Effect of cell adhesion on PDGF-induced superoxide production in cells reconstituted with PDGFβ receptor and the components of NADPH oxidase

Abstract

활성산소(superoxide anion, 과산화수소)는 대개 호흡의 부산물로 생성되며 생체에 유해한 물질로 간주되었으나, 근래 연구결과에 따르면 활성산소는 세포막수용체 신호전달에 주요한 역할을 할 것이라고 보여진다. 또한 산화환원에 의한 신호전달은 성장인자와 세포부착물질인 integrin의 부착의존성 세포성장에 중요한 역할을 한다. Integrin이 세포외기질에 붙으면 Src, FAK, ERK, PI3K, Rho 과 등 여러 종류의 생화학적 신호전달이 유도된다. 이러한 신호는 수용성 성장인자인 EGF 수용체, PDGF 수용체에 의해 활성화되는 신호와 상당히 일치한다. 우리는 본 논문에서 Nox1과 Nox5 그리고 PDGF 수용체로 재구성된 HepG2와 HelaS3 세포를 이용하여 이러한 세포들이 세포외기질에 부착하는 것이 PDGF 자극에 의존하는 superoxide 생성에 미치는 영향을 확인하였다. 우리는 재구성된 세포들을 Poly-D-lysine 또는 fibronectin이 처리된 plate에 부착시켜 superoxide가 integrin 신호전달 활성화에 의해 증가시키는 것을 확인하였다. 그래서 integrin a5b1 항체로 fibronectin이 매개하는 integrin 신호전달을 막았을 때와 siRNA실험을 통해 FAK 단백질의 발현을 억제시켰을 때, PP2로 억제시켰을 때의 superoxide 생성이 감소하는지 살펴보았다. 또한, 세포골격 재구성을 방해하기 위해 세포골격 배열 억제제를 처리하여 세포부착을 통한 신호체계가 아니라 세포부착이 superoxide 생성에 영향을 미치는지 살펴보았다. 더불어, 같은 실험 방법을 PDGF자극 대신 AngⅡ 자극을 주어 수행하여 유사한 결과를 얻었다.;Although superoxide anions (O2._) and hydrogen peroxide (H2O2) are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. And redox signaling is a key integration point for growth factor and integrin collaboration and plays an essential role in anchorage-dependent growth. Integrin binding to the extracellular matrix (ECM) elicits a series of biochemical signals, such as activation of Src and focal adhesion kinases (FAK), activation of the Ras/extracellular signal regulated kinase (ERK) cascade and activation of phosphatidylinositol 3-kinase (PI3K) and Rho family proteins. These signals significantly overlap with those activated by soluble GFs such as epidermal growth factor receptor (EGF-R), insulin receptor and platelet-derived growth factor receptor (PDGF-R). In this study, we investigated the mechanism by which ECM binding affects the PDGF-dependent superoxide generation using HepG2 and HelaS3 cells reconstituted with Nox 1 along with a pair of adaptor subunits (NOXO1 with NOXA1) and with the PDGFβR and Nox5. We allowed reconstituted cells to adhere Poly-D-lysine or Fibronectin and treated integrin antibody to block the fibronectin-mediated integrin signaling and suppressed FAK protein expression by siRNA experiment. We also treated cytoskeleton arrangement inhibitor to disturb cytoskeleton reorganization. Additionally, Angiotensin II (Ang II) ?stimulated experiments were performed in the same way. We concluded that contacts with poly-D-lysine or fibronectin significantly enhanced than anchorage-independent growth in the reconstituted HelaS3 cells. But, inhibition of integrin signaling by FAK depletion, PP2 and cytoskeleton arrangement did not abolish PDGF or Ang II-induced superoxide production in the fibronectin attached, reconstituted HelaS3 cells completely. So we suggest that there would be other molecules which are activated by cell adhesion and could be a major factor of superoxide generation via NOX enzymes.