Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase

Abstract

Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H₂O₂ and TNFα was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38α or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.

Introduction

Vascular Endothelial Growth Factor (VEGF or VEGF-A) is essential for angiogenesis during development and in the pathogenesis of human pathologies including cancer and eye diseases [1], [2]. VEGF exerts its diverse biological effects in endothelial cells through high affinity binding to two tyrosine-kinase receptors, Flt-1 (VEGFR1) and KDR (VEGFR2) [3], [4]. KDR is activated through ligand-stimulated receptor dimerisation and trans(auto)phosphorylation of multiple tyrosine residues in the cytoplasmic domain [5], [6], triggering an array of early signaling events followed by short- and long-term cellular biological effects including production of prostacyclin and nitric oxide, increased cell survival, migration, proliferation and angiogenesis [4], [6], [7], [8], [9], [10], [11], [12], [13], [14]. The function of Flt-1 in the endothelium is unclear, but it is thought to regulate the activity of VEGF partly by acting as a decoy receptor, and in part though direct regulatory effects on KDR [1]. VEGF has been reported to induce Heat Shock Protein (HSP) 27 phosphorylation via a p38 kinase-dependent pathway [15], but the mechanisms involved in VEGF regulation of HSP27 phosphorylation and the role of this protein in VEGF function remain poorly understood.

HSP27 is a ubiquitous and abundantly expressed member of the small heat shock protein family, phosphorylated in response to diverse stress stimuli, including osmotic stress, reactive oxygen species, and inflammatory cytokines. HSP27 is thought to play major roles in the regulation of apoptosis, organisation of the actin cytoskeleton, and cell migration [16], [17], and also protects cardiomyocytes against ischaemic injury [18]. The major pathway through which HSP27 phosphorylation is regulated is the p38 kinase cascade [19], [20]. HSP27 is directly phosphorylated at serines 15, 78 and 82 by the p38 kinase substrate, MAPK-activated protein kinase-2 (MAPKAPK-2). Large HSP27 multimers are formed by unphosphorylated HSP27, and phosphorylation at serine residue 82 in particular, promotes dissociation of multimers, whereas phosphorylation at serine 15 is implicated in dimer interaction with actin [21].

Despite the potential importance of HSP27 for migration and survival, key functions involved in VEGF-dependent angiogenesis, previous studies have not directly examined the role of HSP27 in these or other functions of VEGF. In the present study, we investigated both the signal transduction mechanisms mediating VEGF-induced HSP27 phosphorylation and the role of HSP27 in endothelial biological responses to VEGF. Our findings identify a novel pathway mediated via Protein Kinase D (PKD) and independent of p38 kinase that plays the major role in VEGF regulation of phosphorylation at serine 82, whereas phosphorylation at serines 15 and 78 occurs predominantly via p38 kinase. We further demonstrate that while inhibition of p38 kinase has little effect on the migratory response to VEGF, knockdown of PKDs and HSP27 inhibits VEGF-mediated cell migration and tubulogenesis.