안지오텐신 II 자극에 의한 심근세포의 비대기전

Dept. of Medical Science/박사[한글]심근비대는 심근압력 과부하에 의해 발생하여 세포분열 없이 세포의 크기만 증가하는 현상으로서 비대 후 부정맥이나 심근경색 등의 과정을 겪게 된다. 8개의 아미노산으로 구성된 안지오텐신 II는 NF-κB의 활성을 증가시킴으로써 심근비대를 초래하며 동시에 세포사멸 (apoptosis)을 유도하는 것으로 알려져 있다. 열 충격 단백질들 가운데 하나인 Hsp90은 세포사멸에 대한 보호 기전과 동시에 심근비대 과정에서 NF-κB 활성을 유지하는데 기여한다고 알려져 있다. 따라서 본 연구에서는 안지오텐신 II에 의한 심근비대 과정에서 NF-κB 신호전달 과정에 대한 Hsp90의 영향과 이의 분자기전을 규명하고자 하였다. Hsp90의 특이적인 저해제인 geldanamycin과 17-AAG는 안지오텐신 II에 의해 유도되는 심근비대를 억제하였으며, NF-κB의 활성도 억제함을 확인하였다. 심근세포에 Hsp90 저해제를 처리하였을 때 NF-κB의 상위단계인 IKKα/β의 발현 수준과 NF-κB의 억제자인 IκBα의 인산화가 감소되는 것을 확인하였다. 특히 흥미롭게도 Hsp90 저해제에 의한 IκBα 단백질의 절단현상이 새롭게 관찰되었고, 이런 현상은 caspase-8의 억제제인 z-IETD-fmk에 의하여 감소하였으며, caspase-8가 IκBα 단백질의 절단에 직접 관여함을 확인하였다. Caspase-8에 의한 IκBα 단백질의 절단은 IκBα의 아미노 말단에서 유발되고 이는 유비퀴틴화 잔기의 탈락을 유발하여 IκBα가 인산화되더라도 유비퀴틴화 되지 못해 IκBα가 지속적으로 세포 내에 축적됨으로써 NF-κB 활성을 억제하는 것으로 사료된다. Hsp90 저해제에 의해 IκBα의 절단과 동시에 심근세포의 apoptosis가 유발되는데, caspase-8의 활성이 관찰되었다. 이때 FasL의 발현이 증가되고 FLIPS의 발현이 감소함을 확인하였는데, 이를 통해 caspase-8의 활성이 유도되어 IκBα의 절단을 증가시킨 것으로 생각된다. 이와 같은 결과들을 통하여 안지오텐신 II에 의한 심근세포의 사멸과 비대의 두 가지 경로에서 Hsp90은 caspase-8의 활성을 억제함으로써 NF-κB의 활성을 유지하여 apoptosis를 억제하면서 심근비대를 초래하는 것으로 사료된다. [영문]Angiotensin II (AngII), vasoactive octapeptide, has a modulatory role on hypertrophic effect in cardiac cells. Hypertrophy is well characterized by an increased cell size and protein synthesis without cell division. Numerous studies have evidenced that AngII induces nuclear factor-κB (NF-κB) activation in cardiac cells and that activation of NF-κB is required for hypertrophic process. Interestingly, recent reports proposed that Hsp90 function was required for NF-κB activation. In this study, we investigated the role of Hsp90 on AngII-induced cardiac hypertrophy and molecular mechanism NF-κB pathway. Hsp90 inhibitors inhibited AngII-induced cellular hypertrophy and NF-κB luciferase activity in cardiac cells. Hsp90 inhibitor also inhibited the phosphorylation of IκBα and level of IKKα/β induced by AngII. Interestingly, accumulation of IκBα cleavage fragment was found in Hsp90 inhibitor-treated group. This IκBα cleavage, when pretreated with z-IETD-fmk, an inhibitor of caspase-8, was recovered. IκBα is directly cleaved by caspase-8 in vitro. Cleaved IκBα fragment does not contain the amino terminus baring two lysine residues which can be ubiquitinated by proteosome complex. So, even if IκBα is phosphorylated by any kinases, it can not go through proteosomal degradation, resulting in the maintenance of IκBα interaction with NF-κB. Hsp90 inhibitor induces cardiac cell apoptosis with caspase-8 activation and increase of FasL expression. However, the level of c-Flips was decreased by Hsp90 inhibitor. When caspase-8 inhibitor was added into Hsp90 inhibitor-treated cells, NF-κB activation was recovered. These results suggest that Hsp90 sustains NF-κB activity by blocking the caspase-8 activation, thereby representing a potential molecular switch where apoptosis is inhibited without affecting hypertrophy in cardiac cells.ope

