Signals of MLCK and ROCK Pathways Triggered via Lymphotoxin β Receptor are Involved in Stress Fiber Change of Fibroblastic Reticular Cells

FRC에서 Lymphotoxin β receptor의 자극은 MLCK와 ROCK의 이중 신호전달 경로를 통해 stress fiber 변화에 관여

Abstract

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. In contrast, MLCK and ROCK play critical roles in the regulation of stress fiber (SF) formation in cells. To determine whether $LT{\beta}R$ stimulation in fibroblastic reticular cells (FRCs) is involved in these signaling pathways, myosin light chain kinase inhibitor-7 (ML-7) was used to inhibit them. 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. The inhibition of ROCK activity with Y27632-induced changes in actin cytoskeleton organization and cell morphology in FRCs. Actin bundles rearranged into SFs, and phospho-myosin light chain (p-MLC) co-localized in FRCs. We checked the level of Rho-guanosine diphosphate (RhoGDP)/guanosine triphosphate (GTP) exchange activity using FRC lysate. When $LT{\beta}R$ was stimulated with agonistic anti-$LT{\beta}R$ antibodies, Rho-GDP/GTP exchange activity was markedly reduced. Regarding $LT{\beta}R$ signaling with a focus on MLCK inhibition, we showed that the phosphorylation of MLCs was reduced by $LT{\beta}R$ stimulation in FRCs. Cytoskeleton components, such as tubulin, b-actin, and phospho-ezrin proteins acting as membrane-cytoskeleton linkers, were produced in de-phosphorylation, and they reduced expression in agonistic anti-$LT{\beta}R$ antibody-treated FRCs. Collectively, the results suggested that MLCK and ROCK were simultaneously responsible for SF regulation triggered by $LT{\beta}R$ signaling in FRCs.

Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$)는 TNF 계열로 림프조직의 미세구조와 기관형성에 중요한 역할을 한다. MLCK와 ROCK는 세포의 stress fiber 형성조절에 관여하는 주요 신호전달자이다. Fibroblastic reticular cell (FRC)에서 $LT{\beta}R$ 자극을 통한 이런 신호전달자들의 관련성을 알아보기 위해 ML-7 (MLCK 저해제)이 사용되었다. ML7 처리된 FRC에서 SF가 완전히 파괴되었고 anti-$LT{\beta}R$ antibody 처리 세포와 유사하게 ML7 처리 FRC에서 응축된 세포형태를 관찰 할 수 있었다. Y27632로 ROCK를 저해 했을 때 FRC의 액틴 세포골격과 세포형태 변화가 유도 되었다. FRC에서 p-MLC가 액틴과 함께 SF 구성성분을 이루었다. FRC세포 추출물로 Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) 교환활성을 확인했다. Agonistic anti-$LT{\beta}R$ antibody로 $LT{\beta}R$을 자극 했을 때 Rho-GDP/GTP 교환활성이 크게 감소했다. MLCK 저해처럼 $LT{\beta}R$ 자극은 MLC의 인산화를 감소시켰다. Agonistic anti-$LT{\beta}R$ antibody-treated FRC에서 세포골격 구성요소인 세포막과 세포골격 링커 역할을 하는 p-ezrin의 인산화는 감소 되었고 b- actin, 그리고 tubulin 발현도 줄었다. 이런 결과는 FRC의 $LT{\beta}R$ 신호전달을 통한 SF 조절에는 MLCK와 ROCK가 관여하고 있다는 것을 알 수 있었다.

Fig. 2. pMLC and actin co-localized in normal FRC. FRCs were treated with phalloidin and anti-pMLC antibody for 1 hr and examined for changes in F-actin distribution (phalloidin staining) and p-MLC. Bar is 10 μm.

Fig. 1. FRC preserves cortical contractile ring and central stress fibers. Representative images of rhodamine-phalloidin staining for F-actin (red), and DAPI staining (blue) of FRC. Actin cytoskeletons in FRC are indicated with white arrows and head-arrow. Bar is 10 μm.

Fig. 4. LTβR controls FRC spreading, elongation, and actomyosin contractility. FRC were treated with 5 μM ML7 incubated for indicated times. Actin SF completely abrogated in ML7 treated FRC. Bar is 10 μm.

Fig. 5. Roles of ROCK on SF alignment in FRCs. Representative images of FRCs adhered on slide chamber subjected for 12 h, 24 hr and 36 hr after treatment with 10 μM Y27632. Bar is 10 μm.

Fig. 6. Effect of LTβR signal on MLC phosphorylation and RhoA activation. (A) ML7, MLCK inhibitor, decreased MLC phosphorylation (p-MLC) over the control, which was abolished by 100 ng/ml agonistic anti-LTβR antibody (C). (B) LTβR stimulation induces a significant decrease in active RhoA and p-MYPT compared with control. MLC, total RhoA and MYPT were used as a loading control.

Fig. 7. Protein level of p-ezrin, f-actin, and tubulin as cytoskeletal markers were significantly reduced in agonistic anti-LTβR antibody treated-FRCs. FRC was incubated with anti-LTβR antibody for 24 hr. After incubation, cell was lyzed with RIPA buffer and protein concentration of FRC lysate was measured by BCA method. FRCs were treated with mouse anti-cytoskeletal marker antibody followed by anti-LTβR antibody for 24 hr. The expression degree of p-ezrin, β-actin, a-tubulin and GAPDH was detected by Western blot. GAPDH was used as a loading control.

Fig. 3. Agonistic anti-LTβR antibody disrupted the formation of SF in FRC. FRCs on chamber slides were treated with agonistic anti-LTβR antibody (25, 50 mg/ml) for 24 hr and examined for changes in F-actin distribution (phalloidin staining). Bar is 10 μm.