Decrease of P-Glycoprotein Activity in K562/ADR Cells by MβCD and Filipin and Lack of Effect Induced by Cholesterol Oxidase Indicate That This Transporter Is Not Located in Rafts

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl--cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, light, heavy, and extra heavy fractions we show that no P-gp is found in the light fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.     

The C2238/αANP Variant Is a Negative Modulator of Both Viability and Function of Coronary Artery Smooth Muscle Cells

Background

Abnormalities of vascular smooth muscle cells (VSMCs) contribute to development of vascular disease. Atrial natriuretic peptide (ANP) exerts important effects on VSMCs. A common ANP molecular variant (T2238C/αANP) has recently emerged as a novel vascular risk factor.

Objectives

We aimed at identifying effects of CC2238/αANP on viability, migration and motility in coronary artery SMCs, and the underlying signaling pathways.

Methods and Results

Cells were exposed to either TT2238/αANP or CC2238/αANP. At the end of treatment, cell viability, migration and motility were evaluated, along with changes in oxidative stress pathway (ROS levels, NADPH and eNOS expression), on Akt phosphorylation and miR21 expression levels. CC2238/αANP reduced cell vitality, increased apoptosis and necrosis, increased oxidative stress levels, suppressed miR21 expression along with consistent changes of its molecular targets (PDCD4, PTEN, Bcl2) and of phosphorylated Akt levels. As a result of increased oxidative stress, CC2238/αANP markedly stimulated cell migration and increased cell contraction. NPR-C gene silencing with specific siRNAs restored cell viability, miR21 expression, and reduced oxidative stress induced by CC2238/αANP. The cAMP/PKA/CREB pathway, driven by NPR-C activation, significantly contributed to both miR21 and phosphoAkt reduction upon CC2238/αANP. miR21 overexpression by mimic-hsa-miR21 rescued the cellular damage dependent on CC2238/αANP.

Conclusions

CC2238/αANP negatively modulates viability through NPR-C/cAMP/PKA/CREB/miR21 signaling pathway, and it augments oxidative stress leading to increased migratory and vasoconstrictor effects in coronary artery SMCs. These novel findings further support a damaging role of this common αANP variant on vessel wall and its potential contribution to acute coronary events.     

High Expression of IGFBP7 in Fibroblasts Induced by Colorectal Cancer Cells Is Co-Regulated by TGF-β and Wnt Signaling in a Smad2/3-Dvl2/3-Dependent Manner

Fibroblasts in the tumor microenvironment are a key determinant in cancer progression and may be a promising target for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is known as a tumor suppressor in colorectal cancer (CRC). The present study investigated the inductive mechanism of IGFBP7 expression in fibroblasts by supernatant from the CRC cell line, SW620. The results showed that the expression of IGFBP7 was up-regulated in the fibroblasts when treated with SW620 supernatant and exogenous TGF-β1. The IGFBP7 induced by SW620 supernatant or TGF-β1 was partially inhibited by the TGF-β1 specific antibody AF and TGF-β1 receptor antagonist SB431542. The Wnt signaling-targeted genes, c-Myc, CCND1 and the proteins Dvl2/3, were all up-regulated in fibroblasts expressing high levels of IGFBP7, and the up-regulation could be inhibited both by the Wnt signaling antagonist Dickkopf-1 (DKK1) and by the TGF-β1 receptor antagonist SB431542. In conclusion, CRC cells promote the high expression of IGFBP7 in fibroblasts, most likely through the co-regulation of TGF-β and Wnt signaling in a Smad2/3-Dvl2/3 dependent manner. Taken together, these data suggest that the fibroblasts could be a novel therapeutic target in tumor therapy.     

The Effects of NF-κB and c-Jun/AP-1 on the Expression of Prothrombotic and Proinflammatory Molecules Induced by Anti-β2GPI in Mouse

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β₂GPI/β₂GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β₂GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β₂GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β₂GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β₂GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.     

The Long Non-Coding HOTAIR Is Modulated by Cyclic Stretch and WNT/β-CATENIN in Human Aortic Valve Cells and Is a Novel Repressor of Calcification Genes

Aortic valve calcification is a significant and serious clinical problem for which there are no effective medical treatments. Individuals born with bicuspid aortic valves, 1–2% of the population, are at the highest risk of developing aortic valve calcification. Aortic valve calcification involves increased expression of calcification and inflammatory genes. Bicuspid aortic valve leaflets experience increased biomechanical strain as compared to normal tricuspid aortic valves. The molecular pathogenesis involved in the calcification of BAVs are not well understood, especially the molecular response to mechanical stretch. HOTAIR is a long non-coding RNA (lncRNA) that has been implicated with cancer but has not been studied in cardiac disease. We have found that HOTAIR levels are decreased in BAVs and in human aortic interstitial cells (AVICs) exposed to cyclic stretch. Reducing HOTAIR levels via siRNA in AVICs results in increased expression of calcification genes. Our data suggest that β-CATENIN is a stretch responsive signaling pathway that represses HOTAIR. This is the first report demonstrating that HOTAIR is mechanoresponsive and repressed by WNT β-CATENIN signaling. These findings provide novel evidence that HOTAIR is involved in aortic valve calcification.     

The β-1,3-Exoglucanase Gene exgA (exg1) of Aspergillus oryzae Is Required to Catabolize Extracellular Glucan,and Is Induced in Growth on a Solid Surface

The biological role of ExgA (Exg1), a secretory β-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when β-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall β-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in β-glucan utilization, but is not much involved in cell wall β-glucan remodeling.     

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (?)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells

The histamine H₁ receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (−)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (−)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (−)-maackiain. A pull-down assay revealed that (−)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr³¹¹ on PKCδ. These data suggest that (−)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (−)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.