Korean J Physiol Pharmacol.  2008 Aug;12(4):171-176. 10.4196/kjpp.2008.12.4.171.

Roles of ERK and NF-kappa B in Interleukin-8 Expression in Response to Heat Shock Protein 22 in Vascular Smooth Muscle Cells

Affiliations
  • 1Department of Pharmacology, School of Medicine, Pusan National University, Busan, Korea. koanhoi@pusan.ac.kr

Abstract

Heat shock proteins (HSPs) serve as molecular chaperones and play a role in cell protection from damage in response to stress stimuli. The aim of this article is to investigate whether HSP22 affects IL-8 expression in vascular smooth muscle cells (VSMCs), and which cellular factors are involved in the HSP-mediated IL-8 induction in that cell type in terms of mitogen activated protein kinase (MAPK) and transcription element. Exposure of aortic smooth muscle cells (AoSMCs) to HSP22 not only enhanced IL-8 release but also induced IL-8 transcript via promoter activation. HSP22 activated ERK and p38 MAPK in AoSMCs. HSP22-induced IL-8 release was inhibited by U0126, but not by SB202190. A mutation in the IL-8 promoter region at the binding site of NF-kappa B, but not AP-1 or C/EBP, impaired promoter activation in response to HSP22. Delivery of I kappa B, but not dominant negative c-Jun, lowered HSP22-induced IL-8 release from AoSMCs. These results suggest that HSP22 induces IL-8 in VSMCs via ERK1/2, and that transcription factor NF-kB may be required for the HSP22-induced IL-8 up-regulation.

Keyword

Heat shock protein; Interleukin-8; Vascular smooth muscle cell

MeSH Terms

Binding Sites
Butadienes
Cytoprotection
Heat-Shock Proteins
Hot Temperature
I-kappa B Proteins
Imidazoles
Interleukin-8
Molecular Chaperones
Muscle, Smooth, Vascular
Myocytes, Smooth Muscle
NF-kappa B
Nitriles
p38 Mitogen-Activated Protein Kinases
Promoter Regions, Genetic
Protein Kinases
Proteins
Pyridines
Shock
Transcription Factor AP-1
Butadienes
Heat-Shock Proteins
I-kappa B Proteins
Imidazoles
Interleukin-8
Molecular Chaperones
NF-kappa B
Nitriles
Protein Kinases
Proteins
Pyridines
Transcription Factor AP-1
p38 Mitogen-Activated Protein Kinases

Figure

  • Fig. 1. Induction of IL-8 in VSMCs by HSP22. (A) AoSMCs were treated with 100 ng/ml HSP22 for indicated periods, and IL-8 transcript was amplified by RT-PCR. (B) IL-8 transcript was quantified by quantitative real-time PCR with BSA a control after AoSMCs were treated with 100 ng/ml of HSP22, HSP90 and BSA (con02) for 12 h.

  • Fig. 2. Effects of HSP22 on IL-8 release and IL-8 promoter activity in VSMCs. (A) AoSMCs (1×106 cells/100-mm culture dish) cultured in growth media were incubated for 12 h in the presence of BSA (control) or HSP22 (100 ng/ml). The culture media were harvested. IL-8 secreted in the culture medium was measured by ELISA. Data are expressed as mean±SD (n=3 replicates/group). ∗p<0.01 vs. control. (B) A7r5 cells were transfected with the wild-type pIL-8-Luc651 construct. The transfected cells were incubated in the presence of BSA (control) or HSP22 (100 ng/ml, for 8 h), and processed for luciferase and β-galactosidase assays. Luciferase activity (×106) was normalized to β-galactosidase activity. Induction was calculated relative to the activity of control cells. Data are expressed as mean±SD (n=3 replicates/group). ∗p <0.01 vs. control.

  • Fig. 3. Effects of MAPKs inhibitors on HSP22-induced IL-8 release. (A) Human aorta smooth muscle cells were exposed to HSP22 (100 ng/ml) for the indicated time periods, and cell lysates were then prepared. An equal amount of protein was subjected to Western blot analysis with antibodies for α-tubulin, phospho-ERK2 (p-ERK2), phospho-p38 MAPK (p-p38 MAPK) and α-tubulin. (B) AoSMCs were pre-treated with U0126 or SB202190 (10 μM each, for 2 h) and stimulated with (+) or without (–) HSP22 (100 ng/ml, for 12 h). Secreted IL-8 was measured by ELISA. Data are expressed as mean±SD (n=3 replicates/group). ∗p<0.01.

  • Fig. 4. Effects of transcriptional elements on HSP22-induced IL-8 up-regulation. (A) A7r5 cells were transfected with the wild-type pIL-8-Luc651 construct or the indicated mutant construct. The transfected cells were stimulated with (+) or without (–) HSP22 (100 ng/ml) for 8 h and processed for luciferase and β-galactosidase assays. Induction was calculated relative to the activity of cells incubated in the absence of HSP22. Data are expressed as mean±SD (n=3 replicates/group). ∗p<0.01, ∗∗p<0.05. (B) AoSMCs were infected with indicated recombinant adenoviruses (MOI: 100) and stimulated with (+) or without (–) HSP22 (100 ng/ml, for 12 h). The amount of secreted IL-8 was measured by ELISA. Data are expressed as mean±SD (n=3 replicates/group). ∗p<0.01.


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