RESUMEN
Left ventricular hypertrophy (LVH) is a major risk factor for adverse cardiovascular events. Recently, a novel candidate gene encoding the carboxypeptidase X member 2 (CPXM2) was found to be associated with hypertension-induced LVH. CPXM2 belongs to the M14 family of metallocarboxypeptidases, yet it lacks detectable enzyme activity, and its function remains unknown. Here, we investigated the impact of micro (mi)RNA-29b, miRNA-195, and miRNA-497 on the posttranscriptional expression control of CPXM2. Candidate miRNAs for CPXM2 expression control were identified in silico. CPXM2 expression in rat cardiomyocytes (H9C2) was characterized via real-time PCR, Western blotting, and immunofluorescence. Direct miRNA/target mRNA interaction was analysed by dual luciferase assay. CPXM2 was expressed in H9C2 and co-localised with z-disc associated protein PDZ and LIM domain 3 (Pdlim3). Transfection of H9C2 with miRNA-29b, miRNA-195, and miRNA-497 led to decreased levels of CPXM2 mRNA and protein, respectively. Results of dual luciferase assays revealed that miRNA-29b and miRNA-497, but not miRNA-195, directly regulated CPXM2 expression on a posttranscriptional level via binding to the 3'UTR of CPXM2 mRNA. We identified two miRNAs capable of the direct posttranscriptional expression control of CPXM2 expression in rat cardiomyocytes. This novel data may help to shed more light on the-so far-widely unexplored expression control of CPXM2 and its potential role in LVH.
Asunto(s)
Carboxipeptidasas/genética , Hipertrofia Ventricular Izquierda/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Animales , Células Cultivadas , Regulación de la Expresión Génica/genética , Hipertensión/genética , Miocitos Cardíacos/patología , ARN Mensajero/genética , RatasRESUMEN
INTRODUCTION: Transmembrane protein (TMEM) 63C is a member of the TMEM gene family and was recently linked to glomerular filtration barrier function and albuminuria. Its molecular function and expression regulation are largely unknown. OBJECTIVE: In this study, we set out to characterize the regulating impact of microRNAs (miRNAs) such as miRNA-564 (miR-564) on TMEM63C expression in renal cells. Also, we examined the influence of transforming growth factor beta (TGF-ß) on TMEM63C expression and the potential impact of TMEM63C inhibition on epithelial-mesenchymal transition (EMT) in renal cells and on cell viability in human embryonic kidney 293 cells (HEK 293). METHODS: Expression analyses were done using real-time PCR and Western blot. Dual luciferase assay was performed to determine the miRNA-mediated expression control. Cell viability was assessed via trypan blue exclusion staining. RESULTS AND CONCLUSIONS: MiR-564 reduced TMEM63C expression in HEK 293 and human podocytes (hPC). The treatment of renal cells with TGF-ß led to an increased expression of TMEM63C. Moreover, a reduced TMEM63C expression was associated with a changed ratio of EMT marker proteins such as α-smooth muscle actin versus E-cadherin in HEK 293 and decreased nephrin expression in hPC. In addition, cell viability was reduced upon inhibition of TMEM63C expression in HEK 293. This study demonstrates first mechanisms involved in TMEM63C expression regulation and a link to EMT in renal cells.
Asunto(s)
Albuminuria/genética , Canales de Calcio/genética , MicroARNs/genética , Podocitos/metabolismo , Factor de Crecimiento Transformador beta/genética , Albuminuria/metabolismo , Canales de Calcio/metabolismo , Línea Celular , Supervivencia Celular , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Células HEK293 , Humanos , MicroARNs/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
AIMS: Human podocytes (hPC) play an important role in the pathogenesis of renal diseases. In this context, angiotensin II (Ang II) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) play a crucial role in podocyte injury. Recently, transmembrane protein (Tmem) 63c, a member of the Tmem-family was found to be expressed in kidney and associated with podocyte function. In this study, we analysed the expression regulation and functional impact of Tmem63c on cell viability and apoptosis in hPC in the context of Ang II activation. MATERIALS AND METHODS: Expression of Tmem63c in response to Ang II and the NFκB inhibitor Bay 11-7082 was analysed by Real-Time PCR and Western blotting. Cellular functions were determined by functional assays. KEY FINDINGS: We found Ang II to induce Tmem63c expression in hPC in a concentration-dependent manner. Inhibition of NFκB by Bay 11-7082 reduced basal as well as Ang II-induced Tmem63c expression. SiRNA-mediated down-regulation of Tmem63c diminished cell viability and protein kinase B (Akt) signaling and increased cell apoptosis of resting as well as Ang II-activated hPC. SIGNIFICANCE: These data show that Ang II induced the expression of Tmem63c in hPC, possibly via NFκB-dependent mechanisms. Moreover, down-regulation of Tmem63c was associated with reduced cell viability, indicating Tmem63c to be a potential pro-survival factor in hPC.
Asunto(s)
Angiotensina II/farmacología , Canales de Calcio/metabolismo , Podocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Podocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Human podocytes (hPC) are essential for maintaining normal kidney function and dysfunction or loss of hPC play a pivotal role in the manifestation and progression of chronic kidney diseases including diabetic nephropathy. Previously, α-Lipoic acid (α-LA), a licensed drug for treatment of diabetic neuropathy, was shown to exhibit protective effects on diabetic nephropathy in vivo. However, the effect of α-LA on hPC under non-diabetic conditions is unknown. Therefore, we analyzed the impact of α-LA on cell viability and expression of nephrin and zinc finger protein 580 (ZNF580) in normal hPC in vitro. Protein analyses were done via Western blot techniques. Cell viability was determined using a functional assay. hPC viability was dynamically modulated via α-LA stimulation in a concentration-dependent manner. This was associated with reduced nephrin and ZNF580 expression and increased nephrin phosphorylation in normal hPC. Moreover, α-LA reduced nephrin and ZNF580 protein expression via 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) inhibition. These data demonstrate that low α-LA had no negative influence on hPC viability, whereas, high α-LA concentrations induced cytotoxic effects on normal hPC and reduced nephrin and ZNF580 expression via NF-κB inhibition. These data provide first novel information about potential cytotoxic effects of α-LA on hPC under non-diabetic conditions.