Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2β to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.NHLBI, National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [HL140411]; NCI, National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [CA78383-20, CA187035, CA200572]; Florida Department of Health Cancer Research Chair Fund Florida Grant [3J-02]; American Heart AssociationAmerican Heart Association [13POST14510025, 19CDA34700013]; Mayo Clinic Ted and Loretta Rogers Cardiovascular Career Development Award Honoring Hugh C. SmithThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Vasodilator-Stimulated Phosphoprotein Activity Is Required for <i>Coxiella burnetii</i> Growth in Human Macrophages

<div><p><i>Coxiella burnetii</i> is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and <i>C</i>. <i>burnetii</i> initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). <i>C</i>. <i>burnetii</i> manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during <i>C</i>. <i>burnetii</i> infection. Avirulent and virulent <i>C</i>. <i>burnetii</i> triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in <i>C</i>. <i>burnetii</i>-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</p></div

VASP activity is required for optimal PV formation.

<p>THP-1 cells were transfected with non-targeting (NT) or VASP-specific siRNA and knockdown confirmed by immunoblot. Cells were processed for confocal microscopy at 72 hpi. DNA was labeled with DAPI (blue), and CD63 (green) and <i>C</i>. <i>burnetii</i> (red) were detected with antibodies. Bar, 10 μm. The scatter plot displays PV diameter in NT or VASP siRNA-transfected cells. PV measurements were taken from at least 15 randomly selected fields. When multiple vacuoles were visible, the two largest vacuoles were measured in a single cell. The horizontal bar indicates average vacuole size. <b>*</b> indicates p < 0.0001 according to a Student’s <i>t</i> test comparing vacuole size in NT and VASP siRNA-transfected cells. VASP knockdown results in formation of smaller PV.</p

VASP activity is required for optimal <i>C</i>. <i>burnetii</i> intracellular replication.

<p>THP-1 cells were transfected with non-targeting (NT) or VASP-specific siRNA and harvested at 24–144 hpi. (A) Immunoblot analysis confirmed >80% knockdown of VASP production in VASP siRNA-transfected cells. (B) A viability assay demonstrated that no significant cell death occurred in VASP-depleted cells. (C) siRNA-transfected THP-1 cells were infected with <i>C</i>. <i>burnetii</i> expressing mCherry. Fluorescence was measured for 6 days after infection using a microplate reader (585/620 nm excitation/emission). (D) Bacterial genome equivalents were determined at 24 and 96 hpi using quantitative PCR. Each data point represents an average of seven replicates collected from two independent experiments, and error bars indicate standard deviation from the mean. <b>*</b> indicates p < 0.05, <b>**</b> indicates p < 0.0005, and *** indicates p < 0.0001 according to a Student’s <i>t</i> test comparing NT and VASP-silenced cells infected with <i>C</i>. <i>burnetii</i>-mCherry. Depletion of VASP significantly impairs <i>C</i>. <i>burnetii</i> intracellular growth.</p

VASP (S239E) expression prevents phagosome trafficking to the PV.

<p>THP-1 cells were transfected with wild type (WT) GFP-VASP (GFP-VASP), GFP-VASP (S157E), or GFP-VASP (S239E), then infected with <i>C</i>. <i>burnetii</i>. Cells were incubated with fluorescent beads (violet) overnight and processed for confocal microscopy at 72 hpi. DNA was stained with DAPI (blue). Bar, 10 μm. N = nucleus and * indicates the PV in micrographs. The scatter plot displays the percentage of beads present within PV in GFP-VASP-, GFP-VASP (S157E)-, or GFP-VASP (S239E)-expressing cells. At least 20 cells from randomly selected fields were used for quantification. The horizontal bar indicates average percentage. <b>*</b> indicates p < 0.0001 according to a Student’s <i>t</i> test comparing wild type- and mutant VASP-expressing cells. Bead-containing phagosomes were directed to the PV in GFP-VASP- and GFP-VASP (S157E)-expressing cells. However, GFP-VASP (S239E) expression impaired bead trafficking to the PV.</p

VASP activity is required for virulent <i>C</i>. <i>burnetii</i> PV formation.

