Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression

Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.     

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including Acetazolamide in human Glioblastoma

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24 h at 0.1%, O₂). Aza. application was at a range between 250 and 8000 nM and the HIF-1α inhibitor Chetomin at a concentration range of 150–500 nM. Cell culture plates were incubated for 24 h under hypoxia (0.1% O₂). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24 h of hypoxia (0.1% O₂) at concentrations between 3500 and 8000 nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150–500 nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.     

PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells

Background

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.

Methodology/Principal Findings

In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.

Conclusions/Significance

Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.     

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.