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1.
FASEB J ; 34(11): 14602-14614, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32941657

RESUMO

Melanocyte survival is mediated by diverse signaling pathways. However, the molecular mechanisms they use and molecules that they target are incompletely understood. Here, we show that melanocyte survival is mediated by diverse, nonredundant signaling pathways, including ERK1/2, AKT, PKA, and PKC. Each of these pathways is exerting prosurvival effects by phosphorylating the BAD. While Ser112-BAD phosphorylation is regulated by pERK, pPKA and pPKC, Ser136 and Ser155 phosphorylation are exclusively controlled by pAKT and pPKA, respectively. Inhibition of these pathways individually resulted in only modest apoptosis; however, most significant apoptosis, as a result of BAD dephosphorylation, was seen when all pathways were inhibited concurrently. BAD phosphorylation was essential for survival of melanocytes as cells expressing phosphorylation-deficient BAD were not rescued by any of the identified pathway. Furthermore, melanocytes became insensitive to kinase inhibitor-induced apoptosis when BAD expression was knocked down by BAD-shRNA. Overexpression of BAD in melanocytes stimulated faster apoptosis in response to kinase inhibitors. Taken together, our results show that BAD is acting as a convergence point for diverse survival pathways in melanocytes. Understanding the molecular mechanisms of melanocyte survival provides fundamental new insights into physiological mechanisms involved in the development of various melanocyte pathologies such as melanoma and vitiligo.


Assuntos
Apoptose , Melanócitos/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo , Sobrevivência Celular , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
2.
J Cell Mol Med ; 24(19): 11294-11306, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32853466

RESUMO

Familial Mediterranean fever (FMF) is the most common auto-inflammatory disease. It is transmitted as autosomal recessive trait with mutations in MEditerranean FeVer (MEFV) gene. Despite a typical clinical expression, many patients have either a single or no mutation in MEFV. The current work is aimed to revisit the genetic landscape of FMF disease using high-coverage whole genome sequencing. In atypical patients (carrying a single or no mutation in MEFV), we revealed many rare variants in genes associated with auto-inflammatory disorders, and more interestingly, we discovered a novel variant ( a 2.1-Kb deletion) in exon 11 of IL1RL1 gene, present only in patients. To validate and screen this patient-specific variant, a tandem of allele-specific PCR and quantitative real-time PCR was performed in 184 FMF patients and 218 healthy controls and we demonstrated that the novel deletion was absent in controls and was present in more than 19% of patients. This study sheds more light on the mutational landscape of FMF. Our discovery of a disease-specific variant in IL1RL1 gene may constitute a novel genetic marker for FMF. This finding suggesting a potential role of the IL33/ST2 signalling in the disease pathogenicity highlights a new paradigm in FMF pathophysiology.


Assuntos
Febre Familiar do Mediterrâneo/genética , Genoma Humano , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Mutação/genética , Análise de Sequência de DNA , Transdução de Sinais , Adolescente , Estudos de Casos e Controles , Variações do Número de Cópias de DNA/genética , Feminino , Deleção de Genes , Genes Modificadores , Predisposição Genética para Doença , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Pirina/genética
3.
J Transl Med ; 18(1): 192, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393282

RESUMO

BACKGROUND: Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. METHODS: DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. RESULTS: Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. CONCLUSION: Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/farmacologia
4.
Skin Pharmacol Physiol ; 31(2): 74-86, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29306952

RESUMO

BACKGROUND: Vitamin D is a secosteroid, which was initially known for its skeletal role; however, in recent years, its functions in different organs have been increasingly recognized. In this review, we will provide an overview of vitamin D functions in the skin physiology with specific focus on its role in certain inflammatory skin conditions such as psoriasis and atopic dermatitis. METHODS: A comprehensive literature search was carried out in PubMed and Google Scholar databases using keywords like "vitamin D," "skin," "atopic dermatitis," and "psoriasis." Only articles published in English and related to the study topic were included in this review. RESULTS: Vitamin D is integrally connected to the skin for its synthesis, metabolism, and activity. It regulates many physiological processes in the skin ranging from cellular proliferation, differentiation, and apoptosis to barrier maintenance and immune functions. Vitamin D deficiency is associated with the risk of psoriasis and atopic dermatitis, and several clinical/observational studies have suggested the beneficial effect of vitamin D in the therapy of these 2 inflammatory skin disorders. CONCLUSIONS: Vitamin D exerts a pleiotropic effect in the skin and could be an important therapeutic option for psoriasis and atopic dermatitis.


