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1.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(9): 958-971, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28599891

RESUMO

Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.


Assuntos
Membrana Celular/metabolismo , Queratinócitos/metabolismo , Lipídeos/fisiologia , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Reepitelização/fisiologia , Esfingomielinas/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Colesterol/metabolismo , Humanos , Toxinas Biológicas/metabolismo
2.
Saudi Dent J ; 36(1): 117-122, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38375373

RESUMO

Background: This study investigates the prevalence, distribution and risk indicators of buccal gingival recessions (GRs) in periodontitis patients. Methods: A retrospective examination of 400 periodontitis patients files was performed using an operating sheet. Univariate logistic regression analysis was performed to identify risk indicators of GRs. Multivariate regression analysis was conducted for selected variables with p < 0.05. Results: 354/400 (88.5 %) patients have at least one GR ≥ 1 mm. The prevalence of recession type (RT) at the patient level was 0.5 %, 2.25 % and 85.75 % for RT1, RT2 and RT3 respectively. Lower incisors are the most affected teeth (79.8 %). Upper canines present the lowest frequency (41.8 %). The univariate logistic regression showed that age (SE = 0.021; 95 % CI 1.01-1.10; p = 0.006), plaque index (SE = 0.50; 95 % CI 1.49-10.50; p = 0.006), level of plaque control (SE = 0.529; 95 % CI 0.90-0.72; p = 0.010) and periodontitis stage (SE = 0.41; 95 % CI 1.41-7.07; p = 0.005) were significantly associated with the presence of GR. In the multivariate regression model, significant results were confirmed only for age (SE = 0.021; 95 % CI 1.02-1.17; p = 0.006) and periodontitis stage (SE = 0.41; 95 % CI 1.35-6.75; p = 0.007). Conclusion: The cross-sectional study showed a high prevalence of GRs. Lower incisors were the most affected teeth. Most patients have GRs with advanced interproximal attachment loss (RT3 GRs). Age, plaque index, level of plaque control and periodontitis stage resulted as risk indicators of GRs.

4.
PLoS One ; 18(1): e0279028, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662875

RESUMO

Nod-Like Receptor Pyrin domain-containing protein 6 (NLRP6), a member of the Nucleotide-oligomerization domain-Like Receptor (NLR) family of proteins, assembles together with the ASC protein to form an inflammasome upon stimulation by bacterial lipoteichoic acid and double-stranded DNA. Besides its expression in myeloid cells, NLRP6 is also expressed in intestinal epithelial cells where it may contribute to the maintenance of gut homeostasis and negatively controls colorectal tumorigenesis. Here, we report that NLRP6 is very faintly expressed in several colon cancer cell lines, detected only in cytoplasmic small dots were it colocalizes with ASC. Consequently, it is very hardly detected by standard western-blotting techniques by several presently available commercial antibodies which, in contrast, highly cross-react with a protein of 90kDa that we demonstrate to be unrelated to NLRP6. We report here these results to caution the community not to confuse the 90kDa protein with the endogenous human NLRP6.


Assuntos
Inflamassomos , Neoplasias , Humanos , Inflamassomos/metabolismo , Homeostase , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular
6.
Arch Dermatol Res ; 309(1): 63-69, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27833999

RESUMO

Atopic dermatitis (AD) skin is characterized by over-expression of interleukin (IL)-4, IL-13 and IL-25. When methyl-ß-cyclodextrin (MßCD) treatment preceded exposure to these interleukins, combination of both treatments was found to mimic hallmarks of AD in vitro, such as barrier weakening, histological alterations and typical signaling responses in a reconstructed human epidermis (RHE). However, the respective role of each IL and whether any of them is critical when combined with MßCD treatment was unknown. Therefore, this work aimed to distinguish RHE responses after exposure to MßCD and each one of the three IL reported to mimic typical features of AD. IL-4 incubation preceded by MßCD was found responsible for altered histology, as well as for barrier alterations, evidenced by electrical resistance and dye permeation measurements. This combination further decreased loricrin (LOR) immunoreactivity, whereas mainly IL-25, combined to MßCD treatment, was able to downregulate filaggrin (FLG) mRNA level. Carbonic anhydrase II (CA2) and hyaluronan synthase 3 (HAS3), two other markers up-regulated in AD, were also induced when MßCD treatment was followed by IL-4, whilst the expression of neural epidermal growth factor-like 2 (NELL2) was up-regulated by paired IL-4 and IL-13. In conclusion, multiple features of AD were found in this in vitro model mainly when treatment of RHE by IL-4 was conducted after preliminary MßCD incubation.


