with activity in preclinical models and target inhibition in surrogate and tumour tissue. Purpose: To determine the mechanism of action of JNJ-26481585 and to study factors determining sensitivity to the drug in-vitro in the setting of melanoma cell lines. Experimental Procedures: Array comparative genomic hybridization (array CGH) was carried out to study gene copy number. The growth inhibitory (GI50) concentrations of JNJ-26481585 and 17-allylamino, 17-demethoxygeldanamyin (17-AAG) were determined by sulforhodamine (SRB) assays. A panel of melanoma cell lines was exposed to GI50 and 5 X GI50 for 24hrs and gene expression profiling was performed using gene expression microarrays. Changes in protein expression were studied by western blot analysis. Results: Cells exposed to GI50 and 5X GI50 of 17-AAG for 24hrs showed a molecular signature of HSP90 inhibition including C-RAF depletion and HSP70 induction while this was not the case in equitoxic concentrations of JNJ-26481585. In addition, JNJ-26481585 did not reproducibly induce acetylation of tubulin which is a surrogate for cytoplasmic acetylation of proteins such as HSP90. JNJ-26481585 however induced robust acetylation of histone H3 suggestive of its mechanism of action predominantly as a class I HDAC inhibitor. The micropthalmia associated transcription factor (MITF) locus was amplified in the SKMEL-28 cells. The GI50 concentrations of JNJ-26481585 in the cell lines SKMEL-28, WM266.4, A2058, SKMEL-2 and SKMEL-5 were 5.4 (SD 0.86) nM, 20 (SD 0.58) nM, 28.1 (SD 0.7) nM, 30.4 (SD 3.6) nM, 33.4 (SD 3.6) nM respectively. The cell line panel was exposed to GI50 and 5 X GI50 of JNJ-26481585 for 24hrs. Protein levels of MITF was depleted in addition to MITF m-RNA levels being significantly reduced; p=0.002 (Welch T test with Benjamini and Hochberg correction for multiple testing; FDR 5%) at the time points studied, suggesting down regulation of MITF gene expression as a possible mechanism of action of JNJ-26481585. All the cell lines studied underwent apoptosis as evidenced by demonstration of cleaved PARP on western blotting. Conclusions: In this model, the mechanism of action of JNJ-26481585 is not predominantly due to HSP90 inhibition caused by acetylation of HSP90 in melanoma cells. Melanoma cell lines which have an amplification of MITF are sensitive to the HDAC inhibitor JNJ-26481585. Further exploration of MITF protein to evaluate pharmacodynamic response of JNJ-26481585 is warranted. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5440.

Validation of Methods for Isolation and Culture of Alpaca Melanocytes: A Novel Tool for In vitro Studies of Mechanisms Controlling Coat Color

The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of α-melanocyte stimulating hormone. Treatment with α-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.

MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca ( Lama pacos) skin melanocytes

Although the influence of endocrine factors is well established, the molecular and cellular mechanisms controlling coat color are not completely understood. A major mechanism for post-transcriptional regulation of gene expression is through the action of microRNAs (miRNAs), which anneal to the 3’ untranslated region of mRNAs in a sequence-specific fashion and either block translation or promote transcript degradation. In this study, we investigated the expression of miRNAs in the skin of alpacas with brown vs white coat color using a microarray screen; identified potential mRNA targets for identified miRNAs among coat color genes; and subsequently determined the ability of a specific, differentially expressed miRNA (miR-25) to suppress expression of micropthalmia-associated transcription factor (MITF), a predicted miR-25 target gene that regulates genes linked to coat color. Expression of 10 different miRNA species in the skin of alpacas with brown vs white coat color was identified from microarray screens. Of the 10 alpaca skin miRNAs identified, predicted binding sites in the 3′ untranslated region of RNAs encoding for known genes linked to coat color were primarily for miR-25, but sites were also identified for miR-129 and miR-377. Potential miR-25 binding sites were present in transcripts encoding for 11 coat color genes, including MITF. An inverse relationship between transcript abundance for MITF and miR-25 was observed in skin samples collected from alpacas with white vs brown coat color. Furthermore, overexpression of miR-25 in cultured melanocytes reduced MITF mRNA and protein abundance and corresponding mRNA abundance for the MITF-regulated enzymes tyrosinase and tyrosinase-related protein 1. Results support a novel functional role for miRNA-25 in the regulation of gene expression linked to coat color.

The use and application of special techniques in assessing melanocytic tumours

To improve diagnostic accuracy, a number of novel techniques have been developed to assist in the evaluation of melanocytic tumours. Dematologists have begun to explore the use of new instruments, such as dermoscopy or confocal scanning laser microscopy, in their clinical assessment of pigmented lesions. They have also benefited from advances in digital photography and applied this technique to monitor patients with numerous atypical naevi. Likewise, the diagnostic armamentarium of pathologists has been enriched over the past decade. A number of new reagents have become available for immunohistochemical analysis of melanocytes in archival material, such as antibodies to tyrosinase, Melan-A/Mart-1, micropthalmia-associated transcription factor and PNL2. These markers have allowed improvements in the sensitivity and specificity in the diagnosis of melanoma and its distinction from histological mimics. However, some pitfalls in the use of various reagents have also become apparent. Most recently, molecular techniques are being explored for their potential use in the diagnosis of melanoma. In this review, various modalities are discussed with regard to their application in the evaluation of melanocytic neoplasms.

CAMP Follows a New Path to Melanogenesis

In mammals, adrenocorticotropic hormone and α-melanocyte-stimulating hormone regulate melanogenesis, and thus skin pigmentation, by activating the adenosine 3′,5′ monophosphate (cAMP) pathway through the melanocortin type 1 receptor. One limb of this pathway involves a protein kinase A (PKA)-dependent stimulation in the expression of the micropthalmia-associated transcription factor (MITF). MITF, in turn, stimulates expression of tyrosinase, the rate-limiting enzyme in melanogenesis. Khaled et al. report an alternative pathway whereby cAMP stimulates melanogenesis through a PKA-independent mechanism. Using Western analysis and in vitro assays on immunoprecipitates of proteins transfected into a melanoma cell line, the authors demonstrated that either elevating cAMP or inhibiting phosphatidylinositol 3-kinase (PI3K) reduced phosphorylation and activity of the serine-threonine kinase AKT. Elevated cAMP decreased phosphorylation of glycogen synthase kinase-3β (GSK-3β), a substrate of AKT, and stimulated GSK-3β activity. Pharmacological analyses indicated that the effects of cAMP on AKT and GSK-3β were independent of both PKA and the Ras to extracellular signal-regulated kinase (ERK) pathways and were mediated through a PI3K-dependent mechanism. GSK-3β overexpression had no effect on basal or cAMP-induced transcription of MITF, but dramatically enhanced MITF stimulation of a reporter gene containing the tyrosinase promoter. Gel shift assays indicated that cAMP stimulated MITF binding to the M box, a target sequence found in the tyrosinase promoter, suggesting that GSK-3β promotes melanogenesis by increasing MITF binding to the tyrosinase promoter. The PKA-independent pathway should thus be able to effectively cooperate with the previously described PKA-dependent pathway, which stimulates MITF synthesis, to enhance melanogenesis. M. Khaled, L. Larribere, K. Bille, E. Aberdam, J.-P. Ortonne, R. Ballotti, C. Bertolotto, Glycogen synthase kinase 3β is activated by cAMP and plays an active role in the regulation of melanogenesis. J. Biol. Chem. 277 , 33690-33697 (2002). [Abstract] [Full Text]