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. 2003 Mar;138(5):950-8.
doi: 10.1038/sj.bjp.0705088.

Inhibition by pentoxifylline of TNF-alpha-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-kappa B down-regulation

Yung-Ming Chen  1 , Chao-Jung Tu, Kung-Yu Hung, Kwan-Dun Wu, Tun-Jun Tsai, Bor-Shen Hsieh
Affiliations

Inhibition by pentoxifylline of TNF-alpha-stimulated fractalkine production in vascular smooth muscle cells: evidence for mediation by NF-kappa B down-regulation

Yung-Ming Chen et al. Br J Pharmacol. 2003 Mar.

Abstract

(1) Fractalkine is a CX(3)C chemokine for mononuclear leukocytes that is expressed mainly by vascular cells, and regulated by pro-inflammatory cytokines. This study investigated signal transduction mechanisms by which tumor necrosis factor (TNF)-alpha stimulated fractalkine expression in cultured rat vascular smooth muscle cells (VSMCs), and the modulatory effect of a haemorrheologic agent, pentoxifylline, on its production. (2) TNF-alpha (1-50 ng ml(-1)) stimulated fractalkine mRNA and protein expression in concentration- and time-dependent manners. Pretreatment with calphostin C (0.4 micro M, a selective inhibitor of protein kinase C (PKC), and PD98059 (40 micro M), a specific inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase, attenuated TNF-alpha-stimulated fractalkine mRNA and protein expression. In contrast, H-89 (2 micro M), a selective inhibitor of cAMP-dependent protein kinase, wortmannin (0.5 micro M), a selective inhibitor of phosphatidylinositol 3-kinase, and SB203580 (40 micro M), a specific inhibitor of p38 MAPK, had no discernible effect. (3) The ubiquitin/proteosome inhibitors, MG132 (10 micro M) and pyrrolidine dithiocarbamate (200 micro M), suppressed activation of NF-kappaB as well as stimulation of fractalkine mRNA and protein expression by TNF-alpha. (4) TNF-alpha-activated phosphorylation of PKC was blocked by calphostin C, whereas TNF-alpha-augmented phospho-p42/44 MAPK and phospho-c-Jun levels were reduced by PD98059. Neither calphostin C nor PD98059 affected TNF-alpha-induced degradation of I-kappaBalpha or p65 nuclear translocation. (5) Pretreatment with pentoxifylline (0.1-1 mg ml(-1)) decreased TNF-alpha-stimulated fractalkine mRNA and protein expression, which was preceded by a reduction in TNF-alpha-activated phosphorylation of PKC, p42/44 MAPK and c-Jun as well as degradation of I-kappaBalpha and p65/NF-kappaB nuclear translocation. (6) These data indicate that activation of PKC, p42/44 MAPK kinase, and NF-kappaB are involved in TNF-alpha-stimulated fractalkine production in VSMCs. Down-regulation of the PKC, p42/44 MAPK, and p65/NF-kappaB signals by PTX may be therapeutically relevant and provide an explanation for the anti-fractalkine effect of this drug.

