• Journal of Life Science
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• v.27 no.10
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• pp.1199-1206
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• 2017

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.

• Journal of Life Science
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• v.29 no.2
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• pp.256-264
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• 2019

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.

• Journal of Life Science
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• v.23 no.10
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• pp.1199-1208
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• 2013

Fibroblastic reticular cells (FRCs) form the structural backbone of the T zone provide a guidance path for immigrating T cells in the lymph node (LN). FRCs may contribute directly to developing T-cell biology in the LN and allow analyses of fundamental aspects of FRC biology related to T cells. FRCs inhibited T-cell apoptosis, and FRC culture supernatants strongly induced the expression of Bcl-xL in T cells against doxorubicin. Coculture of FRC and T cells resulted in rearrangements of the actin cytoskeleton, as well as global changes in the morphology of the FRCs. In addition, when cocultured, the T cells adhered to the FRC monolayer, and the membrane intercellular adhesion molecule (ICAM)-1 was slightly increased by day-dependent manner. In contrast, the expression of soluble ICAM-1 was dramatically increased in a day-dependent manner. Several chemokines, such as CCL5, CXCL1, CXCL5, CXCL16, CCL8, CXCL13, and ICAM-1, and MMPs were expressed in FRCs sensed by tumor necrosis factor (TNF) families. Nuclear factor kappa B ($NF{\kappa}B$)-RelA of the $NF{\kappa}B$ canonical pathway was translocated into FRC nuclear by $TNF{\alpha}$. In contrast, p52 proteolyzed from p100, a counterpart of RelB of the noncanonical $NF{\kappa}B$ pathway, accumulated in the peripheral FRC nucleus by agonistic anti-$LT{\beta}R$ antibody. In summary, we propose a model in which FRCs engage in bidirectional crosstalk to increase the efficiency of T-cell biology. This cooperative feedback loop may help to maintain tissue integrity and function during immune responses.