Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression

Abstract

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

De Novo Ceramide Synthesis Is Required for N-Linked Glycosylation in Plasma Cells

“…Thus, evidence linking sphingolipid biosynthesis to the machinery of N-linked glycosylation is only beginning to accumulate. Concerning the second possibility, protein kinase A was recently demonstrated to regulate N-linked glycosylation (48). Ceramides activate protein kinase A (49), but addition of an inhibitor of protein kinase A did not exhibit a phenotype similar to that evoked by treatment with FB1 (not shown).…”

Antiangiogenic Activity of 2-Deoxy-D-Glucose

“…The importance of surface glycoproteins and N-linked glycosylation on endothelial functions in vitro and in vivo has been reported [46], [47], [48]. Pili et al, showed that Castanospermine (CST), an alpha glucosidase inhibitor, which prevents the synthesis of complex oligosaccharides, inhibits bovine pulmonary artery ECs and bovine aortic ECs in vitro , and inhibits in vivo angiogenesis and tumor growth [48].…”

“…Pili et al, showed that Castanospermine (CST), an alpha glucosidase inhibitor, which prevents the synthesis of complex oligosaccharides, inhibits bovine pulmonary artery ECs and bovine aortic ECs in vitro , and inhibits in vivo angiogenesis and tumor growth [48]. Martinez et al, demonstrated that N-glycosylation is critical for EC proliferation [46], [47], and that tunicamycin, an inhibitor of N-linked glycosylation, induces apoptosis in bovine adrenal microvascular endothelial cells [49]. The work presented here differs from the above studies in several aspects.…”

Cloning and expression of mannosylphospho dolichol synthase from bovine adrenal medullary capillary endothelial cells

“…Therefore, it is expected that up- or down-regulation of the DPMS activity should reflect appropriately to the level of LLO and the glycoproteins. In fact, phosphorylation up-regulation of DPMS has increased the LLO biosynthesis and turnover [39]. These are significantly reduced in PKA-deficient CHO cells [38].…”