3',4'-Dihydroxyflavonol down-regulates monocyte chemoattractant protein-1 in smooth muscle: role of focal adhesion kinase and PDGF receptor signalling

Abstract

Background and purpose: We investigated the effects of a synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF) on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat vascular smooth muscle cells.

Experimental approach: MCP-1 expression was assessed by quantitative real-time PCR and protein phosphorylation by immunoprecipitation and Western blots.

Key results: DiOHF (1-30 micromol x L(-1)) concentration-dependently reduced MCP-1 expression in both quiescent cells and cells stimulated with platelet-derived growth factor (PDGF) or interleukin 1-beta. The effect of DiOHF was associated with a suppression of focal adhesion kinase (FAK)-mediated signalling. In vitro kinase assays demonstrated that DiOHF is a potent inhibitor of FAK kinase activity (EC(50)= 2.4 micromol x L(-1)). Expression of FAK-related non-kinase reduced basal MCP-1 expression, but not that induced by PDGF or interleukin 1-beta. DiOHF also inhibited autophosphorylation of PDGF receptors. The PDGF receptor inhibitor AG-1296 potently suppressed basal and PDGF-induced MCP-1 expression. Inhibition of extracellular signal-regulated kinase activation by DiOHF, either directly or indirectly, may also be involved in its effects on MCP-1 expression. DiOHF had no inhibitory effect on either p38 or nuclear factor-kappaB activation. Moreover, DiOHF inhibited smooth muscle cell spreading (a FAK-mediated response) and proliferation.

Conclusions and implications: This is the first report on a flavonoid compound (DiOHF) that is a potent FAK inhibitor. DiOHF also inhibits PDGF receptor autophosphorylation. These effects underlie the inhibitory action of DiOHF on MCP-1 expression in smooth muscle cells. Our results suggest that DiOHF might be a useful tool for dissection of the (patho)physiological roles of FAK signalling.

Figure 1

Quantitative real-time PCR analysis of monocyte chemoattractant protein-1 (MCP-1) mRNA expression in growth arrested rat aortic smooth muscle cells treated with 3′,4′-dihydroxyflavonol (DiOHF), quercetin (Quer), Tempo (T, 1 mmol·L⁻¹), H₂O₂ (H, 100 µmol·L⁻¹), the catalase inhibitor (C-I) 3-amino-1, 2, 4-triazole (10 mmol·L⁻¹), genistein (Gen, 100 µmol·L⁻¹), PD-98059 (PD, 25 µmol·L⁻¹) and SB-202190 (SB, 10 µmol·L⁻¹). The duration of treatment for all agents was 6 h. The results are expressed as fold of control (Con). *P < 0.05 versus control, n= 3–6.

Figure 8

Effects of 3′,4′-dihydroxyflavonol (DiOHF) on extracellular signal-regulated kinase (ERK)/p38 phosphorylation and nuclear factor-κB (NF-κB) activation. (A) Time course of ERK and p38 phosphorylation induced by platelet-derived growth factor (PDGF) (20 ng·mL⁻¹) and interleukin 1-β (IL-1β) (10 ng·mL⁻¹). Example from two independent experiments. (B) Concentration-dependent effects of DiOHF on basal and PDGF-induced ERK1/2 and p38 phosphorylation. Cells were pretreated with DiOHF for 1 h followed by PDGF stimulation (for 20 min). (C) Effects of DiOHF on IL-1β-induced ERK1/2 and p38 phosphorylation. Cells were pretreated with DiOHF for 1 h followed by IL-1β (for 20 min). All Western blot experiments in (B) and (C) were repeated three times. (D) Effects of DiOHF on IL-1β-induced p50 NF-κB activation. Cells were pretreated with DiOHF for 1 h followed by IL-1β for another hour. The DNA binding activity of p50 was assessed with a colorimetric transcription factor assay kit from Millipore (Billerica, MA, USA). The results were expressed as the difference of absorbance at 450 and 650 nm normalized to protein content. *P < 0.05 versus control, one-way anova, n= 3.

Figure 2

The effects of 3′,4′-dihydroxyflavonol (DiOHF) on monocyte chemoattractant protein-1 (MCP-1) mRNA expression in the presence of interleukin 1-β (IL-1β) (10 ng·mL⁻¹) or platelet-derived growth factor (PDGF)-BB (20 ng·mL⁻¹). The duration of treatment for all agents was 6 h. The results are expressed as fold of control (Con). *P < 0.05 versus control; #P < 0.05 versus cells treated with IL-1β or PDGF respectively; n= 3–6.