Assuntos
Inflamação/metabolismo , Inflamação/patologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Pele/metabolismo , Pele/patologia , Vitamina D/metabolismo , Animais , Dermatite Atópica/patologia , Humanos , Psoríase/metabolismo , Psoríase/patologia
5.
Int J Mol Sci ; 19(6)2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29849001

RESUMO

The classical function of Vitamin D, which involves mineral balance and skeletal maintenance, has been known for many years. With the discovery of vitamin D receptors in various tissues, several other biological functions of vitamin D are increasingly recognized and its role in many human diseases like cancer, diabetes, hypertension, cardiovascular, and autoimmune and dermatological diseases is being extensively explored. The non-classical function of vitamin D involves regulation of cellular proliferation, differentiation, apoptosis, and innate and adaptive immunity. In this review, we discuss and summarize the latest findings on the non-classical functions of vitamin D at the cellular/molecular level and its role in complex human diseases.


Assuntos
Vitamina D/metabolismo , Imunidade Adaptativa/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Receptores de Calcitriol/metabolismo
6.
BMC Med Genet ; 17(1): 42, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27282200

RESUMO

BACKGROUND: KCNH1 encodes a voltage-gated potassium channel that is predominantly expressed in the central nervous system. Mutations in this gene were recently found to be responsible for Temple-Baraitser Syndrome (TMBTS) and Zimmermann-Laband syndrome (ZLS). METHODS: Here, we report a new case of TMBTS diagnosed in a Lebanese child. Whole genome sequencing was carried out on DNA samples of the proband and his parents to identify mutations associated with this disease. Sanger sequencing was performed to confirm the presence of detected variants. RESULTS: Whole genome sequencing revealed three missense mutations in TMBTS patient: c.1042G > A in KCNH1, c.2131 T > C in STK36, and c.726C > A in ZNF517. According to all predictors, mutation in KCNH1 is damaging de novo mutation that results in substitution of Glycine by Arginine, i.e., p.(Gly348Arg). This mutation was already reported in a patient with ZLS that could affect the connecting loop between helices S4-S5 of KCNH1 with a gain of function effect. CONCLUSIONS: Our findings demonstrate that KCNH1 mutations cause TMBTS and expand the mutational spectrum of KCNH1 in TMBTS. In addition, all cases of TMBTS were reviewed and compared to ZLS. We suggest that the two syndromes are a continuum and that the variability in the phenotypes is the result of the involvement of genetic modifiers.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades Craniofaciais/genética , Fibromatose Gengival/genética , Hallux/anormalidades , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Unhas Malformadas/genética , Polegar/anormalidades , Anormalidades Múltiplas/diagnóstico , Anormalidades Craniofaciais/diagnóstico , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Mutacional de DNA , Canais de Potássio Éter-A-Go-Go/genética , Fibromatose Gengival/diagnóstico , Deformidades Congênitas da Mão/diagnóstico , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Masculino , Mutação de Sentido Incorreto , Unhas Malformadas/diagnóstico , Proteínas Serina-Treonina Quinases/genética , Polegar/diagnóstico por imagem , Dedos do Pé/diagnóstico por imagem
7.
JCI Insight ; 6(5)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33529170

RESUMO

The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome coronavirus (MERS-CoV) seropositive but MERS-CoV free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer and M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of Betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in nonimmunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Camelus/imunologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , COVID-19/imunologia , Camelus/virologia , Reações Cruzadas , Epitopos , Feminino , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia
8.
Oxid Med Cell Longev ; 2019: 2841814, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31871544

