PDIA4, a novel ER stress chaperone, modulates adiponectin expression and inflammation in adipose tissue.

Increasing evidence supporting a causal link between obesity and endoplasmic reticulum (ER) stress in adipose tissue is being reported. Protein disulfide isomerase 4 (PDIA4) is a novel ER chaperone involved in the pancreatic ß-cells pathogenesis in diabetes. However, the role of PDIA4 in obesity progression remains poorly understood. To assess the relationship between PDIA4, adiponectin, and metformin, we used the palmitate-induced inflammation in hypertrophic adipocytes and the high-fat diet-induced obesity mouse model. Our results revealed that palmitate-induced hypertrophic adipocytes exhibit obesity-associated conditions such as increased lipid accumulation, inflammation, and reduced glucose uptake. Pharmacological and genetic inhibition of PDIA4 significantly reverses these obesity-associated conditions in adipocytes. PDIA4 mechanistically promotes obesity progression via adiponectin downregulation. Furthermore, metformin modulates PDIA4 and adiponectin expression and improves obesity-associated conditions in both in vitro adipocytes and in vivo mouse models. Serum PDIA4 concentrations are also associated with body mass index, adiponectin, triglycerides, and inflammatory cytokines in humans. This is the first study demonstrating that PDIA4 modulates adipocytes by downregulating adiponectin. Moreover, metformin may serve as a potential therapeutic for preventing obesity via PDIA4-targeting.

Circulating protein disulfide isomerase family member 4 is associated with type 2 diabetes mellitus, insulin sensitivity, and obesity.

AIMS: Endoplasmic reticulum (ER) stress is associated with obesity and type 2 diabetes mellitus (T2DM) and increasing evidence demonstrates that some ER stress markers can represent the severity of metabolic dysfunction in either cellular or animal models. However, no appropriate molecule has been identified to demonstrate these relationships in clinical practice. METHODS: To determine whether the serum level of the ER chaperone, protein disulfide isomerase family A, member 4 (PDIA4), is associated with type 2 diabetes mellitus, obesity, and insulin sensitivity, we conducted a cross-sectional study for which a total of 553 adults, including 159 with normal glucose tolerance (NGT), 169 with prediabetes (Pre-DM), and 225 with newly diagnosed T2DM, were recruited. RESULTS: Serum PDIA4 levels were significantly higher in patients with T2DM than in those with NGT (P < 0.001), even after adjustment for potential confounders. These levels correlated positively with fasting plasma glucose, BMI, waist circumference as well as high-sensitivity C-reactive protein levels, and negatively and strongly correlated with insulin sensitivity. In a multivariate logistic regression analysis, higher serum PDIA4 concentration was observed to be significantly associated with an increased risk of T2DM. CONCLUSIONS: Our findings provide new mechanistic insights linking ER stress, T2DM, insulin sensitivity, and obesity, which may, in part, account for the ER chaperone properties associated with PDIA4. The results suggest that PDIA4 may serve as a potential instigator of and a putative therapeutic target for T2DM.

Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation.

Obesity results from an imbalance between caloric intake and energy expenditure, and it is highly associated with colorectal carcinogenesis and therapeutic resistance in patients with colorectal cancer (CRC). Dysregulation of adipokine production in obesity has been reported to cause malignant behaviors in CRC. Leptin, which is the principal hormone secreted by adipocytes and an obesity-associated adipokine, is significantly overexpressed in CRC tissues. However, the effect of leptin on chemoresistance in CRC is unclear. Therefore, the aim of this study was to clarify the role of leptin and the underlying mechanisms in mediating 5-fluorouracil (5-FU) resistance in CRC. We used palmitate to artificially generate obese adipocytes. As expected, lipid accumulation was significantly increased in obese adipocytes. We demonstrated that CRC cells incubated with conditioned media (CM) harvested from obese adipocytes were associated with increased resistance to 5-FU. Notably, this increase in resistance to 5-FU was through the elevated production and secretion of leptin. Leptin could further stimulate the expression of AXL and activate its downstream signaling molecule, PLCγ, thereby resulting in an increased expression of p-glycoprotein (P-gp) in CRC cells. Mechanistically, leptin induced AXL expression via the inhibition of AMPK and subsequent increase in YAP activation and nuclear translocation. In addition, nuclear YAP interacted with TEAD and promoted the occupancy of TEAD on the AXL promoter, thereby stimulating AXL promoter activity after leptin treatment. Furthermore, leptin neutralization rescued the sensitivity of CRC tumors to 5-FU in mice fed on a high-fat diet (HFD). These results indicated that leptin mediated 5-FU resistance through YAP-dependent AXL overexpression in CRC.

