Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport.

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field

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

Hypoxia-inducible factor 1α is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

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

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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

IGF1 Promotes Resistance to Apoptosis in Melanoma Cells through an Increased Expression of BCL2, BCL-X(L), and Survivin

International audienceIGF1 plays a key role in the development and growth of multiple tumors and in the prevention of apoptosis. In melanoma cells, IGF1 has been shown to mediate resistance to anoikis-induced apoptosis. However, the effect of IGF1 on other proapoptotic stimuli has never been reported. Further, the molecular mechanisms by which IGF1 mediates its prosurvival properties in melanoma cells remain unknown. Here, we demonstrate that IGF1 impairs the onset of tumor necrosis factor-related apoptosis-inducing ligand and staurosporine-induced apoptosis in melanoma cells expressing either wild-type or oncogenic B-Raf. Further, we show that IGF1 inhibits mitochondrial damage that occurs during apoptosis, thereby indicating that IGF1 acts at the level of mitochondria to mediate its antiapoptotic stimuli. Accordingly, IGF1 increases the mRNA levels and protein expression of antiapoptotic members of the BCL2 family--BCL2 and BCL-X(L)--and that of the inhibitor of apoptosis protein, survivin. Further, their specific silencing by small interfering RNA prevents the protective effect of IGF1. These findings therefore delineate the molecular mechanisms by which IGF1 mediates its prosurvival properties and provide a basis for clinical strategies designed to neutralize IGF1 or its target genes

Glycogen Synthase Kinase 3β Is Activated by cAMP and Plays an Active Role in the Regulation of Melanogenesis

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Up-regulation of MET Expression by α-Melanocyte-stimulating Hormone and MITF Allows Hepatocyte Growth Factor to Protect Melanocytes and Melanoma Cells from Apoptosis

International audienceThe MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. In mouse melanoma cells, Met expression is increased by alphaMSH via the activation of the cAMP pathway. However, the mechanism by which cAMP regulates MET and the biological consequences of this increase were not known. In the present report, we show that alphaMSH regulates MET expression in both human melanocytes and mouse melanoma cells through a transcriptional mechanism that requires MITF. Furthermore, the adenovirus driven expression of MITF is sufficient to increase MET in melanoma cells. Functional analysis of the MET promoter allows us to identify an E-box motif conserved in both human and mouse promoter that mediates the effect of MITF. Interestingly, up-regulation of MET expression by cAMP leads to an exacerbated HGF signaling and allows HGF to protect melanocytes and melanoma cells from apoptosis. Thus, physiological stimuli or pathological events that would induce MITF expression may lead to increased MET expression thereby favoring melanoma survival. These observations strengthen the roles of MITF and MET in melanoma development