Search

The development of nephrotoxicity largely limits the clinical use of chemotherapy. MiRNAs are able to target various genes and involved in the regulation of diverse cellular processes, including cell apoptosis and death. Our study showed that miR-181a expression was significantly increased after 5-fluorouracil (5-FU) treatment in renal mesangial cells and kidney tissue, which was associated with decreased baculoviral inhibition of apoptosis protein repeat-containing 6 (BIRC6) expression and increased apoptotic rate. Enforced miR-181a expression enhanced 5-FU-induced p53-dependent mitochondrial apoptosis, including declined Bcl-2/Bax ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase-9 and caspase-3 activation. However, inhibition of miR-181a was associated with reduced p53-mediated mitochondrial apoptosis induced by 5-FU. Moreover, miR-181a increased BIRC6 downstream gene p53 protein expression and transcriptional activity by reducing ubiquitin-mediated protein degradation. We found that miR-181a directly targeted 3′-UTR of BIRC6 mRNA and negatively regulated BIRC6 expression. In vivo study, knockdown of miR-181a with adeno-associated virus harboring miR-181a-tough decoy attenuated 5-FU-induced renal cell apoptosis, inflammation and kidney injury. In conclusion, these results demonstrate that miR-181a increases p53 protein expression and transcriptional activity by targeting BIRC6 and promotes 5-FU-induced apoptosis in mesangial cells. Inhibition of miR-181a ameliorates 5-FU-induced nephrotoxicity, suggesting that miR-181a may be a novel therapeutic target for nephrotoxicity treatment during chemotherapy.

Orai1-dependent Ca2+ entry plays an essential role in inflammatory response through regulating T cell and macrophage activation and neutrophil infiltration. However, whether Orai1 Ca2+ entry contributes to endothelial activation, one of the early steps of vascular inflammation, remains elusive. In the present study, we observed that knockdown of Orai1 reduced, whereas overexpression of Orai1 potentiated, TNFα-induced expression of adhesion molecules such as ICAM-1 and VCAM-1 in HUVECs, and subsequently blocked adhesion of monocyte to HUVECs. In vivo, Orai1 downregulation attenuated TNFα-induced ICAM-1 and VCAM-1 expression in mouse aorta and the levels of pro-inflammatory cytokines in the serum. In addition, Orai1 knockdown also dramatically decreased the expression of pro-inflammatory cytokines and neutrophil infiltration in the lung after TNFα treatment, and thus protected lung tissue injury. Notably, among all isoforms of nuclear factor of activated T cells (NFATs), TNFα only triggered NFATc4 nuclear accumulation in HUVECs. Knockdown of Orai1 or inhibition of calcineurin prevented TNFα-induced NFATc4 nuclear translocation and reduced ICAM-1 and VCAM-1 expression in HUVECs. Overexpression of NFATc4 further enhanced ICAM-1 and VCAM-1 expression induced by TNFα. Our study demonstrates that Orai1-Ca2+-calcineurin-NFATc4 signaling is an essential inflammatory pathway required for TNFα-induced endothelial cell activation and vascular inflammation. Therefore, Orai1 may be a potential therapeutic target for treatment of inflammatory diseases.

MicroRNAs have emerged as important post-transcriptional regulators of gene expression and are involved in diverse diseases and cellular process. Decreased expression of miR-181a has been observed in the patients with coronary artery disease, but its function and mechanism in atherogenesis is not clear. This study was designed to determine the roles of miR-181a-5p, as well as its passenger strand, miR-181a-3p, in vascular inflammation and atherogenesis. We found that the levels of both miR-181a-5p and miR-181a-3p are decreased in the aorta plaque and plasma of apoE−/− mice in response to hyperlipidemia and in the plasma of patients with coronary artery disease. Rescue of miR-181a-5p and miR-181a-3p significantly retards atherosclerotic plaque formation in apoE−/− mice. MiR-181a-5p and miR-181a-3p have no effect on lipid metabolism but decrease proinflammatory gene expression and the infiltration of macrophage, leukocyte and T cell into the lesions. In addition, gain-of-function and loss-of-function experiments show that miR-181a-5p and miR-181a-3p inhibit adhesion molecule expression in HUVECs and monocytes-endothelial cell interaction. MiR-181a-5p and miR-181a-3p cooperatively receded endothelium inflammation compared with single miRNA strand. Mechanistically, miR-181a-5p and miR-181a-3p prevent endothelial cell activation through blockade of NF-κB signaling pathway by targeting TAB2 and NEMO, respectively. In conclusion, these findings suggest that miR-181a-5p and miR-181a-3p are both antiatherogenic miRNAs. MiR-181a-5p and miR-181a-3p mimetics retard atherosclerosis progression through blocking NF-κB activation and vascular inflammation by targeting TAB2 and NEMO, respectively. Therefore, restoration of miR-181a-5p and miR-181a-3p may represent a novel therapeutic approach to manage atherosclerosis.

Increasing evidence supports that activation of store‐operated Ca2+ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca2+‐dependent autophagy. [Ca2+]i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca2+ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.

