Abstract
The lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. We found that $LT{\beta}R$ stimulation induced changes in stress fibers (SFs) in fibroblastic reticular cells (FRCs). MLCK and ROCK play critical roles in the regulation of SF formation in cells. The present study was performed to investigate the antifibrotic effects on SF regulation of $LT{\beta}R$ signaling, with a focus on MLCK inhibition. The effect of $LT{\beta}R$ on the SF change was analyzed using immunoblot and fluorescence assays and agonistic $anti-LT{\beta}R$ antibody-treated FRCs. In addition, we checked the level of Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange activity with FRC lysate. Phospho-ezrin proteins acting as membrane-cytoskeleton linkers completely de-phosphorylated in agonistic $anti-LT{\beta}R$ antibody-treated FRCs. The actin bundles rearranged into SFs, where phospho-myosin light chain (p-MLC) co-localized in FRCs. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic $anti-LT{\beta}R$ antibody-treated cells. Inhibition of ROCK activity induced changes in the actin cytoskeleton organization; however, some SFs remained in the cells, while they were completely disrupted by MLCK inhibition with ML7. We showed that the phosphorylation of MLC was completely abolished with $LT{\beta}R$ stimulation in FRCs. When $LT{\beta}R$ was stimulated with the agonistic $anti-LT{\beta}R$ antibody, the Rho-GDP/GTP exchange activity was reduced, however, the activity was not completely abolished. Collectively, the results illustrated that MLCK was potently responsible for the SF regulation triggered via $LT{\beta}R$ signaling in FRCs.
종양괴사인자 수용체 일종인 Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$)은 림프 구조와 기관 형성에 중요한 역학을 한다. 우리는 fibroblastic reticular cell (FRC)에서 agonistic $anti-LT{\beta}R$ antibody로 $LT{\beta}R$을 자극하면 stress fiber (SF)에 변화가 생긴다는 것을 알았다. MLCK와 ROCK는 세포에서 SF 형성 기여에 중요한 역할을 한다. 본 연구는 MLCK 저해에 초점을 맞추어 $LT{\beta}R$ 신호 전달은 SF 조절로 항섬유화 효과에 대하여 조사하였다. SF 변화에 대한 $LT{\beta}R$의 기능 조사를 위해 agonistic $anti-LT{\beta}R$ antibody로 처리된 FRC와 세포 추출물을 이용하여 immunoblot, fluorescence assay와 Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange 활성 분석법으로 분석하였다. 세포막과 세포골격 연결자 ezrin은 agonistic $anti-LT{\beta}R$ antibody 처리된 FRC에서 완전히 탈인사화가 유도되었다. Actomysoisn에 의한 SF를 확인하였고 인산화 myosin light chain (p-MLC)인 함께 co-localization 되는 것도 확인하였다. ML7 처리된 FRC에서 agonistic $anti-LT{\beta}R$ antibody 처리된 세포에서 관찰되는 유사한 현상인 SF분해, 세포막 응축과 쇠퇴 현상이 나타났다. ROCK 활성저해는 액틴 골격 변화는 유도하였으나 부분적으로 SF가 세포에 남아 있었다. 반면, ML7에 의한 MLCK저해는 SF를 완전히 분해하였다. 또한, $LT{\beta}R$ 자극은 MLC 인산화를 완전히 억제하였지만, Rho-GDP/GTP exchange 활성변화에서는 감소는 되었으나 활성이 완전히 없어지지는 않았다. 결론적으로 이런 결과들은 FRC에서 $LT{\beta}R$신호전달을 통해 유도되는 SF 조절에는 MLCK가 보다 더 강력한 역할을 한다는 것을 제시하고 있다.
