G6PD-knockdown Cells Research Articles

Glucose-6-phosphate dehydrogenase is indispensable in embryonic development by modulation of epithelial-mesenchymal transition via the NOX/Smad3/miR-200b axis

Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme involved in the pentose phosphate shunt for producing nicotinamide adenine dinucleotide phosphate (NADPH). Severe G6PD deficiency leads to embryonic lethality, but the underlying mechanism is unclear. In the current study, the effects of G6PD on epithelial–mesenchymal transition (EMT), especially during embryonic development, were investigated. The knockdown of G6PD induced morphological changes, accompanied by the suppression of epithelial markers, E-cadherin and β-catenin, in A549 and MDCK cells. Such modulation of EMT was corroborated by the enhancement of migration ability in G6PD-knockdown A549 cells. Zebrafish embryos with g6pd knockdown exhibited downregulation of the E-cadherin/β-catenin adhesion molecules and impaired embryonic development through reduction in epiboly rate and increase in cell shedding at the embryo surface. The dysregulation in zebrafish embryonic development caused by g6pd knockdown could be rescued through human G6PD or CDH1 (E-cadherin gene) cRNA coinjection. The Smad3/miR-200b axis was dysregulated upon G6PD knockdown, and the reconstitution of SMAD3 in G6PD-knockdown A549 cells restored the expression of E-cadherin/β-catenin. The inhibition of NADPH oxidase (NOX) activation through the loss of p22phox signaling was involved in the dysregulation of the Smad3/miR-200b axis upon G6PD knockdown. The reconstitution of G6PD led to the recovery of the regulation of NOX/Smad3/miR-200b signaling and increased the expression of E-cadherin/β-catenin in G6PD-knockdown cells. Thus, these results suggest that in the EMT process, G6PD plays an important regulatory role as an integral component of the NOX/Smad3/miR-200b axis.

G6PD promotes renal cell carcinoma proliferation through positive feedback regulation of p-STAT3.

Ectopic Glucose 6-phosphate dehydrogenase (G6PD) expression plays important role in tumor cell metabolic reprogramming and results in poor prognosis of multiple malignancies. Our previous study indicated that G6PD is overexpressed in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC. However, its role in RCC is still unclear. Here, we demonstrate that G6PD is not only up-regulated in all types of RCC specimens but also displays higher activities in RCC cell lines. G6PD overexpression promoted RCC cell proliferation, altered cell cycle distribution, and enhanced xenografted RCC development. G6PD up-regulated ROS generation by facilitating NADPH-dependent NOX4 activation, which led to increased expression of p-STAT3 and CyclinD1. Enhanced ROS generation rescued the p-STAT3 and CyclinD1 expression reduction in G6PD-knockdown cells, while ROS scavengers reversed the up-regulated p-STAT3 and CyclinD1 expression in G6PD-overexpressing cells. Furthermore, p-STAT3 activated G6PD gene expression via binding to the G6PD promoter, demonstrating that p-STAT3 forms a positive feedback regulatory loop for G6PD overexpression. G6PD expression was up or down-regulated in response to the impact of p-STAT3 activators or inhibitors. Therefore, G6PD may be an effective RCC therapeutic target.

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

Impaired dephosphorylation renders G6PD-knockdown HepG2 cells more susceptible to H 2O 2-induced apoptosis

Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H 2O 2-elicited signaling in HepG2 cells. H 2O 2 was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H 2O 2 signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H 2O 2 on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H 2O 2 signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H 2O 2-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H 2O 2. Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H 2O 2-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise.

Glucose-6-Phosphate Dehydrogenase Deficiency Enhances Human Coronavirus 229E Infection

The host cellular environment is a key determinant of pathogen infectivity. Viral gene expression and viral particle production of glucose-6-phosphate dehydrogenase (G6PD)–deficient and G6PD-knockdown cells were much higher than their counterparts when human coronavirus (HCoV) 229E was applied at 0.1 multiplicity of infection. These phenomena were correlated with increased oxidant production. Accordingly, ectopic expression of G6PD in G6PD-deficient cells or addition of antioxidant (such as α-lipoic acid) to G6PD-knockdown cells attenuated the increased susceptibility to HCoV 229E infection. All experimental data indicated that oxidative stress in host cells is an important factor in HCoV 229E infectivity