Dysregulation of the miR-30a/BiP axis by cigarette smoking accelerates oral cancer progression.

BACKGROUND: Cigarette smoking is the most significant cause of oral cancer progression. Cigarette smoke condensate (CSC) has been shown to induce endoplasmic reticulum (ER) stress. Binding immunoglobulin protein (BiP) being as an ER stress regulator, has been reported to be implicated in malignant behaviors. Therefore, the aim of this study was to investigate the role of the ER stress-responsive protein, BiP, in CSC-induced oral squamous cell carcinoma (OSCC) malignancy. METHODS: The biological role of BiP in CSC-induced tumor progression was investigated in OSCC cells (YD38 and SCC25) and in a tumor xenograft mouse model. The expressions of related genes were investigated using quantitative RT-PCR and Western blot analysis. Cell migration and invasion were assessed using scratch wound healing and Transwell invasion assays. The effects of conditioned media from OSCC cells on the angiogenic activities of endothelial cells were analyzed using a tube formation assay. The interaction between miR-30a and BiP mRNA was detected using a luciferase reporter assay. RESULTS: Our results demonstrated that CSC increased the expression of BiP in time- and dose-dependent manners in YD38 and SCC25 cells, and that silencing BiP abrogated CSC-induced cell invasion and tumor-associated angiogenesis. Notably, the putative miR-30a binding site was observed in the 3'untranslated region (UTR) of BiP mRNA, and miR-30a suppressed BiP expression by targeting 3'UTR of BiP transcript. In addition, CSC increased the expression of BiP in OSCC cells by downregulating miR-30a. We also showed that BiP promoted invasion and tumor-associated angiogenesis by increasing the production and secretion of vascular endothelial growth factor in CSC-exposed OSCC cells. Moreover, BiP inhibition suppressed OSCC growth and reduced tumor vessel density in tumor-bearing mice administered with CSC. CONCLUSIONS: These observations suggest that epigenetic regulation of BiP via miR-30a downregulation is involved in CSC-induced OSCC progression.

YAP-Dependent BiP Induction Is Involved in Nicotine-Mediated Oral Cancer Malignancy.

Cigarette smoking is a significant risk factor for the development and progression of oral cancer. Previous studies have reported an association between nicotine and malignancy in oral cancer. Recent studies have also demonstrated that nicotine can induce endoplasmic reticulum (ER) stress in tumor cells. Binding immunoglobulin protein (BiP) acts as a master regulator of ER stress and is frequently overexpressed in oral cancer cell lines and tissues. However, the effect of nicotine on BiP in oral cancer is unknown. Therefore, this study aimed to evaluate the role of BiP and its underlying regulatory mechanisms in nicotine-induced oral cancer progression. Our results showed that nicotine significantly induced the expression of BiP in time- and dose-dependent manners in oral squamous cell carcinoma (OSCC) cells. In addition, BiP was involved in nicotine-mediated OSCC malignancy, and depletion of BiP expression remarkably suppressed nicotine-induced malignant behaviors, including epithelial-mesenchymal transition (EMT) change, migration, and invasion. In vivo, BiP silencing abrogated nicotine-induced tumor growth and EMT switch in nude mice. Moreover, nicotine stimulated BiP expression through the activation of the YAP-TEAD transcriptional complex. Mechanistically, we observed that nicotine regulated YAP nuclear translocation and its interaction with TEAD through α7-nAChR-Akt signaling, subsequently resulting in increased TEAD occupancy on the HSPA5 promoter and elevated promoter activity. These observations suggest that BiP is involved in nicotine-induced oral cancer malignancy and may have therapeutic potential in tobacco-related oral cancer.