RESUMO

Oxidative stress is known to induce melanocyte death, but the underlying mechanisms are incompletely understood. To identify oxidative stress-induced global gene expression changes in melanocytes, we treated PIG1 melanocytes with H2O2 in a dose- and time-dependent manner and performed RNA-seq. This approach allowed us to capture the events occurring early as well as late phase after treatment with H2O2. Our bioinformatics analysis identified differentially expressed genes involved in various biological processes of melanocytes which are known to contribute to the vitiligo development, such as apoptosis, autophagy, cell cycle regulation, cell adhesion, immune and inflammatory responses, melanocyte pluripotency, and developmental signaling such as WNT and NOTCH pathways. We uncovered several novel genes that are not previously described to be involved in melanocytic response to stress nor in vitiligo pathogenesis. Quantitative PCR and western blot analysis of selected proteins, performed on PIG1 and primary human epidermal melanocytes, confirmed the RNA-seq data. Interestingly, we discovered an aberrant regulation of several transcription factors that are involved in diabetes, neurological, and psychiatric diseases, all of which are comorbid conditions in patients with vitiligo. Our results may lead to a better understanding of the molecular mechanisms underlying vitiligo pathogenesis and help developing new drug targets for effective treatment.


Assuntos
Melanócitos/metabolismo , Vitiligo/metabolismo , Vitiligo/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Biologia Computacional , Humanos , Peróxido de Hidrogênio/farmacologia , Melanócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Cell Death Dis ; 8(6): e2844, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569785

RESUMO

Cancer stem cells (CSCs) are increasingly considered to be responsible for tumor initiation, metastasis and drug resistance. The drug resistance mechanisms activated in CSCs have not been thoroughly investigated. Although neuropeptides such as vasoactive intestinal peptide (VIP) can promote tumor growth and activate antiapoptotic signaling in differentiated cancer cells, it is not known whether they can activate antiapoptotic mechanisms in CSCs. The objectives of this study are to unravel the cytoprotective effects of neuropeptides and identify antiapoptotic mechanisms activated by neuropeptides in response to anticancer drug treatment in CSCs. We enriched and purified CSCs (CD44+/high/CD24-/low or CD133+ population) from breast and prostate cancer cell lines, and demonstrated their stemness phenotype. Of the several neuropeptides tested, only VIP could protect CSCs from drug-induced apoptosis. A functional correlation was found between drug-induced apoptosis and dephosphorylation of proapoptotic Bcl2 family protein BAD. Similarly, VIP-induced cytoprotection correlated with BAD phosphorylation at Ser112 in CSCs. Using pharmacological inhibitors and dominant-negative proteins, we showed that VIP-induced cytoprotection and BAD phosphorylation are mediated via both Ras/MAPK and PKA pathways in CSCs of prostate cancer LNCaP and C4-2 cells, but only PKA signaling was involved in CSCs of DUVIPR (DU145 prostate cancer cells ectopically expressing VIP receptor) and breast cancer MCF7 cells. As each of these pathways partially control BAD phosphorylation at Ser112, both have to be inhibited to block the cytoprotective effects of VIP. Furthermore, VIP is unable to protect CSCs that express phosphorylation-deficient mutant-BAD, suggesting that BAD phosphorylation is essential. Thus, antiapoptotic signaling by VIP could be one of the drug resistance mechanisms by which CSCs escape from anticancer therapies. Our findings suggest the potential usefulness of VIP receptor inhibition to eliminate CSCs, and that targeting BAD might be an attractive strategy for development of novel therapeutics.


Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Proteína de Morte Celular Associada a bcl/genética , Antineoplásicos/farmacologia , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Niacinamida/análogos & derivados , Niacinamida/antagonistas & inibidores , Niacinamida/farmacologia , Compostos de Fenilureia/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais , Sorafenibe , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína de Morte Celular Associada a bcl/antagonistas & inibidores , Proteína de Morte Celular Associada a bcl/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética , Proteínas ras/metabolismo
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