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Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis

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Abstract

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms \(\alpha\) and \(\delta\) revealed that these proteins are uniquely regulated. Characterization of an additional PMA target, protein kinase D (PKD) demonstrated that this enzyme becomes phosphorylated in HUVECs, and may therefore be involved in proangiogenic signaling. Further examination of downstream effectors of PKC showed that extracellular signal-regulated kinase (ERK) is critical for angiogenesis, and is accordingly phosphorylated in response to PMA. Surprisingly however, phosphorylation of ERK is independent of PKC activity. In addition, we show that the PKC target sphingosine kinase (SPK) is required for vessel formation. These findings illustrate the complexities of blood vessel formation, and suggest that activators utilize multiple independent pathways to invoke a complete angiogenic response.

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Abbreviations

bFGF:

basic fibroblast growth factor

BisI:

bisindolylmaleimide I

CalC:

calphostin C

ERK:

extracellular signal-regulated kinase

FBS:

fetal bovine serum

HUVECs:

human umbilical vein endothelial cells

MAPK:

mitogen activated protein kinase

PKC:

protein kinase C

PKD:

protein kinase D

PMA:

phorbol-12-myristate-13-acetate

SPK:

sphingosine kinase

SPKI:

sphingosine kinase inhibitor

S1P:

sphingosine 1-phosphate

VEGF:

vascular endothelial growth factor

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Acknowledgements

The authors thank Robert Vernon for invaluable advice on tube assays. We also wish to thank David Fulton for providing caveolin and phospho p44/p42 MAP kinase antibodies, Mario Marrero for providing the cleaved caspase-3 antibody and Katina Corley for technical assistance. This work was supported in part␣by grants from the Georgia Cancer Coalition␣(GCC00023) and National Institute of Health (K01-CA089689) to K.M. and American Heart Association (AHA00063) and Georgia Cancer Coalition (GCC00022) to B.L.

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Authors and Affiliations

  1. Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, USA

    Caroline J. Taylor, Kouros Motamed & Brenda Lilly

  2. Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia, USA

    Brenda Lilly

  3. Department of Pathology, Medical College of Georgia, Augusta, Georgia, USA

    Kouros Motamed

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  1. Caroline J. Taylor
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  2. Kouros Motamed
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Correspondence to Brenda Lilly.

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Taylor, C.J., Motamed, K. & Lilly, B. Protein kinase C and downstream signaling pathways in a three-dimensional model of phorbol ester-induced angiogenesis. Angiogenesis 9, 39–51 (2006). https://doi.org/10.1007/s10456-006-9028-y

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