Role of a Rho A-specific guanine nucleotide exchange factor, p190RhoGEF, in mouse dendritic cells

Abstract

Dendritic cells (DCs) are professional antigen (Ag)-presenting cells (APCs) and play essential roles in innate and adaptive immunity. The processes of Ag uptake and the movement to the area for Ag presentation, have been shown to require rearrangement of cytoskeletal protein, actin. The Rho family members of GTPases have also been shown to regulate cell polarization, motility, and chemotaxis in many cell types including DCs. Specifically, RhoA regulates the maturation of focal complexs into focal adhesions and the formation of associated stress fibers. Previous studies in our laboratory demonstrated that the expression of p190RhoGEF, a guanine nucleotide exchange factors (GEF) specific for RhoA, was remarkably increased following CD40 stimulation in B cells. Moreover, the CD40-mediated structural change of B cells was found to be meidiated by the activation of p190RhoGEF-induced RhoA activity. Our studies in B cells also showed that the cellular morphology changes by p190RhoGEF during CD40-mediated B cell activation correlate with B cell differentiation. In this study, the function of p190RhoGEF in DCs was examined in transgenic (Tg) mice that express a wild-type (WT) or a dominant-negative (DN) form of p190RhoGEF under the control of an invariant chain (li) promoter, which allows a specific expression of the p190RhoGEF transgene in APCs. In these mice, the development of myeloid and lymphoid DC subsets was found normal. However, upon comparison of the responses of isolated CD11c^(high) cells to LPS stimulation, the expression of surface markers, such as, CD86 (B7.2) and CD40, and the ability of Ag uptake were reduced in cells from WT-Tg mouse compared to the cells from littermate control mouse. LPS stimulation-dependent polymerization of the cytoskeletal protein, actin, was also inhibited in CD11c^(high) cells isolated from WT-Tg mouse. Morever, IL-6 cytokine secretion and localization of activated DCs to T cell area after LPS stimulation were inhibited in WT-Tg mouse. In contrast, the ability of CCL21-dependent migration was not affected in CD11c^(high) DCs from WT-Tg mouse. The responses of DN-Tg mouse DCs were shown similar to those of the littermate control mouse DCs. Collectively, these results may suggest that over-expression of p190RhoGEF negatively affects various functions of DCs after LPS stimulation. Further studies are needed to elucidate mechanisms how p190RhoGEF regulates the responses of DCs to LPS.;항원제시세포의 일종인 수지상 세포는 선천성 면역반응과 후천성 면역반응을 매개 하는 세포로 알려져 있다. Rho famliy member인 GTPase는 여러 가지 세포 내에서 방향성, 운동성, 및 케모카인에 의한 이동성에 중요한 작용을 한다. 우리는 이전 연구에서 B 임파구 분화에 중요한 CD40 수용체의 자극을 통해 RhoA를 특이적으로 활성화 시키는 p190RhoGEF 단백질의 발현이 증가하는 것을 발견하였으며, B임파구에서 p190RhoGEF의 기능은 세포의 morphology 변화 와 F-actin의 재배열에 관여하며, 최종적으로 활성화 및 분화과정에 관여함을 확인하였다. 이번연구에서 항원제시세포에 특이적으로 발현하는 p190RhoGEF의 과 발현 (WT) 또는 점 돌연변이 (DN) 형태의 형질전환 마우스를 이용하여 수지상 세포와 p190RhoGEF 단백질의 역할을 연구하였다. p190RhoGEF 형질전환 마우스의 골수에서부터 분화되어 나타나는 수지상 세포의 림프계와 골수계의 subtypes를 확인하였고, 박테리아 감염에 의한 수지상 세포의 활성화 및 기능 그리고 비장 내에서 이들을 이동성을 확인한 결과, subtypes의 표현형과 케모카인에 의한 이동성에 차이는 없지만, WT 형질전환 마우스에서 항원 uptake 능력, 비장 내 수지상 세포의 이동성이 감소하였음을 확인하였다. 이러한 결과들을 토대로 p190RhoGEF의 과 발현이 세포골격 단백질인F-actin형성을 조절하여 수지상 세포의 분화 및 성숙, 활성화를 조절할 것으로 예상되어진다.