ZAKα/P38 kinase signaling pathway regulates hematopoiesis by activating the NLRP1 inflammasome.

Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress-induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.

Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment.

BACKGROUND: Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. METHODS: Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. RESULTS: We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1ß cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. CONCLUSIONS: Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.

Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 in fish granulocytes.

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.

Gasdermin E mediates pyroptotic cell death of neutrophils and macrophages in a zebrafish model of chronic skin inflammation.

Chronic diseases and hematopoietic disorders are associated with dysregulation of the inflammasome. Our group has recently reported the relevance of the inflammasome in the differentiation of hematopoietic stem and progenitor cells. However, the impact of the inflammasome of myeloid cells in the regulation of hematopoiesis is largely unknown. In this study, we used the unique advantages of the zebrafish model to demonstrate that genetic inhibition of macrophage inflammasome resulted in increased number of macrophages in larvae with skin inflammation without affecting erythrocyte and neutrophil counts. Similarly, the inhibition of the neutrophil inflammasome by the same strategy resulted in increased number of neutrophils in larvae with skin inflammation but did not affect erythrocytes and macrophages. Consistently, hyperactivation of the inflammasome in neutrophils in this model promoted neutrophil death, which was recovered by pharmacological inhibition of Gasdermin E. We conclude that the myeloid inflammasome autonomously regulates pyroptotic cell death in chronic inflammation through a Gasdermin E-dependent pathway in zebrafish.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA (terc) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.

Bcl-xL: A Focus on Melanoma Pathobiology.

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors.

Zebrafish Models to Study Inflammasome-Mediated Regulation of Hematopoiesis.

Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

The vitamin B6-regulated enzymes PYGL and G6PD fuel NADPH oxidases to promote skin inflammation.

Psoriasis is a skin inflammatory disorder that affects 3% of the human population. Although several therapies based on the neutralization of proinflammatory cytokines have been used with relative success, additional treatments are required. The in silico analysis of gene expression data of psoriasis lesional skin and an analysis of vitamin B6 metabolites in the sera of psoriasis patients point to altered vitamin B6 metabolism at both local and systemic levels. Functional studies showed that vitamin B6 vitamers reduced skin neutrophil infiltration, oxidative stress and Nfkb activity in two zebrafish models of skin inflammation. Strikingly, inhibition of glycogen phosphorylase L (Pygl) and glucose-6-phosphate dehydrogenase (G6pd), two vitamin B6-regulated enzymes, alleviated oxidative-stress induced inflammation in zebrafish skin inflammation models. Despite the central role of G6pd in antioxidant defenses, the results of the study demonstrate that glycogen stores and G6pd fuel NADPH oxidase to promote skin inflammation, revealing novel targets for the treatment of skin inflammatory disorders.

Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1.

Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

USP45 and Spindly are part of the same complex implicated in cell migration.

Ubiquitylation is a protein modification implicated in several cellular processes. This process is reversible by the action of deubiquinating enzymes (DUBs). USP45 is a ubiquitin specific protease about which little is known, aside from roles in DNA damage repair and differentiation of the vertebrate retina. Here, by using mass spectrometry we have identified Spindly as a new target of USP45. Our data show that Spindly and USP45 are part of the same complex and that their interaction specifically depends on the catalytic activity of USP45. In addition, we describe the type of ubiquitin chains associated with the complex that can be cleaved by USP45, with a preferential activity on K48 ubiquitin chain type and potentially K6. Here, we also show that Spindly is mono-ubiquitylated and this can be specifically removed by USP45 in its active form but not by the catalytic inactive form. Lastly, we identified a new role for USP45 in cell migration, similar to that which was recently described for Spindly.

An Adult Zebrafish Model Reveals that Mucormycosis Induces Apoptosis of Infected Macrophages.

Mucormycosis is a life-threatening fungal infection caused by various ubiquitous filamentous fungi of the Mucorales order, although Rhizopus spp. and Mucor spp. are the most prevalent causal agents. The limited therapeutic options available together with a rapid progression of the infection and a difficult early diagnosis produce high mortality. Here, we developed an adult zebrafish model of Mucor circinelloides infection which allowed us to confirm the link between sporangiospore size and virulence. Transcriptomic studies revealed a local, strong inflammatory response of the host elicited after sporangiospore germination and mycelial tissue invasion, while avirulent and UV-killed sporangiospores failed to induce inflammation and were rapidly cleared. Of the 857 genes modulated by the infection, those encoding cytokines, complement factors, peptidoglycan recognition proteins, and iron acquisition are particularly interesting. Furthermore, neutrophils and macrophages were similarly recruited independently of sporangiospore virulence and viability, which results in a robust depletion of both cell types in the hematopoietic compartment. Strikingly, our model also reveals for the first time the ability of mucormycosis to induce the apoptosis of recruited macrophages but not neutrophils. The induction of macrophage apoptosis, therefore, might represent a key virulence mechanism of these fungal pathogens, providing novel targets for therapeutic intervention in this lethal infection.

Interleukin 8 mediates bcl-xL-induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model.

The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma.

Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization.

