RESUMEN
We investigated whether docosahexaenoic acid (DHA), a dietary n-3 fatty acid, modulates calcium (Ca2+) signaling and cell cycle progression in human Jurkat T-cells. Our study demonstrates that DHA inhibited Jurkat T-cell cycle progression by blocking their passage from S phase to G2/M phase. In addition, DHA decreased the plasma membrane expression of TRPC3 and TRPC6 calcium channels during T-cell proliferation. Interestingly, this fatty acid increased plasma membrane expression of TRPC6 after 24 h of mitogenic stimulation by phorbol-13-myristate-12-acetate (PMA) and ionomycin. These variations in the membrane expression of TRPC3 and TRPC6 channels were not directly correlated with the mRNA expression, indicating that it was a post-translational phenomenon. DHA increased free intracellular calcium concentrations, [Ca2+]i, via opening TRPC3 and TRPC6 channels. We conclude that the anti-proliferative effect of DHA might involve the modulation of TRPC3 and TRPC6 channels in human T-cells.
Asunto(s)
Membrana Celular/metabolismo , Ácidos Docosahexaenoicos/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Linfocitos T/metabolismo , Canales Catiónicos TRPC/biosíntesis , Canal Catiónico TRPC6/biosíntesis , Humanos , Ionomicina/farmacología , Células Jurkat , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
OBJECTIVE: Transient receptor potential canonical (TRPC) channels are involved in neovascularization repairing after vascular injury in many tissues. However, whether TRPCs play a regulatory role in the development of diabetic retinopathy (DR) has rarely been reported. In the present study, we selected TRPC1, 3, and 6 to determine their roles and mechanism in human retina vascular endothelial cells (HREC) under high glucose (HG) conditions. METHODS: HRECs were cultured in vitro under HG, hyper osmosis, and normal conditions. The expression of TRPC1, 3, and 6 in the cells at 24 and 48 h were detected by RT-polymerase chain reaction (PCR), Western blot and cell immunohistochemistry (IHC); In various concentrations, SKF96365 acted on HG cultured HRECs, the expression of vascular endothelial growth factor (VEGF) were detected by the same methods above; and the CCK-8, Transwell, cell scratch assay, and Matrigel assay were used to assess cell proliferation, migration, and lumen formation. RESULTS: The RT-PCR, Western blot, and IHC results showed that TRPC1 expression was increased, and TRPC6 mRNA expression was increased under high-glucose conditions. SKF96365 acted on HG cultured HRECs that VEGF expression was significantly decreased. The CCK-8 assay, Transwell assay, cell scratch assay, and Matrigel assay showed that cell proliferation, migration, and lumen formation were downregulated by SKF96365. CONCLUSION: HG can induce increased expression of TRPC1 and 6 in HRECs. Inhibition of the TRPC pathway not only can decrease VEGF expression but also can prevent proliferation, migration, and lumen formation of HRECs induced by HG. Inhibition of TRPC channels is expected to become a drug target for DR.
Asunto(s)
Endotelio Vascular/patología , Regulación de la Expresión Génica/efectos de los fármacos , Neovascularización Retiniana/genética , Vasos Retinianos/metabolismo , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6/genética , Animales , Western Blotting , Movimiento Celular , Proliferación Celular , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Glucosa/farmacología , Humanos , ARN/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Canales Catiónicos TRPC/biosíntesis , Canal Catiónico TRPC6/biosíntesisRESUMEN
The lamina cribrosa (LC) in glaucoma is with augmented production of extracellular matrix proteins (ECM) and connective tissue fibrosis. Fundamental pathological mechanisms for this fibrosis comprise fibrotic growth factors and oxidative stress. Transient receptor potential canonical channels (TRPC) channels play a key role in ECM fibrosis. Here, we study TRPC expression in glaucomatous LC cells, and investigate the role of TRPC in oxidative stress induced-profibrotic ECM gene transcription and cell proliferation in normal LC cells. Age-matched human LC cells (normal, n = 3 donors; glaucoma, n = 3 donors) were used. Hydrogen peroxide (H2O2, 100 µM), was used to induce oxidative stress in LC cells in the presence or absence of the pan TRPC inhibitor SKF96365 (10 µM) or knockdown of TRPC1/6 with siRNA. After treatments, ECM gene transcription, LC cell viability and proliferation and the phosphorylation of the transcription factor NFATc3, were measured using real time RT-PCR, colorimetric cell counting with the methyl-thiazolyl tetrazolium salt (MTS) assay, and Western immunoblotting, respectively. Results showed that TRPC1/C6 transcript and protein expression levels were significantly (p < 0.05) enhanced in glaucoma LC cells. Both SKF96365 and siRNA-TRPC1/C6 treatments significantly reduced the oxidative stress induced-ECM gene expression (transforming growth factor-ß1 (TGFß1), alpha smooth muscle actin (α-SMA), and collagen type 1A1 (Col1A1)), and cell proliferation in normal and glaucoma LC cells. Also, SKF96365 treatment inhibited the H2O2-induced NFATc3 protein dephosphorylation in LC cells. In conclusion, TRPC1/C6 expression is enhanced in glaucoma LC cells. These channels may contribute to oxidative stress-induced ECM gene transcription and cell proliferation in normal and glaucoma LC cells through Ca2+-NFATc3 signaling pathway mechanism. TRPC1 and TRPC6 channels could be important therapeutic targets to prevent ECM remodeling and fibrosis development in glaucoma optic neuropathy.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Glaucoma/genética , Disco Óptico/patología , ARN/genética , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6/genética , Western Blotting , Proliferación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Glaucoma/metabolismo , Glaucoma/patología , Humanos , Disco Óptico/metabolismo , Canales Catiónicos TRPC/biosíntesis , Canal Catiónico TRPC6/biosíntesis , Transcripción GenéticaRESUMEN
The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes.