Antiproliferative mechanisms of raxofelast (IRFI-016) in H2O2-stimulated rat aortic smooth muscle cells

Reactive oxygen species-mediated cellular injury is involved in the pathogenesis of many diseases, including those affecting the cardiovascular system, such as myocardial ischemia-reperfusion injury, inflammation, and atheroscleosis. Raxofelast (IRFI-016; (+/-)-5-acetoxy-2, 3-dihydro-4, 6, 7-trimethyl-2-benzofuran-acetic acid) was designed with the aim of maximizing the antioxidant potency of phenols chemically related to vitamin E. The antioxidant activity of raxofelast has been convincingly demonstrated in several in vitro studies and in various models of ischemia-reperfusion injury. In this study, the antiproliferative effects of raxofelast were investigated to determine whether transduction signals and protooncogenes are affected in H(2)O(2)-stimulated rat aortic smooth muscle cells. In a tetrazolium-based colorimetric assay, the proliferation of rat aortic smooth muscle cells was increased by 3-fold in 0.1% fetal bovine serum/Dulbecco's modified Eagle's medium (DMEM) containing 500 microM H(2)O(2), indicating that exogenous 500 microM H(2)O(2) was a growth stimulator of rat aortic smooth muscle cells. Exogenous H(2)O(2) significantly activated extracellular signal-regulated kinases (ERKs) activity within 30 min and raxofelast inhibited the ERKs activation dose dependently in 500 microM H(2)O(2)-stimulated rat aortic smooth muscle cells (IC(50): 200 microM). Raxofelast reduced the intracellular reactive oxygen species generated by exogenous H(2)O(2) in a dose-dependent manner. In 500 microM H(2)O(2)-stimulated rat aortic smooth muscle cells, raxofelast dramatically attenuated the activation of mitogen-activating protein kinase (MAPK)/ERK kinase 1, 2 (MEK1,2) and protein kinase C (PKC) without affecting Ras expression. Induction of c-myc mRNA was significantly reduced dose dependently up to 100 microM by raxofelast in concentrations. These data indicate that the antiproliferative effects of raxofelast in H(2)O(2)-stimulated rat aortic smooth muscle cells may involve the suppression of intracellular reactive oxygen species formation and the inhibition of ERKs by inactivation through PKC and MEK1,2 and down-regulation of c-myc expression, regardless of Ras activation.ope

C-reactive protein induces p53-mediated cell cycle arrest in H9c2 cardiac myocytes

C-reactive protein (CRP) is one of the most important biomarker for cardiovascular diseases. Recent studies have shown that CRP affects cell survival, differentiation and apoptosis. However, the effect of CRP on the cell cycle has not been studied yet. We investigated the cell cycle alterations and cellular mechanisms induced by CRP in H9c2 cardiac myocytes. Flow cytometry analysis showed that CRP-treated H9c2 cells displayed cell cycle arrest in G0/G1 phase. CRP treatment resulted in a significant reduction in the levels of CDK4, CDK6 and cyclin D1 in a concentration-dependent manner. Interestingly, CRP caused an increase in the p53 accumulation and its phosphorylation on Ser15, leading to induce p21 upregulation. Treatment with a specific p53 inhibitor, PFT-α restored the levels of CDK4 and CDK6. A significant increase of ERK1/2 phosphorylation level was detected in CRP-treated cells. Furthermore, pretreatment of a specific ERK inhibitor resulted in decreased p53 phosphorylation and p21 induction. ERK inhibitor pretreatment induced significant restoration of protein levels of CDK4 and CDK6, leading to re-entry into the cell cycle. In addition, increased phosphorylation of p53 and ERK induced by CRP was considerably reversed by Fc gamma receptor IIIa (FcγRIIIa) knock-down using siRNA. FcγRIIIa siRNA transfection also restored the levels of cell cycle proteins. Our study has provided the first proposal on the novel insights into how CRP directly affects cell cycle in cells.ope