Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells

Abstract

Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis.

Fig. 1.

Histamine induces activation of protein kinase D (PKD) 1 and PKD2 in human aortic smooth muscle cells (HASMCs). A: Western blot analysis of the expression of PKD isoforms in HASMCs using antibodies against PKD1, PKD2, and PKD3. Lysates of HEK293 cells were used as a positive control. B: Western blot analysis of the time course of histamine stimulation of PKD phosphorylation in HASMCs. Antibodies against phospho (p)-PKD1 (Ser⁹¹⁰), p-PKD2 (Ser⁸⁷⁶), and p-PKD activation loop were used. C: time-dependent PKD phosphorylation was quantified by densitometry. Data are means ± SE from three experiments. D: Western blot analysis of the concentration dependence of histamine induction of PKD phosphorylation. HASMCs were stimulated with histamine for 5 min. E: concentration-dependent PKD phosphorylation induced by histamine was quantified by densitometry. Data are means ± SE from three experiments. F and G: immunofluorescence data of histamine-induced phosphorylation of PKD1 and PKD2. HASMCs were treated with histamine (10 μM) for 5 min. DAPI was used for nuclei staining. Specific antibodies against p-PKD1 (Ser⁹¹⁰), p-PKD2 (Ser⁸⁷⁶), and α-actin were used, followed by a secondary antibody: goat anti-rabbit IgG Alexa Fluor 488 or rhodamine red-X-conjugated goat anti-mouse IgG.

Fig. 2.

Role of histamine receptors in histamine-induced PKD activation. PKD phosphorylation induced by histamine was detected by Western blot analysis. HASMCs were pretreated with the histamine receptor 1 (H₁) antagonist mepyramine or the histamine receptor 2 (H₂) antagonist cimetidine of indicated concentrations for 40 min; then cells were stimulated with histamine (10 μM) for 5 min. A: pretreatment with mepyramine dose-dependently blocked histamine-induced PKD phosphorylation. B: results of Western blot analysis were quantified by densitometry. C: pretreatment with cimetidine had no effect on PKD activation induced by histamine. D: Western blot analysis of the effect of cimetidine on histamine-induced PKD phosphorylation quantified by densitometry. Data are means ± SE from three experiments. * and #P < 0.05 and ** and ##P < 0.01 vs. histamine alone.

Fig. 3.

Effect of the inhibitors of G_i/o proteins and protein kinase C (PKC) on histamine-induced PKD activation. HASMCs were pretreated with the G_i/o protein inhibitor pertussis toxin (PTX) overnight or the PKC inhibitor Ro-31–8220 for 40 min; then cells were stimulated with histamine (10 μM) for 5 min. A: G_i/o proteins are dispensable in histamine-induced PKD activation. Western blot analysis of the PTX effect on PKD phosphorylation induced by histamine. B: results of Western blot analysis of PKD phosphorylation were quantified by densitometry. Data are means ± SE from three experiments. C: PKC mediates histamine-induced PKD phosphorylation. Western blot analysis of the effect of PKC inhibitor Ro-31–8220 on PKD phosphorylation induced by histamine. D: results of Western blot analysis were quantified by densitometry. Data are means ± SE from three experiments. * and #P < 0.05 and ** and ##P < 0.01 vs. histamine alone.

Fig. 4.

Effect of PKD on histamine-induced mitogen-activated protein kinase (MAPK) activation in HASMCs. A: top, effects of specific small-interfering RNA (siRNA) of PKD isoforms on histamine-induced MAPK activation. The specific PKD siRNAs were transfected into HASMCs for 48 h followed by serum starvation for 24 h; then cells were stimulated with histamine (10 μM) for 5 min. Expression of PKD1, PKD2, and PKD3 proteins and phosphorylation of PKDs and MAPKs were detected by Western blot analysis. Middle and bottom, Western blotting results were quantified by densitometry. Data are means ± SE from three experiments. **P < 0.01 vs. histamine alone. B: effect of MAPK inhibitors on phosphorylation of PKDs was detected by Western blot analysis. HASMCs were pretreated with the extracellular signal-regulated kinase (ERK) inhibitor U-0126, p38 MAPK inhibitor SB-203580, or c-Jun NH₂-terminal kinase (JNK) inhibitor SP-600125 for 40 min; then cells were stimulated with histamine (10 μM) for 5 min.

Fig. 5.

Effect of PKD on tissue factor (TF) expression and TF surface activity induced by histamine in HASMCs. The specific PKD siRNAs were transfected into HASMCs for 48 h followed by serum starvation for 24 h; then cells were stimulated with histamine (10 μM) for 5 h. A: top, effect of knockdown of the expression of PKD isoforms with PKD siRNAs on histamine-induced TF protein expression. Expression of PKDs and TF proteins was examined by Western blot analysis. Bottom, results were quantified by densitometry. B: effect of knockdown of the expression of PKD isoforms with PKD siRNAs on histamine-induced TF surface activity. Data are means ± SE from three experiments. **P < 0.01 vs. histamine alone.

Fig. 6.

Effect of PKC-specific inhibitor on p38 MAPK activity and TF protein expression. A: effect of PKC-specific inhibitor Ro-31–8220 on p38 MAPK activation. Top, phosphorylation of PKD2 and p38 MAPK was detected by Western blot analysis. HASMCs were pretreated with Ro-31–8220 for 40 min, followed by histamine (10 μM) stimulation for 5 min. Bottom, results were quantified by densitometry. B: effects of PKC-specific inhibitor Ro-31–8220 and p38 MAPK inhibitor SB-203580 on histamine-induced TF expression. Top, TF protein expression was examined by Western blot analysis. HASMCs were pretreated with inhibitors for 40 min and then stimulated with histamine (10 μM) for 5 h. Bottom, results were quantified by densitometry. Data are means ± SE from three experiments. **P < 0.01 vs. histamine alone.