Correction to: Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy.

[This corrects the article DOI: 10.1186/s12935-020-01401-w.].

Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy.

BACKGROUND: The mechanisms of neuronal protein γ-synuclein (SNCG) in the malignancy of oral squamous cell carcinoma (OSCC) are not clear. This study tested the hypothesis that SNCG is involved in nicotine-induced malignant behaviors of OSCC. The effect of nicotine on SNCG expression and epithelial-to-mesenchymal transition (EMT) markers were examined. METHODS: Short hairpin RNA (shRNA) and an antagonist specific for α7-nicotine acetylcholine receptors (α7-nAChRs) were used to examine the role of α7-nAChRs in mediating the effects of nicotine. Knockdown of SNCG in nicotine-treated cells was performed to investigate the role of SNCG in cancer malignancy. The in vivo effect of nicotine was examined using a nude mouse xenotransplantation model. RESULTS: Nicotine increased SNCG expression in a time- and dose-dependent manner. Nicotine treatment also increased E-cadherin and ZO-1 and decreased fibronectin and vimentin expression. After specific knockdown of α7-nAChRs and inhibition of the PI3/AKT signal, the effect of nicotine on SNCG expression was attenuated. Silencing of SNCG abolished nicotine-induced invasion and migration of OSCC cells. The xenotransplantation model revealed that nicotine augmented tumor growth and SNCG expression. CONCLUSION: Nicotine upregulated SNCG expression by activating the α7-nAChRs/PI3/AKT signaling that are participated in nicotine-induced oral cancer malignancy.

α7-Nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer.

OBJECTIVE: To investigate the effect of nicotine on cell survival and cisplatin resistance in oral cancer and the possible involvement of α7-nicotinic acetylcholine receptors (α7-nAChRs). DESIGN: The effects of nicotine on cell survival and cisplatin-induced apoptosis were assessed. Knockdown of α7-nAChRs by short hairpin RNA and the specific antagonist methyllycaconitine (MLA) was used to examine the involvement of α7-nAChRs in modulating the effects of nicotine. Apoptosis signal molecules were examined in nicotine- and cisplatin-treated cells. RESULTS: Nicotine increased the survival of the oral cancer cells YD8 and OEC-M1 in a dose- and time-dependent manner. Nicotine treatment accelerated cell cycle progression in the oral cancer cells, and significantly reduced cisplatin-induced cell apoptosis. In the α7-nAChR-silenced cells, the prosurvival effect of nicotine in the cisplatin-treated cells was attenuated. Co-treatment of cisplatin and nicotine attenuated the effect of cisplatin on Bcl-2 expression. In addition, the effect of nicotine on cell survival under cisplatin treatment was attenuated with the addition of the Bcl-2 inhibitor ABT-737. CONCLUSIONS: Treating oral cancer cells with nicotine increased cell survival and cisplatin resistance, in which α7-nAChRs were involved.

Cilostazol inhibits hyperglucose-induced vascular smooth muscle cell dysfunction by modulating the RAGE/ERK/NF-κB signaling pathways.

