Heparan sulfate proteoglycan deficiency up‐regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines

Abstract

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

Endocytosis and the Participation of Glycosaminoglycans Are Important to the Mechanism of Cell Death Induced by β-Hairpin Antimicrobial Peptides

“…We used 50 nM sodium chlorate (that reduces GAG sulfation) for 48 h, 10 μM Heparin (unfractioned heparin from porcine intestinal mucosa Roche, Brazilwas used as sulfated proteoglycan negative charge competitor) for 5 min, or 2 mM xylopyranoside (compromises proteoglycan biosynthesis) for 5 days. 25 Next, cells were stimulated with AMP cytotoxic EC 50 (half maximal effective concentration) for 24 h. Further 50 μL of MTT solution from the Stock (5 mg/mL) was added, and cells were incubated in a CO 2 incubator in the dark for 4 h. The medium was removed and formazan crystals formed by the cells were dissolved using DMSO. The absorbance was read at 570 nm using 630 nm as reference wavelength on a multiwell plate reader.…”

Identification of potential ferroptosis hub genes in acute-on-chronic liver failure based on bioinformatics analysis and experimental verification

“…When cells are separated from the ECM, it can lead to a series of deleterious metabolic changes, including the triggering of ferritin deposition and the induction of ferroptosis [36]. Sulfated glycosaminoglycans (GAGs), especially chondroitin sulfate and heparan sulfate, have antioxidant effects [37,38]. Sulfation plays an important role in maintaining cell survival and preventing oxidative stress-induced cell death [39].…”

Cell-surface glycosaminoglycans regulate the cellular uptake of charged polystyrene nanoparticles

“…13 Herein, we investigate the influence of two major molecular components of the glycocalyx -HS and CSon the cellular uptake of anionic (carboxyl-terminated) and cationic (amineterminated) polystyrene (PS) NPs of 50 nm in size. To this end, we perform uptake studies in wild-type and GAG-deficient Chinese hamster ovary (CHO) cells, 40,41 as well as in wild-type cells after the selective enzymatic cleavage of their GAG species. We also investigate the major endocytic mechanisms by which the NPs are taken up in the wild-type and mutant cell lines.…”