Uvabain Aracılıklı Apoptotik Hücre Ölümleri ve Rho-Kinaz: Bir Proteomik Çalışma

Yıl 2010, Cilt: 3 Sayı: 1, - , 01.03.2010

Emrah Bayram Aysun Özdemir Ayla Cihan Duygu ÖZEL Demiralp Belgin Polat Mustafa Ark

Öz

Abstract Ouabain-Induced Apoptotic Cell Death and Rho Kinase: A Proteomics Study Objective: In the current work, we aimed to evaluate the possible involvement of Rho kinase in the ouabain-induced apoptotic cell death, using a proteomic approach. Method: Human umbilical vein endothelial cells were exposed to 10 μM ouabain, 10 μM Y27632 (30 min before the ouabain treatment)+10 μM ouabain, and 10 μM Y27632 alone. Cell homogenates were separated by 2D gel electrophoresis and stained with Coomassie blue. Results: It was detected that 54 spots were changed with the ouabain treatment. Seven spots, changed with ouabain plus Y27632 treatment, were excised and subjected to MALDI-TOF MS to identify the proteins of interest. The levels of caspase-7 and protein unc-119 homolog B proteins increased with ouabain treatment (p<0.05) and this increase has decreased by the pretreatment with Y27632 (p<0.05) . In addition, the levels of neurensin-2, C-type lectin domain family-2 member D and putative FERM domain-containing protein FKSG43 proteins were decreased with ouabain treatment (p<0.05). This decrease has increased by the pretreatment with Y27632 (p<0.05). Conclusion: These results indicate that Rho kinase pathway may involve in the changes of expression of these proteins. Caspase-7, protein unc-119 homolog B, neurensin-2 and C-type lectin domain family-2 member D proteins are involved in apoptosis, neurotransmitter release, tumor suppression and the inhibition of osteoclast formation, respectively. However, further studies are needed to confirm our results and to determine the importance of these proteins.

Anahtar Kelimeler

ouabain, Rho-kinase, proteomics, apoptosis, sodium pump.

Uvabain Aracılıklı Apoptotik Hücre Ölümleri ve Rho-Kinaz: Bir Proteomik Çalışma

Öz

Türkçe Özet: Amaç: Bu çalışmada, uvabain ile indüklenmiş apoptotik hücre ölümlerinde, Rho-kinazın olası rolünün proteomik yaklaşımlar kullanılarak değerlendirilmesi amaçlandı. Yöntem: İnsan umbilikal ven endotel hücreleri, 10 μM uvabain, 10 μM Y27632 (uvabain uygulamasından 30 dk önce)+10 μM uvabain ve yalnız 10 μM Y27632\'ye maruz bırakılmıştır. Hücre homojenatları, iki boyutlu jel elektroforezi ile ayrılmış ve Coomassie mavisi ile boyanmıştır . Bulgular: Uvabain uygulaması sonucunda, 54 spotun değiştiği tespit edilmiştir. Uvabain + Y27632 uygulamasında değiştiği gözlenmiş olan 7 protein, MALDI-TOF MS yöntemi ile tanımlanmak üzere seçilmiştir. Kaspaz-7 ve protein-unc-119 homolog B proteinlerinin miktarları uvabain uygulaması ile artmış (p<0.05) ve bu artış Y27632 ön uygulaması ile azalmıştır(p<0.05). Buna ek olarak nörensin-2, C-type lectin domain family-2 member D ve putative FERM domain-containing protein FKSG-43 proteinleri uvabain uygulaması ile azalmış (p<0.05) ve bu azalış Y27632 ön uygulaması ile artmıştır (p<0.05). Sonuç: Bu sonuçlar, bu proteinlerin ekspresyonlarındaki değişiklikler üzerinde Rho-kinaz yolağının önemini göstermektedir. Kaspaz-7, protein unc-119 homolog B, nörensin-2 ve C-tipi lektin bölgesi ailesi-2 üyesi D proteinleri sırasıyla, apoptoz, nörotransmitter salınımı, tümör süpresyonu ve osteoklast oluşumunun inhibisyonunda yer almaktadırlar. Bununla birlikte sonuçlarımızın doğrulanması ve bu proteinlerin öneminin belirlenmesi için daha başka çalışmalara ihtiyaç vardır.

Anahtar Kelimeler

uvabain, Rho-kinaz, proteomik, apoptoz, sodyum pompası.

Kaynakça

• Skuo JC. The influence of some eations on an adenosine triphosphatase from peripheral nerves. J Am Soc Nephrol 1957;23:394—401.
• Apeıia A. New roles for an old enzyme: Na+l K+— ATPase emerges as an interesting drug target. J Intern Med 2007;261:44-52.
• Skou JC, Esınann M. The Nal" K+—ATPase. J Bioenerg Biomembr 1992;24:249-61.
• Xie Z, Cai T. Na(+)-K(+)-ATPase-mediated signal transduction: from protein interaction to cellular function. Mol lnterv 2003;3:157-68.
• Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Haris DW, Mandel F, Mathews WR, Ludens JH. Identification and characterization of ouahain—like compound from human plasma. Proc Natl Acad Sci USA ;88:6259—63.
• Ludens JH, Clark MA, DuCharme DW, Harris DW, Lutzke BS. Maııdel F, Mathews WR, Sutter DM, Hamlyn JM. Prutîcation of an endogenous digitalis-like factor from human plasma for structural analysis. Hypertension 1991 ;171923—9.
• Hamlyn JM, Lu ZR, Manunta P., Ludens JH, Kimura K, Shah JR, Laredo J, Hamilton JP, Hamilton MJ, Hamilton BP. Observations on the nature, biosynthesis, secretion and significance of endogenous ouabain. Clin Exp Hypertens 1998;20:523—33.
• Manunta P, Rogowski AC, Hamilton BP. Hamlyn JM. Ouabain—induced hypertesion in the rat: relationships among plasma and tissue ouaban and blood prssure. J Hypertens 1994;12:549-60.
• Khan MI, Chesney JA, Laber DA. Miller DM. Digitalis, a . Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A targeted therapy for cancer? AmJ Med Sci 2009;52355—9. protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2007 ;2:481—5.
• Ark M, Özdemir A, Polat B. Ouabain—induced apoptosis and rho kinase. A novel caspase—2 clevage site and fragment of . Özel Demiralp D, Haznedaroğlu İC, Akar N. Functional rock—Z.Apapmsı's 2010 (DOI: 10.1007/s10495—010—0529—l). proteomic analysis of Ankaferd® Blood Stoper Turk J Hematol 2010; 27: 70—7.
• Takai Y, Sasaki T, Matozaki T. Small GTP—binding proteins. Physiol Rev 2001;81:153-208. . Yu SP. Na(+)K(+)—ATPase: the new face of an old player in pathogenesis and apoptotic/hybrid cell death.
• Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Biochem Pharmacol 2003;66(8):1601-9. Nature 2002;420:629-35
• Qiu J, Gao HQ, Li BY, Shen L. Proteomics investigation Fukata Y, Amano M, Kaibuchi K. Rho—Rho—kinase pathway of protein expression changes in ouabain induced in smooth muscle contraction and cytoskeletal reorganization apoptosis in human umbilical vein endothelial cells. ] of non—muscle cells. Trends Pharmacol Sci 2001(1);22:32—9. Cell Biochem 2008;104(3):1054—64.
• Ark M, Kubat H, Beydaği H, Ergenoğlu T, Songu—Mize E. Involvement of rho kinase in the ouabain—induced contractions of the rat renal arteries. Biochem Biophys Res Commim 2006;340(2):4l7—21.