Abstract
The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNL-HSP27-EGFP-2 (all P < 0.01). To conclude, these data indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases.
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References
Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316
Owens GK, Wise G (1997) Regulation of differentiation/maturation in vascular smooth muscle cells by hormones and growth factors. Agents Actions Suppl48:3–24
Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:767–780
Davis-Dusenbery BN, Wu C, Hata A (2011) Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation. Arterioscler Thromb Vasc Biol 31:2370–2377
Rensen SS, Doevendans PA, van Eys GJ (2007) Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 15:100–108
Tallquist M, Kazlauskas A (2004) PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15:205–213
Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, Fuqua SA (1993) Biological and clinical implications of heat shock protein 27,000 (HSP27): a review. J Natl Cancer Inst 85(19):1558–1570
Benndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, Lutsch G (1994) Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. J Biol Chem 269(32):20780–20784
Miron T, Vancompernolle K, Vandekerckhove J, Wilchek M, Geiger BA (1991) 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. J Cell Biol 114:255–261
Bitar KN (2002) HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction. Am J Physiol Gastrointest Liver Physiol 282(5):G894–G903
Chen HF, Xie LD, Xu CS (2009) Role of heat shock protein 27 phosphorylation in migration of vascular smooth muscle cells. Mol Cell Biochem 327(1–2):1–6
Chen HF, Xie LD, Xu CS (2010) The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells. Mol Cell Biochem 333:49–56
Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59:75–86
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Sohail M, Doran G, Riedemann J, Macaulay V (2003) Southern EM.A simple and cost-effective method for producing small interfering RNAs with high efficacy. Nucleic Acids Res 31(7):e38
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double stranded RNA. Nature 431:343–349
McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737–747
Huang J, Xie LD, Xu CS, Wang HJ (2009) Construction and identification of a recombinant lentiviral vector harbouring RNAi targeting rat HSP27 gene. Chin Pharmacol Bull 25(4):488–492
Huang J, Xie LD (2008) Expression of human apM1 lentiviral vector in 293 T cell and it’s significance. Chin Pharmacol Bull 24(7):928–932
Yu HZ, Xie L, Zhu P, Xu C, Wang H-J (2012) Human tissue kallikrein 1 gene delivery inhibits PDGF-BB-induced vascular smooth muscle cells proliferation and upregulates the expressions of p27Kip1 and p2lCip1. Mol Cell Biochem 360:363–371
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Ross R (1999) Atherosclerosis is an inflammatory disease. Am Heart J 138:S419–S420
Braun-Dullaeus RC, Mann MJ, Dzau VJ (1998) Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation 98(1):82–89
Sriram V, Patterson C (2001) Cell cycle in vasculoproliferative diseases: potential interventions and routes of delivery. Circulation 103(19):2414–2419
Rousseau S, Houle F, Landry J, Huot J (1997) p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15:2169–2177
Park HK, Park EC, Bae SW et al (2006) Expression of heat shock protein 27 in human atherosclerotic plaques and increased plasma level of heat shock protein 27 in patients with acute coronary syndrome. Circulation 114:886–893
Tallot P, Grongnet JF, David JC (2003) Dual perinatal and developmental expression of the small heat shock proteins [FC12] aB-crystallin and HSP27 in different tissues of the developing piglet. Biol Neonate 83:281–288
Tartakover-Matalon S, Cherepnin N, Kuchuk M et al (2007) Impaired migration of trophoblast cells caused by simvastatin is associated with decreased membrane IGF-I receptor, MMP-2 activity and HSP27 expression. Hum Reprod 22(4):1161–1167
Lee CK, Lee HM, Kim HJ et al (2007) Syk contributes to PDGFBB-mediated migration of rat aortic smooth muscle cells via MAPK pathways. Cardiovasc Res 74(1):159–168
Purushothaman KR, Meerarani P, Moreno PR (2007) Inflammation and neovascularization in diabetic atherosclerosis. Indian J Exp Biol 45(1):93–102
Somara S, Bitar KN (2004) Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle. Am J Physiol Cell Physiol 286:C1290–C1301
Chen Y, Currie RW (2006) Small interfering RNA knocks down heat shock factor-1(HSF-1) and exacerbates pro-inflammatory activation of NF-kB and AP-1 in vascular smooth muscle cells. Cardiovasc Res 69:66–75
Huot J, Houle F, Spitz DR, Landry J (1996) HSP27 phosphorylation mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 56:273–279
Doshi BM, Hightower LE, Lee J (2009) The role of HSP27 and actin in the regulation of movement in human cancer cells responding to heat shock. Cell Stress Chaperones 14:445–457
Hedges JC, Dechert MA, Yamboliev IA et al (1999) A role for p38MAPK/HSP27 pathway in smooth muscle cell migration. Biol Chem 274(34):24211–24219
Rosner D, McCarthy N, Bennett M (2005) Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53. Cardiovasc Res 66(3):601–610
Sun J, Zheng J, Ling KH et al (2012) Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA. J Transl Med 10:2
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This work was supported by Grants from the Clinical Key Program of the Fujian Medical University (XK201107) and the Fujian Natural Science Foundation (2011J01160).
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Huang, J., Xie, Ld., Luo, L. et al. Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro. Mol Cell Biochem 390, 115–121 (2014). https://doi.org/10.1007/s11010-014-1962-1
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DOI: https://doi.org/10.1007/s11010-014-1962-1