The deubiquitylating enzyme OTUB1 is present in all tissues and targets many substrates, in both the cytosol and nucleus. We found that casein kinase 2 (CK2) phosphorylated OTUB1 at Ser(16) to promote its nuclear accumulation in cells. Pharmacological inhibition or genetic ablation of CK2 blocked the phosphorylation of OTUB1 at Ser(16), causing its nuclear exclusion in various cell types. Whereas we detected unphosphorylated OTUB1 mainly in the cytosol, we detected Ser(16)-phosphorylated OTUB1 only in the nucleus. In vitro, Ser(16)-phosphorylated OTUB1 and nonphosphorylated OTUB1 exhibited similar catalytic activity, bound K63-linked ubiquitin chains, and interacted with the E2 enzyme UBE2N. CK2-mediated phosphorylation and subsequent nuclear localization of OTUB1 promoted the formation of 53BP1 (p53-binding protein 1) DNA repair foci in the nucleus of osteosarcoma cells exposed to ionizing radiation. Our findings indicate that the activity of CK2 is necessary for the nuclear translocation and subsequent function of OTUB1 in DNA damage repair.

USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses.

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair.

Differential and competitive regulation of human melanocortin 1 receptor signaling by ß-arrestin isoforms.

The melanocortin 1 receptor (MC1R) is a G-protein-coupled receptor (GPCR) crucial for the regulation of melanocyte proliferation and differentiation. MC1R activation by melanocortin hormones triggers the cAMP pathway and stimulates the extracellular-signal-regulated protein kinases ERK1 and ERK2 to promote synthesis of photoprotective eumelanin pigments, among other effects. Signaling from most GPCRs is regulated by the ß-arrestin (ARRB) family of cytosolic multifunctional adaptor proteins, which mediate signal termination and endocytosis of GPCR-agonist complexes. The ubiquitously expressed non-visual ß-arrestin1 (ARRB1) and ß-arrestin2 (ARRB2) are highly similar but not functionally equivalent. Their role in the regulation of MC1R is unknown. Using a combination of co-immunoprecipitation, gel filtration chromatography, confocal microscopy, siRNA-mediated knockdown and functional assays, we demonstrated agonist-independent competitive interactions of ARRB1 and ARRB2 with MC1R, which might also be independent of phosphorylation of Ser/Thr residues in the C-terminus of the MC1R. The effects of ARRBs were isoform specific; ARRB2 inhibited MC1R agonist-dependent cAMP production but not ERK activation, stimulated internalization and showed prolonged co-localization with the receptor in endocytic vesicles. By contrast, ARRB1 had no effect on internalization or functional coupling, but competed with ARRB2 for binding MC1R, which might increase signaling by displacement of inhibitory ARRB2. These data suggest a new mechanism of MC1R functional regulation based on the relative expression of ARRB isoforms, with possible activatory ARRB1-dependent effects arising from partial relief of inhibitory ARRB2-MC1R interactions. Thus, competitive displacement of inhibitory ARRBs by functionally neutral ARRB isoforms might exert a paradigm-shifting signal-promoting effect to fine-tune signaling downstream of certain GPCRs.

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells.

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.

Mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase inhibits signaling from melanocortin receptor by competition with Galphas.

Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R-MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on beta(2)-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Galpha(s) binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Galpha(s) abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Galpha(s) for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Galpha(s), thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.

Identification and functional analysis of novel variants of the human melanocortin 1 receptor found in melanoma patients.

The melanocortin 1 receptor, a Gs protein-coupled receptor expressed in epidermal melanocytes, is a major determinant of skin pigmentation and phototype and an important contributor to melanoma risk. MC1R activation stimulates synthesis of black, strongly photoprotective eumelanin pigments. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation, and increased skin cancer risk. The MC1R gene is highly polymorphic, but only a few naturally occurring alleles have been functionally characterized, which complicates the establishment of accurate correlations between the signaling properties of mutant alleles and defined cutaneous phenotypes. We report the functional characterization of six MC1R alleles found in Spanish melanoma patients. Two variants (c.152T>C, p.Val51Ala and c.865T>C, p.Cys289Arg) have never been described, and the others (c.112G>A, p.Val38Met; c.122C>T, p.Ser41Phe; c.383T>C, p.Met128Thr; and c.842A>G, p.Asn281Ser) have not been analyzed for function. p.Asn281Ser corresponds to a functionally silent polymorphism. The other mutations are associated with varying degrees of loss of function (LOF), from moderate decreases in coupling to the cAMP pathway (p.Val38Met and p.Val51Ala) to nearly complete absence of functional coupling (p.Ser41Phe, p.Met128Thr, and p.Cys289Arg). The LOF p.Met128Thr and p.Cys289Arg mutants are trafficked to the cell surface, but are unable to bind agonists efficiently. Conversely, LOF of p.Val38Met, p.Ser41Phe, and p.Val51Ala is due to reduced cell surface expression as a consequence of retention in the endoplasmic reticulum (ER). Therefore, LOF of MC1R alleles is frequently associated with aberrant forward trafficking and accumulation within the ER or with inability to bind properly the activatory ligand.