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Figures

Figure 1
Figure 1
Time-course and dose-response of TNF-α stimulation on fractalkine mRNA and protein expression. VSMCs were incubated with TNF for the given periods. Ten micrograms of total RNA, forty micrograms of cell lysate, and fifty micrograms of concentrated condition media were analysed for fractalkine mRNA and protein expression as described in Methods. (a and b) Representative Northern and Western blots of fractalkine expression in response to TNF-α (5 ng ml−1) at variable timepoints. FKN: fractalkine mRNA, GAPDH: glyceraldehyde-3-phosphate dehydrogenase mRNA, cFKN: cell-bound fractalkine in cell lysate, sFKN: soluble fractalkine in concentrated conditioned media. (c and d) Representative Northern and Western blots of fractalkine expression in response to TNF-α (1–50 ng ml−1) at 4 and 24 h, respectively. Right panels show corresponding quantitative results of FKN/GAPDH mRNA and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 vs control at zero time.
Figure 2
Figure 2
Effects of GM 6001 on TNF-α-stimulated cell-bound and soluble fractalkine expression. VSMCs were incubated with TNF-α (5 ng ml−1) for 24 h, with or without pretreatment with GM (GM 6001, 2 μM) for 30 min. Upper panel shows representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panel shows quantitative results of cFKN (corrected for β-actin) and sFKN relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 between the given experimental conditions.
Figure 3
Figure 3
Effects of protein kinase inhibitors on TNF-α stimulated fractalkine mRNA and protein expression. VSMCs were incubated with TNF-α (5 ng ml−1) for 4 or 24 h, with or without pretreatment with the given pharmacologic inhibitors (H-89, 2 μM; Cal C: calphostin C, 0.4 μM; PD: PD98059, 40 μM; SB: SB203580, 40 μM; WM: wortmannin, 0.5 μM). (a) Representative Northern blots. FKN: fractalkine mRNA. (b) Representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panels show corresponding quantitative results of FKN/GAPDH mRNA and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 vs TNF-α-treated cells.
Figure 4
Figure 4
Effect of NF-κB inhibition on TNF-α stimulated fractalkine mRNA and protein expression. VSMCs were pretreated with MG (MG132, 10 μM) or PDTC (pyrrolidine dithiocarbamate, 200 μM) for 1.5 and 1 h, respectively, followed by TNF-α (5 ng ml−1) for 4 or 24 h. (a) Representative Northern blots. FKN: fractalkine mRNA. (b) Representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panels show corresponding quantitative results of FKN/GAPDH and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 vs TNF-α-treated cells.
Figure 5
Figure 5
Effects of MG132, PDTC, calphostin C, and PD98059 on TNF-α-activated phospho-p42/44 MAPK, phospho-PKC, phospho-c-Jun and I-κBα levels. VSMCs were incubated with TNF-α (5 ng ml−1) for varying timepoints, with or without pretreatment with the given pharmacologic inhibitors. Graphs showing representative Western blots from three independent experiments with similar results. The concentrations of inhibitors used in the signalling studies were MG: MG132 (10 μM); PDTC: pyrrolidine dithiocarbamate (200 μM); Cal C: calphostin C (0.4 μM); PD: PD98059 (40 μM). PKC(pan): PKC of classical/novel isoforms; PKCζ/ι: PKC of atypical isoformζ/ι.
Figure 6
Figure 6
Immunostaining of nuclear translocated NF-κB/p65 induced by TNF-α: effects of MG132, PDTC, and pentoxifylline. (a) control. (b) incubation of VSMCs with TNF-α (5 ng ml−1) for 15 min induced nuclear translocation of p65/NF-κB. (c, d, e) MG132 (10 μM), pyrrolidine dithiocarbamate (200 μM), and pentoxifylline (1 mg ml−1) blocked translocation of p65/NF-κB induced by TNF-α. (f, g) Calphostin C (0.4 μM) and PD98059 (40 μM) did not affect NF-κB/p65 nuclear translocation (original magnification×300).
Figure 7
Figure 7
Effects of pentoxifylline on TNF-α stimulated fractalkine mRNA and protein expression as well as TNF-α-activated phospho-p42/44 MAPK, phospho-PKC, phospho-c-Jun and I-κBα levels. VSMCs were incubated with TNF-α (5 ng ml−1) for 4 or 24 h, with or without pretreatment with PTX (pentoxifylline, 1–0.1 mg ml−1) for 45 min. (a) Representative Northern blots. FKN: fractalkine mRNA. (b) Representative Western blots. cFKN: cell-bound fractalkine, sFKN: soluble fractalkine. Lower panels show corresponding quantitative results of FKN/GAPDH mRNA and cFKN/β-actin ratios relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 vs TNF-α-treated cells. (c) Representative Western blots showing the effects of PTX (1 mg ml−1) on TNF-α-activated signalling pathways. These experiments were performed three times, and similar results were obtained. PKC(pan): PKC of classical/novel isoforms; PKCζ/ι: PKC of atypical isoformζ/ι.
Figure 8
Figure 8
Northern blot of TNF-α-stimulated fractalkine mRNA stability. Cells were exposed to TNF-α (5 ng ml−1) for 1 h, followed by actinomycin D (20 μg ml−1) with or without pentoxifylline (1 mg ml−1) to stop further mRNA transcription. Cells were harvested at serial timepoints as indicated to assess the rate of fractalkine mRNA breakdown. Upper panel shows representative Northern blots. Significant fractalkine mRNA transcript breakdown appears to occur between 3 to 7 h after addition of actinomycin D. Lower panel shows quantitative results of FKN/GAPDH mRNA ratios relative to that of control. Values are mean±s.e.mean of three experiments. *P<0.05 vs corresponding TNF-α-treated cells.
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