Assuntos
Dermatite Atópica/patologia , Epiderme/efeitos dos fármacos , Interleucina-4/farmacologia , Queratinócitos/efeitos dos fármacos , beta-Ciclodextrinas/farmacologia , Anidrase Carbônica II/genética , Anidrase Carbônica II/metabolismo , Forma Celular , Células Cultivadas , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Impedância Elétrica , Epiderme/metabolismo , Epiderme/patologia , Proteínas Filagrinas , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Permeabilidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Oncotarget ; 8(42): 72324-72341, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-29069790

RESUMO

Breast cancer remains a research priority due to its invasive phenotype. Although the role of ion channels in cancer is now well established, the role of inositol (1,4,5)-trisphosphate (IP3) receptors (IP3Rs) remains enigmatic. If the three IP3Rs subtypes expression have been identified in various cancers, little is known about their physiological role. Here, we investigated the involvement of IP3R type 3 (IP3R3) in the migration processes of three human breast cancer cell lines showing different migration velocities: the low-migrating MCF-7 and the highly migrating and invasive MDA-MB-231 and MDA-MB-435S cell lines. We show that a higher IP3R3 expression level, but not IP3R1 nor IP3R2, is correlated to a stronger cell line migration capacity and a sustained calcium signal. Interestingly, silencing of IP3R3 highlights an oscillating calcium signaling profile and leads to a significant decrease of cell migration capacities of the three breast cancer cell lines. Conversely, stable overexpression of IP3R3 in MCF-7 cells significantly increases their migration capacities. This effect is completely reversed by IP3R3 silencing. In conclusion, we demonstrate that IP3R3 expression level increases the migration capacity of human breast cancer cells by changing the calcium signature.

8.
Eur J Cancer ; 49(17): 3738-51, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23992640

RESUMO

BACKGROUND: The implication of ion channels and inositol 1,4,5-trisphosphate (IP3)-induced Ca(2+) signalling (IICS) in the carcinogenesis processes, including deregulation of cell proliferation, migration and invasion, is increasingly studied. Studies from our laboratory have shown that type 3 IP3 receptor (IP3R3) and voltage- and Ca(2+)-dependent K(+) channels BKCa channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP3R3 and BKCa channel) in normal and in breast cancer cells. METHODS: MCF-7 and MCF-10A cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay in the presence or absence of adenosine triphosphate (ATP). Furthermore, cell-cycle analysis was carried out and cell cycle protein expression was examined by Western blotting. Immunocytochemistry and co-immunoprecipitation assays were used to check co-localisation of BKCa and IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca(2+) imaging were performed to assess the functional interaction. RESULTS: Our results are in favour of a functional and a molecular coupling between IP3R3 and BKCa channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BKCa and/or IP3R3 has been reduced by specific small interfering RNAs (siRNAs). Flow cytometry experiments showed that both siRNAs led to cell cycle arrest in the G0/G1 phase and these results were confirmed by the analysis of cell cycle protein expression. Specifically, BKCa and IP3R3 silencing decreased both cyclin-D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Furthermore, ATP elicited a phospholipase C (PLC)-dependent elevation of internal Ca(2+) that triggered in turn an iberiotoxin (IbTx)- and a tetra-ethyl-ammonium (TEA)-sensitive membrane hyperpolarisation that was strongly reduced in the cells with silenced IP3R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca(2+) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP3R3 also co-immunoprecipitated and this interaction seemed to occur in cholesterol-enriched microdomains. Conversely, in the normal breast cell line MCF-10A, neither ATP application nor BKCa silencing affected cell proliferation. Furthermore, IP3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line. CONCLUSION: Altogether, our results suggest a molecular and functional link between BKCa channel and IP3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.


Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Proliferação de Células , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Imunoprecipitação , Inositol 1,4,5-Trifosfato/metabolismo , Células MCF-7 , Ligação Proteica
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