Identification of Protein Kinase D2 as a Pivotal Regulator of Endothelial Cell Proliferation, Migration, and Angiogenesis

Abstract

Angiogenesis, the formation of new blood vessels, plays a crucial role in normal physiological processes and in various pathological conditions. In the present study, we have investigated the physiological function of a newly described serine/threonine protein kinase D2 (PKD2) in aspects of endothelial cell biology involved in angiogenesis. We found that PKD2 was expressed in primary human endothelial cells from different tissues and was a critical PKD isoform mediating the phosphorylation of PKD substrates in endothelial cells. By using small interference RNAs that target different PKD2 regions, we found that silencing PKD2, but not PKD1 isoform, markedly inhibited the proliferation, migration, and in vitro angiogenesis of endothelial cells cultured in EGM-2 complete medium. We further showed that PKD2, but not PKD1, was required for the expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 that are two key growth factor receptors involved in angiogenesis. These findings indicate that PKD2 plays a pivotal role in endothelial cell proliferation and migration necessary for angiogenesis at least in part through modulation of the expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1.Angiogenesis is the fundamental physiological process by which new blood vessels are generated from pre-existing vasculature. It plays a crucial role in embryonic development in addition to numerous normal physiological processes. Angiogenesis is critically involved in a wide variety of human diseases including ischemic vascular diseases, tumor growth and metastasis, diabetic retinopathy, age-related macular degeneration, and rheumatoid arthritis (1-3). Emerging evidence indicates that angiogenesis also plays a critical role in the development and progression of atherosclerosis, a chronic inflammatory disease of the vessel wall (4 -7). For these reasons, an understanding of the complex mechanisms that regulate angiogenesis is seen not only as a fundamental problem in human biology but also critical in fulfilling the important goal of biomedical research to develop more specific and efficacious pro-and antiangiogenesis therapies.The process of angiogenesis depends on endothelial cell (EC) 2 proliferation, migration, and differentiation. It is controlled by a variety of positive and negative signals. Vascular endothelial growth factor (VEGF, also termed VEGF-A) is the most critical and potent of all the known proangiogenic factors, and its proangiogenic effect is mediated through the VEGF receptor 2 (VEGFR2, also termed Flk-1 or KDR) that is selectively expressed in vascular ECs (8). Besides VEGF, fibroblast growth factors (FGFs) and their receptors that are expressed on ECs also play a crucial role in angiogenesis (9). ECs express the FGF receptor 1 (FGFR1), and under some circumstances, FGFR2. Activation of FGFR1 or FGFR2 promotes EC proliferation, migration, and angiogenesis (9). Additionally, angiopoietins, transforming growth factors, cytokine...

Protein kinase D in vascular biology and angiogenesis

“…This suggests that PKD2 may be required for nonVEGF-mediated angiogenic mechanisms. In support of this hypothesis they found that PKD2 was also required for fibroblast growth factor receptor expression (33). Further work using PKD isoform specific mutants or endothelial specific knockouts will be required to further clarify the roles of the PKD isoforms in endothelial function.…”

“…PKD2 seems to be more abundantly expressed in HUVECs compared with PKD1, as judged by an antibody that recognized both isoforms, and knockdown of PKD2 appears to have a greater effect on HSP27 phosphorylation (9). In addition, Hao et al showed that only PKD2 knockdown was able to inhibit endothelial migration, proliferation and tube formation stimulated by serum (33). This suggests that PKD2 may be required for nonVEGF-mediated angiogenic mechanisms.…”

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

“…These findings suggest a functional differentia among PKD isoforms. We have reported that PKD1 regulates the production of proinflammatory cytokines by vascular endothelial growth factor (VEGF) in endothelial cells (29) and that PKD2 is pivotal for angiogenesis (30). We also found that both PKD2 and PKD3 were novel growth regulators in triple-negative breast cancer cells (31).…”

“…1B). By using a phospho-Ser/Thr PKD substrate antibody that specifically recognizes the PKD consensus motif LXRXX(S/T) and PKD-mediated protein phosphorylation (30,42), we found that the PKD-dependent protein phosphorylation in 16HBE14oϪ cells was markedly suppressed by the selective PKD inhibitor kb-NB142-70 (Fig. 1C, top panel).…”

The vitamin K–dependent anticoagulant factor, protein S, inhibits multiple VEGF-A–induced angiogenesis events in a Mer- and SHP2-dependent manner

“…Human umbilical vein endothelial cells (HUVECs, Lonza Walkersville) were cultured in endothelial growth medium-2 (EGM-2; cc-3162, Lonza Walkersville) referred to as growth factor containing medium as described previously 24 and used at early passages 1-6. When indicated, HUVEC cultures were switched to EBM-2 medium (Lonza Walkersville) referred to as a growth factor-depleted medium that was supplemented with either 0.5% FCS or ITS (insulin 10 g/mL, transferrin 6.7 ng/mL, selenium 5.5 g/mL).…”