<p>THP-1 cells (A) or primary hAMs (B) were infected with virulent <i>C</i>. <i>burnetii</i> and lysates collected at the indicated times. Immunoblotting was performed using antibodies directed against phosphorylated (S157 and S239) or total VASP (left panels). β-tubulin was used as a loading control. UI = Uninfected cells. Virulent <i>C</i>. <i>burnetii</i> triggered increased levels of phosphorylated VASP from 24–96 hpi in THP-1 cells and hAMs. In the middle and right panels, THP-1 cells (A) or hAMs (B) were transfected with non-targeting (NT) or VASP-specific siRNA. Cells were processed for confocal microscopy at 72 hpi. DNA was labeled with DAPI (blue), and CD63 (green) and <i>C</i>. <i>burnetii</i> (red) were detected with antibodies. Bar, 10 μm. VASP-depleted macrophages contain smaller PV than NT siRNA-transfected cells.</p

VASP phosphorylation levels increase during <i>C</i>. <i>burnetii</i> intracellular growth.

<p>(A) THP-1 cells were infected with <i>C</i>. <i>burnetii</i> for the indicated times and uninfected cells at 6 and 72 h were harvested as controls. (B) THP-1 cells were infected with <i>C</i>. <i>burnetii</i> deficient in CpeD or CpeE and harvested at 72 hpi for immunoblot or immunofluorescence analysis. Cell lysates were subjected to immunoblot analysis using antibodies directed against phosphorylated (S157, S239, S322, or T278) or total VASP. β-tubulin served as a loading control. Densitometric analysis is shown in the graph and represents an average of three independent experiments. Band intensities for all experimental conditions were normalized to β-tubulin levels, then compared to the average of uninfected controls. Error bars indicate the standard error of the mean. ** indicates p < 0.005 and * indicates p < 0.05 according to a Student’s <i>t</i> test. In panel B, cells were processed for microscopy using antibodies directed against <i>C</i>. <i>burnetii</i> (red) and CD63 (green). DAPI was used to stain DNA (blue). Levels of phosphorylated VASP (S157 and S239) increase significantly from 24–96 hpi by wild type and CpeD- (top) or CpeE-deficient (bottom) mutants that form a small, atypical PV, indicating that an expanded PV is not required for VASP phosphorylation.</p

VASP (S157A) expression prevents typical PV formation.

<p>THP-1 cells were transfected with constructs encoding GFP-VASP (S157A) or GFP-VASP (S239A). At 72 hpi with <i>C. burnetii</i> (red), cells were processed for fluorescence microscopy and DNA was stained with DAPI (blue). Bar, 10 μm. N = nucleus and * indicates the PV. Results are representative of at least three separate experiments. GFP-VASP (S239A) expression allowed typical PV expansion, while GFP-VASP (S157A) expression interfered with vacuole expansion, indicating phosphorylation of S157 is critical for PV development.</p

VASP S239E expression interferes with optimal PV formation.

<p>THP-1 cells were transfected with constructs encoding GFP-tagged wild type VASP (GFP-VASP), GFP-VASP (S157E), or GFP-VASP (S239E). At 72 hpi, cells were processed for fluorescence microscopy and DNA was stained with DAPI (blue). Actin was labeled with phalloidin (red). Bar, 10 μm. N = nucleus and * indicates the PV. GFP-VASP and GFP-VASP (S157E) localized around the PV with actin, while GFP-VASP (S239E) expression interfered with vacuole expansion and actin co-localization.</p

VASP is differentially phosphorylated during <i>C</i>. <i>burnetii</i> infection.

<p>THP-1 cells infected for 24 or 96 h and uninfected control cells were harvested and total protein immunoprecipitated with a PKA phospho substrate-specific antibody. (A) Immunoprecipitated proteins were subjected to Coomassie Blue staining and the arrow indicates a ~ 47 kDa protein only in infected cell samples. UI = Uninfected Cells. (B) Schematic of VASP showing five phosphorylation sites (Y39, S157, S239, T278, and S322) and EVH protein-protein interaction domains. PRO = proline rich region. (C) Immunoprecipitated proteins were subjected to immunoblot analysis using antibodies directed against VASP, phosphorylated VASP (S157), or PKA phospho substrates. Lysates were analyzed for β-tubulin to confirm equal protein loading. Agarose bead-bound PKA phospho substrate-specific antibody was eluted, immunoblotted, and probed with anti-IgG antibody to confirm equal amounts of antibody for immunoprecipitations. Densitometry analysis is shown in the graph and represents an average of three independent experiments. Error bars indicate the standard error of the mean. Control = uninfected cells. Increased levels of total and phosphorylated VASP (S157) are present in <i>C</i>. <i>burnetii</i>-infected cells.</p