Mechanism underlying synergic activation of Tyrosinase promoter by MITF and IRF4

Background: The transcription factor interferon regulatory factor 4 (IRF4) was identified to be involved in human pigmentation by genome-wide association studies (GWASs). The rs12203592-[T/C], which is located in intron 4 of IRF4, shows the strongest link to these pigmentation phenotypes including freckling, sun sensitivity, eye and hair color. Previous studies indicated a functional cooperation of IRF4 with Microphthalmia-associated transcription factor (MITF), a causing gene of Waardenburg syndrome (WS), to synergistically trans-activate Tyrosinase (TYR). However, the underlying mechanism is still unknown. Methods: To investigate the importance of DNA binding in the synergic effect of IRF4. Reporter plasmids with mutant TYR promoters was generated to locate the IRF4 DNA binding sites in the Tyrosinase minimal promoter. By building MITF and IRF4 truncated mutations plasmids, the necessary regions of the synergy functions of these two proteins were also located. Results: The cooperative effect between MITF and IRF4 was specific for TYR promoter. The DNA-binding of IRF4 was critical for the synergic function. IRF4 DNA binding sites in TYR promoter were identified. The Trans-activation domains in IRF4 (aa134-207, aa300-420) were both important for the synergic function, whereas the auto-mask domain (aa207-300) appeared to mask the synergic effect. Mutational analysis in MITF indicated that both DNA-binding and transcriptional activation domains were both required for this synergic effect. Conclusions: Here we showed that IRF4 potently synergized with MITF to activate the TYR promoter, which was dependent on DNA binding of IRF4. The synergic domains in both IRF4 and MITF were identified by mutational analysis. This identification of IRF4 as a partner for MITF in regulation of TYR may provide an important molecular function for IRF4 in the genesis of melanocytes and the pathogenic mechanism in WS

SCD5 restored expression favors differentiation and epithelial-mesenchymal reversion in advanced melanoma

Our previous data supported a role for the Stearoyl-CoA desaturase (SCD5) in protection against malignancy, whereby it appears to functionally modify tumor stroma impairing tumor spread. SCD5 is significantly expressed in primary melanoma, but becomes barely detectable at tumor advanced stages. Looking for the regulatory mechanisms underlying SCD5 reduced expression during melanoma progression, we demonstrated a significantly lower stability of SCD5 protein as well as the direct targeting of SCD5 mRNA by the oncogenic miR-221 & 222 in metastatic cell lines. Moreover, our results indicated the existence of a negative feedback loop between SCD5 and miR-221 & 222, in good agreement with their opposite functions. Also, we showed how SCD5 re-expression and the direct supplementation of its main product oleic acid (OA) can drive advanced melanoma cell lines toward differentiation and reversion of the epithelial-mesenchymal (EMT)-like process, eventually inducing a less malignant phenotype. Indeed, SCD5 re-established the sensitivity to all-trans retinoic acid in A375M metastatic melanoma, associated with increased levels of Tyrosinase, melanin production and reduced proliferation. As evidenced by the correct modulation of some key transcription factors, SCD5 managed by favoring a partial mesenchymal-to-epithelial (MET) transition in in vitro studies. Interestingly, a more complete MET, including E-cadherin re-expression correctly localized at cell membranes, was obtained in in vivo xenograft models, thus indicating the requirement of direct contacts between tumor cells and the surrounding microenvironment as well as the presence of some essential factors for SCD5 complete function

Magnetic and quadrupolar order in a one-dimensional ferromagnet with cubic crystal-field anisotropy

The zero temperature phase diagram of a one-dimensional S=2 Heisenberg ferromagnet with single-ion cubic anisotropy is studied numerically using the density-matrix renormalization group method. Evidence is found that although the model does not involve quadrupolar couplings, there is a purely quadrupolar phase for large values of the anisotropy. The phase transition between the magnetic and quadrupolar phases is continuous and it seems to be characterized by Ising critical exponents.Comment: 11 pages, 7 figures, REVTeX, accepted in Phys. Rev. B (scheduled on June 99

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol

Understanding signaling cascades in melanoma

Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-beta pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.Fil: Lopez Bergami, Pablo Roberto. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Fitchmann, B. Sanford-burnham Medical Research Institute; Estados UnidosFil: Ronai, Ze´ev. Sanford-burnham Medical Research Institute; Estados Unido

Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model syste

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease