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

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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 ∼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 Glc3Man9GlcNAc2-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 Glc3Man9GlcNAc2-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.

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Abbreviations

EMEM:

minimal essential medium with Earle’s salt

DMEM:

Dulbeccos’ minimal essential medium

NP-40:

Nonidet P-40

SDS:

sodium dodecyl sulfate

PBS:

phosphate-buffer-saline

PAGE:

polyacrylamide gel electrophoresis

HRP:

horseradish peroxidase

Dol-P:

dolicylmonophosphate

DMSO:

dimethylsolfoxide

Con A:

concanavalin A

WGA:

wheat germ agglutinin

cAMP:

adenosine 3′, 5′-cyclic monophosphate

PKA:

cAMP-dependent protein kinase

ER:

endoplasmic reticulum

LLO:

Glc3Man9GlcNAc2-PP-Dol

Dol-P-Man:

dolichol-P-Mannose

Dol-P-Glc:

dolichol-P-glucose

GPT:

UDP-GlcNAc-dolichol-P GlcNAc-1-P transferase

DPMS:

Dol-P-Man synthase

UPR:

unfolded protein response

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Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR, 00936-5067, USA

    Juan A. Martínez, José J. Tavárez, Caroline M. Oliveira & Dipak K. Banerjee

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  1. Juan A. Martínez
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  2. José J. Tavárez
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  3. Caroline M. Oliveira
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  4. Dipak K. Banerjee
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Correspondence to Dipak K. Banerjee.

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Martínez, J.A., Tavárez, J.J., Oliveira, C.M. et al. Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression. Glycoconj J 23, 209–220 (2006). https://doi.org/10.1007/s10719-006-7926-2

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