Figure 3

Effects of 3′,4′-dihydroxyflavonol (DiOHF), quercetin (Quer) and PP2 on protein tyrosine phosphorylation. (A) Effects of DiOHF (D; 30 µmol·L⁻¹ for 1 h) on total tyrosine-phosphorylated proteins detected by a monoclonal anti-phosphotyrosine antibody in rat aortic smooth muscle cells with or without platelet-derived growth factor (PDGF) (20 ng·mL⁻¹) treatment. The arrows indicate the 130 and 70 kDa bands that were significantly modified by DiOHF treatment (n= 3). Con, vehicle control. The blot strips were from the same membrane, in which equal amounts of protein were loaded for all lanes. (B) Cells were treated with DiOHF (D; 30 µmol·L⁻¹), quercetin (Quer; 30 µmol·L⁻¹) or PP2 (1 µmol·L⁻¹) for 1 h. Total cell lysates were immunoprecipitated with the anti-phosphotyrosine antibody followed by Western blotting with antibodies against paxillin (Pax), pp120 or focal adhesion kinase (FAK). Total paxillin, pp120, FAK and Src were determined by western blot with the cell lysates without immunoprecipitation. n= 3. The lower thin band in total FAK is likely to be an artefact because of the relatively high protein load; in experiments with a lower protein load, this band disappeared. (C) Concentration-dependent effects of DiOHF (D, concentration shown in µmol·L⁻¹) and quercetin (Q) treatment for 1 h on tyrosine phosphorylation of paxillin (Pax) and FAK. n= 3. (D) Summary data of the Western blots in (C) measured by densitometry. *P < 0.05 versus control (normalized to 100%), n= 3.

Figure 4

Effects of the Src inhibitor PP2 (1 µmol·L⁻¹) on monocyte chemoattractant protein-1 (MCP-1) mRNA expression under basal conditions and after platelet-derived growth factor (PDGF) (20 ng·mL⁻¹ for 6 h) or interleukin 1-β (IL-1β) (10 ng·mL⁻¹) stimulation. Data shown as mean ± SEM, *P < 0.05, n= 4–6. The numbers in parenthesis are the results for PP2 alone and show that the low basal expression of MCP-1 mRNA was significantly inhibited by PP2.

Figure 5

Concentration-dependent effects of 3′,4′-dihydroxyflavonol (DiOHF) on the kinase activity of (A) focal adhesion kinase (FAK) and (B) Src measured with an in vitro kinase assay using recombinant human FAK or Src. *P < 0.05 versus control, n= 8 assays.

Figure 6

Effects of focal adhesion kinase-related non-kinase (FRNK) on monocyte chemoattractant protein-1 (MCP-1) expression. (A) Time course of FRNK expression. Rat aortic smooth muscle cells transfected with no vector (N), the control pRK vector (P) or pRK-vesicular stomatitis virus (VSV)-FRNK (F) for different times as indicated were analysed for FRNK expression with Western blot using an anti-VSV-glycoprotein (VSV-G) antibody. (B) Western blots showing that FRNK expression did not affect the total expression levels of focal adhesion kinase (FAK) or paxillin (Pax). (C,D) Effects of FRNK expression on MCP-1 mRNA levels under basal condition and after platelet-derived growth factor (PDGF) or interleukin 1-β (IL-1β) stimulation measured by real-time PCR. *P < 0.05 versus control, n= 6–7.

Figure 7

Role of platelet-derived growth factor receptor (PDGFR) activation in 3′,4′-dihydroxyflavonol (DiOHF)-induced inhibition on monocyte chemoattractant protein-1 (MCP-1) expression. (A) Effects of DiOHF on ligand-induced PDGFR autophosphorylation analysed by immunoprecipitation (IP) with anti-phosphotyrosine (pY) followed by Western blotting (IB) with anti-PDGFR-α. DiOHF alone had little effects on PDGFR phosphorylation but inhibited PDGF-induced PDGFR phosphorylation (n= 3 experiments). (B) Concentration–response effects of DiOHF on PDGFR autophosphorylation induced by PDGF (20 ng·mL⁻¹ for 10 min). The phosphorylation of PDGFR was detected by Western blot with anti-phosphotyrosine (pY) followed by membrane stripping and reprobing with anti-PDGFR-α. *P < 0.05 versus control (open bar); #P < 0.05 versus PDGF alone, n= 3. (C) Effects of the PDGFR inhibitor AG-1296 (10 µmol·L⁻¹ for 6 h) on MCP-1 expression (*P < 0.05 versus control, n= 3). (D) Effects of AG-1296 (10 µmol·L⁻¹) on paxillin tyrosine phosphorylation in resting RASMCs. Paxillin phosphorylation was measured with an anti-phospho-paxillin (phospho Pax) antibody. The same membrane was then stripped and reprobed with anti-paxillin. The summary data are shown on the right (n= 3).

Figure 9

Effects of 3′,4′-dihydroxyflavonol (DiOHF) on cell spreading and proliferation. (A) Phase contrast microscopy showing the morphology of rat aortic smooth muscle cells (RASMCs) 3 h after plating in culture dish without (control) or with DiOHF treatment. Cells that showed spreading were indicated by arrows, while the cells remained round were indicated by arrowheads. (B) Percentage of spread cells at 3 h after plating without (Con) or with DiOHF (D, concentrations in µmol·L⁻¹) treatment. *P < 0.05 versus Con, n= 3. (C) Effects of DiOHF on RASMC proliferation. Cells were cultured in medium containing 10% serum. DiOHF remained in the culture medium throughout the culture period. The data are averaged numbers from triplicate wells. (D) Effects of DiOHF on RASMC viability. Cells were growth arrested by incubating in serum-free medium for 48 h. Then DMSO (control) or DiOHF was added and cultured for another 24 or 48 h period. Cell proliferation was assessed with the CellTiter-96 AQueous One Solution kit (Promega) and measured as absorbance at 490 nm. *P < 0.05 versus control, one-way anova, n= 4.