Korean J Physiol Pharmacol.  2015 Nov;19(6):549-555. 10.4196/kjpp.2015.19.6.549.

Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

• Jeong JY
• Son Y
• Kim BY
• Eo SK
• Rhim BY
• Kim K

• KMID: 2070795
• DOI: http://doi.org/10.4196/kjpp.2015.19.6.549

Abstract

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

Keyword

CCL11; Secretory phenotype; Signaling pathway; Thrombin; Vascular smooth muscle cell

MeSH Terms

Humans
Muscle, Smooth, Vascular*
Myocytes, Smooth Muscle
Pertussis Toxin
Phenotype*
Phosphorylation
Protein Kinase C
Proto-Oncogene Proteins c-raf
Thrombin
Pertussis Toxin
Protein Kinase C
Proto-Oncogene Proteins c-raf
Thrombin

Figure

• 

  Fig. 1 Effects of thrombin on expression of CCL11 in human VSMCs. (A, B) HAoSMCs were treated for the indicated time periods with thrombin (1 U/ml). (A) CCL11 transcripts were amplified by RT-PCR (upper panel) and the levels of CCL11 transcripts were determined by realtime PCR (lower panel). (B) The levels of CCL11 protein secreted into culture media were determined by ELISA. Data are expressed as mean±SD (n=3 replicates for each group). **p<0.01 vs. control cultured in the absence of thrombin (0 h); ***p<0.001 vs. 0 h.

• 

  Fig. 2 Effects of inhibitors of MEK and Akt in thrombin-induced expression of CCL11. (A, B) HAoSMCs were incubated with or without U0126 and Akti IV (10 µM each) for 2 h followed by stimulation with thrombin for 9 h. (A) CCL11 transcripts were amplified by RT-PCR (upper panel) and the levels of CCL11 transcripts were determined by realtime PCR (lower panel). (B) The levels of CCL11 protein secreted into culture media were determined by ELISA. Data are expressed as mean±SD (n=3 replicates for each group). ***p<0.001 vs. control; ###p<0.001 vs. thrombin.

• 

  Fig. 3 Effects of inhibitors of PKC and Raf-1 on phosphorylation of ERK and expression of CCL11 induced by thrombin. (A) HAo-SMCs were stimulated with thrombin for 5 min after incubation with or without GF109203X (3 µM) and GW5074 (25 nM) for 2 h. An equal amount of protein was subjected to Western blot analysis with antibodies against α-tubulin and phosphorylated and unphosphorylated forms of ERK1/2. (B, C) HAoSMCs were incubated with or without GF109203X (3 µM) and GW5074 (25 nM) for 2 h followed by stimulation with thrombin for 9 h. The levels of CCL11 transcripts were determined by realtime PCR (B), and the levels of CCL11 protein secreted into culture media were determined by ELISA (C). Data are expressed as mean±SD (n=3 replicates for each group). ***p<0.001 vs. control; ###p<0.001 vs. thrombin.

• 

  Fig. 4 Effects of pertussis toxin and LY294002 on phosphorylation of Akt and expression of CCL11 induced by thrombin. (A) HAo-SMCs were stimulated with thrombin for 5 min after incubation with or without pertussis toxin (100 ng/ml) and LY294002 (10 µM) for 2 h. An equal amount of protein was subjected to Western blot analysis with antibodies against α-tubulin and phosphorylated and unphosphorylated forms of Akt. The mean band intensity of p-Akt was normalized to that of Akt. (B, C) HAoSMCs were incubated with or without pertussis toxin (100 ng/ml) and LY294002 (10 µM) for 2 h followed by stimulation with thrombin for 9 h. The levels of CCL11 transcripts were determined by realtime PCR (B), and the levels of CCL11 protein secreted into culture media were determined by ELISA (C). Data are expressed as mean±SD (n=3 replicates for each group). ***p<0.001 vs. control; ###p<0.001 vs. thrombin.

• 

  Fig. 5 Effects of ROS quenchers on expression of CCL11 induced by thrombin. (A, B) HAoSMCs were incubated with or without DPI (10 µM) and NAC (5 mM) for 2 h followed by stimulation with thrombin for 9 h. The levels of CCL11 transcripts were determined by realtime PCR (A), and the levels of CCL11 protein secreted into culture media were determined by ELISA (B). Data are expressed as mean±SD (n=3 replicates for each group). ***p<0.001 vs. control; #p<0.05 vs. thrombin; ###p<0.001 vs. thrombin.

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