Asunto(s)
Glucosa/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Podocitos/metabolismo , Canal Catiónico TRPC6/biosíntesis , Animales , Calcio/metabolismo , Línea Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Podocitos/efectos de los fármacos , Ratas , Canal Catiónico TRPC6/efectos de los fármacos , Proteína de la Zonula Occludens-1/biosíntesisRESUMEN
BACKGROUND: Transient receptor potential canonical (TRPC) 6 inhibits Aß in Alzheimer's disease (AD) mouse brain and improves the behavioral performance. AIMS: To evaluate the association of TRPC6 expression in peripheral leucocytes from AD and mild cognitive impairment (MCI) patients and to explore its potential value in early diagnosis of AD. METHODS: TRPC6 mRNA levels in peripheral leucocytes were detected by quantitative real-time PCR. The Spearman correlation test was used to ascertain the associations between TRPC6 and the scores of MMSE, ADL, CSDD, CDR. The Receiver Operating Characteristic (ROC) curve was drawn to evaluate the diagnostic potential of TRPC6 for AD and MCI. RESULTS: There were 108â¯CE, 136 MCI, 164 Con and 60 PD in the study. The expression of TRPC6 mRNA level in peripheral leucocytes was significantly lower: 1) in patients with AD and MCI compared to Con; 2) in AD compared to MCI; 3) in hospitalized AD compared to AD from communities. There was a significantly positive correlation between TRPC6 mRNA and MMSE score (pâ¯=â¯.001, Râ¯=â¯0.327). Significantly inverse correlations were found between TRPC6 and CDR score (pâ¯<â¯0.001, Râ¯=â¯-0.303) as well as between TRPC6 and ADL score (pâ¯=â¯.001, Râ¯=â¯-0.342) for all AD. The area under curve of ROC was 0.881 for the classification of AD, and 0.706 for the classification of MCI, respectively. CONCLUSION: TRPC6 expression is inversely correlated with cognitive performance of AD. TRPC6 in peripheral leucocytes may be a potential biomarker for the diagnosis of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Disfunción Cognitiva/metabolismo , Canal Catiónico TRPC6/biosíntesis , Anciano , Estudios de Casos y Controles , Diagnóstico Precoz , Femenino , Hospitalización , Humanos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/metabolismo , Escalas de Valoración Psiquiátrica/estadística & datos numéricosRESUMEN
PURPOSE: It has been suggested that FK506 could improve some symptoms of OAB in both clinical settings and animal models; however, its mechanism of action is not well-understood. Here, we investigated the effect of FK506 on TRPC6 in bladder smooth muscle, and explored the possible involvement of TRPC6 in OAB. METHODS: FK506 was injected intraperitoneally into rats in which OAB was induced via BOO, and urodynamic indices were recorded. Rats and human bladder smooth muscle tissues with or without OAB were examined for TRPC6 expression by western blot, RT-PCR and IF staining. Cultured BSMCs were treated with PDGF, TRPC6 siRNAs and FK506. Then the TRPC6 expression and cellular proliferation were examined, and the Ca2+ influx and contractility of BSMCs were examined by time-lapse Ca2+ imaging and collagen gel contraction. Finally, IF and Co-IP were performed to test the effects of FK506 on NFAT translocation to the nucleus and the interaction of TRPC6 with FKBP12, respectively. RESULTS: FK506 improved urodynamic indices of OAB rats, and TRPC6 was expressed in rats and human bladder tissues. TRPC6 elevation in OAB rats was inhibited by FK506, and this inhibition coincided with improvements in urodynamic indices. PDGF enhanced TRPC6 expression, cellular proliferation, Ca2+ influx and contractility of BSMCs, and these effects were inhibited by TRPC6 siRNAs and FK506. FK506 inhibited NFAT translocation to the nucleus and disrupted the interaction of TRPC6 with FKBP12. CONCLUSIONS: Our results collectively indicate that FK506 may be used to treat OAB, and that TRPC6 may serve as an attractive target for therapeutic intervention in OAB.