BACKGROUND: Increasing evidence suggests that high glucose (HG) causes abnormalities in endothelial and vascular smooth muscle cell function (VSMC) and contributes to atherosclerosis. Receptor for advanced glycation end-products (RAGE) has been linked to the pathogenesis of both the macrovascular and microvascular complications of diabetes. Cilostazol is used to treat diabetic vasculopathy by ameliorating HG-induced vascular dysfunction. OBJECTIVES: In this study, we investigated whether the cilostazol suppression of HG-induced VSMC dysfunction is through RAGE signaling and its possible regulation mechanism. METHOD: We investigated the effect of HG and cilostazol on RAGE signaling in A7r5 rat VSMCs. Aortic tissues of streptozotocin (STZ) diabetic mice were also collected. RESULTS: Aortic tissue samples from the diabetic mice exhibited a significantly decreased RAGE expression after cilostazol treatment. HG increased RAGE, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions, and was accompanied with increased reactive oxygen species (ROS), cell proliferation, adhesion and migration. Cilostazol significantly reversed HG-induced RAGE, ROS, downstream gene expressions and cell functions. RAGE knockdown significantly reversed the expressions of HG-induced vasculopathy related gene expressions and cell functions. Cilostazol with RAGE knockdown had additive effects on downstream ERK/NF-κB signaling pathways, gene expressions and cell functions of A7r5 rat VSMCs in HG culture. CONCLUSIONS: Both in vitro and in vivo experimental diabetes models showed novel signal transduction of cilostazol-mediated protection against HG-related VSMC dysfunction, and highlighted the involvement of RAGE signaling and downstream pathways.

A novel potential biomarker for metabolic syndrome in Chinese adults: Circulating protein disulfide isomerase family A, member 4.

BACKGROUND/OBJECTIVES: Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins that are involved in the pathogenesis of numerous diseases including neurodegenerative diseases, cancer and obesity. However, the metabolic effects of PDIA4 remain unclear in humans. The aims of this study were to investigate the associations of serum PDIA4 with the metabolic syndrome (MetS) and its components in Chinese adults. SUBJECTS/METHODS: A total of 669 adults (399 men and 270 women) were recruited. Serum PDIA4 concentrations and biochemical variables were recorded. Insulin sensitivity and ß-cell function were examined by homeostasis model assessment. MetS was defined based on the modified National Cholesterol Education Program Adult Treatment Panel III criteria for Asia Pacific. RESULTS: The participants with MetS had significantly higher serum PDIA4 levels than those without MetS (P<0.001). After adjustments, the individuals with the highest PDIA4 tertile were associated with a higher risk of MetS than those with the lowest tertile (OR = 4.83, 95% CI: 2.71-8.60). The concentration of PDIA4 showed a stepwise increase with the components of MetS (P<0.001 for trend). The individuals with the highest PDIA4 tertile were significantly associated with waist circumference (OR = 2.41, 95% CI 1.34-4.32), blood pressure (OR = 2.71, 95% CI 1.57-4.67), fasting glucose concentration (OR = 3.17, 95% CI 1.80-5.57), and serum triglycerides (OR = 4.12, 95% CI 2.30-7.37) than those with the lowest tertile. At cutoff point of 15.24 ng/ml, the diagnostic sensitivity and specificity of PDIA4 for the metabolic syndrome were 67 and 72%, respectively, in male patients and 60 and 78%, respectively, in female patients. Finally, the result showed that PDIA4 had a significantly higher area under the curve compared with blood pressure to detect MetS using receiver operating characteristic analysis. CONCLUSIONS: Serum PDIA4 concentrations are closely associated to MetS and its components in Chinese adults.

Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-κB signaling.

Accumulating evidence is indicating metformin to possess the potential ability in preventing tumor development and suppressing cancer growth. However, the exact mechanism of its antitumorigenic effects is still not clear. We found that metformin suppressed the ability of cancer to skew macrophage toward M2 phenotype. Metformin treated cancer cells increased macrophage expression of M1-related cytokines IL-12 and TNF-α and attenuated M2-related cytokines IL-8, IL-10, and TGF-ß expression. Furthermore, metformin treated cancer cells displayed inhibited secretion of IL-4, IL-10 and IL-13; cytokines important for inducing M2 macrophages. Conversely, M1 inducing cytokine IFN-γ was upper-regulated in cancer cells. Additionally, through increasing AMPK and p65 phosphorylation, metformin treatment activated AMPK-NF-κB signaling of cancer cells that participate in regulating M1 and M2 inducing cytokines expression. Moreover, Compound C, an AMPK inhibitor, significantly increased IL-4, IL-10, and IL-13 expression while BAY-117082, an NF-κB inhibitor, decreased expression. In metformin-treated tumor tissue, the percentage of M2-like macrophages decreased while M1-like macrophages increased. These findings suggest that metformin activates cancer AMPK-NF-κB signaling, a pathway involved in regulating M1/M2 expression and inducing genes for macrophage polarization to anti-tumor phenotype.