Objective— To determine the role of orai1 store–operated Ca 2+ entry in foam cell formation and atherogenesis. Approach and Results— Acute administration of oxidized low-density lipoprotein (oxLDL) activates an orai1-dependent Ca 2+ entry in macrophages. Chelation of intracellular Ca 2+ , inhibition of orai1 store–operated Ca 2+ entry, or knockdown of orai1 dramatically inhibited oxLDL-induced upregulation of scavenger receptor A, uptake of modified LDL, and foam cell formation. Orai1-dependent Ca 2+ entry induces scavenger receptor A expression and foam cell formation through activation of calcineurin but not calmodulin kinase II. Activation of nuclear factor of activated T cells is not involved in calcineurin signaling to foam cell formation. However, oxLDL dephosohorylates and activates apoptosis signal–regulating kinase 1 in macrophages. Orai1 knockdown prevents oxLDL-induced apoptosis signal–regulating kinase 1 activation. Knockdown of apoptosis signal–regulating kinase 1, or inhibition of its downstream effectors, JNK and p38 mitogen-activated protein kinase, reduces scavenger receptor A expression and foam cell formation. Notably, orai1 expression is increased in atherosclerotic plaques of apolipoprotein E −/− mice fed with high-cholesterol diet. Knockdown of orai1 with adenovirus harboring orai1 siRNA or inhibition of orai1 Ca 2+ entry with SKF96365 for 4 weeks dramatically inhibits atherosclerotic plaque development in high-cholesterol diet feeding apolipoprotein E −/− mice. In addition, inhibition of orai1 Ca 2+ entry prevents macrophage apoptosis in atherosclerotic plaque. Moreover, the expression of inflammatory genes in atherosclerotic lesions and the infiltration of myeloid cells into the aortic sinus plaques are decreased after blocking orai1 signaling. Conclusions— Orai1-dependent Ca 2+ entry promotes atherogenesis possibly by promoting foam cell formation and vascular inflammation, rendering orai1 Ca 2+ channel a potential therapeutic target against atherosclerosis.

Background Previous work has demonstrated that the volume-regulated chloride channel is activated during foam cell formation, and inhibition of chloride movement prevents intracellular lipid accumulation. However, the mechanism explaining how chloride movement promotes foam cell formation is not clear. Methods and results Foam cell formation was determined by Oil Red O staining. Western blotting and co-immunoprecipitation were used to examine protein expression and protein-protein interaction. [Cl(-)]iwas measured using 6-methoxy-N-ethylquinolinium iodide dye. The results showed that [Cl(-)]iwas decreased in monocytes/macrophages from patients with hypercholesterolemia and from apoE(-/-)mice fed with a high-fat diet. Lowering [Cl(-)]iupregulated scavenger receptor A (SR-A) expression, increased the binding and uptake of oxLDL, enhanced pro-inflammatory cytokine production and subsequently accelerated foam cell formation in macrophages from humans and mice. In addition, low Cl(-)solution stimulated the activation of JNK and p38 mitogen-activated protein kinases. Inhibition of JNK and p38 blocked Cl(-)reduced medium-induced SR-A expression and lipid accumulation. In contrast, reduction of [Cl(-)]ipromoted the interaction of SR-A with caveolin-1, thus facilitating caveolin-1-dependent SR-A endocytosis. Moreover, disruption of caveolae attenuated SR-A internalization, JNK and p38 activation, and ultimately prevented SR-A expression and foam cell formation stimulated by low Cl(-)medium. Conclusions This data provide strong evidence that reduction of [Cl(-)]iis a critical contributor to intracellular lipid accumulation, suggesting that modulation of [Cl(-)]iis a novel avenue to prevent foam cell formation and atherosclerosis.

Orai1-dependent Ca

Evidence supports an important role of Ca2+ release-activated Ca2+ channel protein 1 (Orai1)-mediated Ca2+ entry in the development of renal fibrosis, a common pathologic feature of CKDs that lead to ESRD, but the molecular mechanisms remain unclear. We determined the role of Orai1 calcium channel in renal fibrosis induced by high-fat diet and by unilateral ureteral obstruction. Mouse kidneys with fibrosis had higher levels of Orai1 protein expression than did kidneys without fibrosis. In vivo knockdown of Orai1 with adenovirus harboring Orai1-short hairpin RNA or inhibition of Orai1 with SKF96365 dramatically prevented renal fibrosis and significantly decreased protein expression of fibronectin, α‑smooth muscle actin, and TGF‑β1 in the kidney cortex of ApoE-/- mice on a high-fat diet and in the obstructed kidneys of mice with unilateral ureteral obstruction. Compared with kidney biopsy specimens of patients with glomerular minimal change disease, those of patients with fibrotic nephropathy had higher expression levels of Orai1. In cultured human proximal tubule epithelial cells (HK2), knockdown of Orai1 Ca2+ channel with adenovirus-Orai1-short hairpin RNA markedly inhibited TGF-β1-induced intracellular Ca2+ influx and phosphorylation of smad2/3. Knockdown or blockade of the Orai1 Ca2+ channel in HK2 cells also prevented epithelial-to-mesenchymal transition induced by TGF‑β1. In conclusion, blockade of the Orai1 Ca2+ channel prevented progression of renal fibrosis in mice, likely by suppressing smad2/3 phosphorylation and TGF-β1-induced epithelial-to-mesenchymal transition. These results render the Orai1 Ca2+ channel a potential therapeutic target against renal fibrosis.