Estrogen modulates vascular smooth muscle cell function through downregulation of SIRT1.

BACKGROUND: There are sex differences in the incidence and severity of cardiovascular disease. Although an estrogen-mediated vasculoprotective effect is widely accepted, clinical trial results have been conflicting and the detailed mechanisms are still unclear. Sirtuin 1 (SIRT1), a class III histone deacetylase, may protect against vascular aging and atherosclerosis; however, the effects of estrogen on SIRT1 expression and vascular smooth muscle cell (VSMC) behavior remain unknown. MATERIALS AND METHODS: We ovariectomized (OVX) female, wild-type, C57BL/6J mice, which were randomized into non-estrogen- and estrogen-supplemented groups. We also treated A7r5 VSMCs with 17-ß-estradiol and resveratrol, a SIRT1 activator, in vitro, and measured the expression of SIRT1 and apoptotic markers, as well as proliferation, viability, and migration. RESULTS: Aortic tissue from OVX mice exhibited marked VSMC hyperplasia and upregulation of SIRT1, which was reversed by 17-ß-estradiol supplementation, as assessed by western blotting and immunohistochemical staining. In vitro, 17-ß-estradiol downregulated SIRT1 expression in a dose- and time-dependent manner, increased apoptosis, and reduced proliferation, viability, and migration. Resveratrol reversed these effects through the activation of SIRT1. Estrogen appeared to mediate its effects through the Akt and ERK pathways. CONCLUSIONS: Estrogen may regulate cardiovascular health via the expression of SIRT1, possibly through the AKT and ERK signaling pathways.

Cilostazol inhibits uremic toxin-induced vascular smooth muscle cell dysfunction: role of Axl signaling.

Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, and vascular smooth muscle cell (VSMC) dysfunction plays a pivotal role in uremic atherosclerosis. Axl signaling is involved in vascular injury and is highly expressed in VSMCs. Recent reports have shown that cilostazol, a phosphodiesterase type 3 inhibitor (PDE3), can regulate various stages of the atherosclerotic process. However, the role of cilostazol in uremic vasculopathy remains unclear. This study aimed to identify the effect of cilostazol in VSMCs in the experimental CKD and to investigate whether the regulatory mechanism occurs through Axl signaling. We investigated the effect of P-cresol and cilostazol on Axl signaling in A7r5 rat VSMCs and the rat and human CKD models. From the in vivo CKD rats and patients, aortic tissue exhibited significantly decreased Axl expression after cilostazol treatment. P-cresol increased Axl, proliferating of cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and matrix metalloproteinase-2 (MMP-2) expressions, decreased caspase-3 expression, and was accompanied by increased cell viability and migration. Cilostazol significantly reversed P-cresol-induced Axl, downstream gene expressions, and cell functions. Along with the increased Axl expression, P-cresol activated PLCγ, Akt, and ERK phosphorylation and cilostazol significantly suppressed the effect of P-cresol. Axl knockdown significantly reversed the expressions of P-cresol-induced Axl-related gene expression and cell functions. Cilostazol with Axl knockdown have additive changes in downstream gene expression and cell functions in P-cresol culture. Both in vitro and in vivo experimental CKD models elucidate a new signal transduction of cilostazol-mediated protection against uremic toxin-related VSMCs dysfunction and highlight the involvement of the Axl signaling and downstream pathways.