Monocarboxylate Transporter-1 Is Required for Cell Death in Mouse Chondrocytic ATDC5 Cells Exposed to Interleukin-1β via Late Phase Activation of Nuclear Factor κB and Expression of Phagocyte-type NADPH Oxidase

Kentaro Yoshimura,Yoichi Miyamoto,Rika Yasuhara,Toshifumi Maruyama,Tomohito Akiyama,Atsushi Yamada,Masamichi Takami,Tetsuo Suzawa,Shoko Tsunawaki,Tetsuhiko Tachikawa,Kazuyoshi Baba,Ryutaro Kamijo

doi:10.1074/jbc.m111.221259

Kentaro Yoshimura, Yoichi Miyamoto + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m111.221259

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Abstract

Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.

Highlights

Introduction of siRNAStealthTM siRNAs for mouse Mct-1, Mct-6, Mct-13, and Nox-2 and their control non-silencing siRNAs were purchased from Invitrogen
NAC suppressed the late phase degradation of I␬B␣ induced after 48 h of treatment with IL-1␤ (Fig. 6B), and NAC suppressed IL-1␤induced expression of Nox-2 mRNA (Fig. 6C), whereas L-NAME did not have effects on either. These results indicate that reactive oxygen species (ROS) play an important role in I␬B␣ degradation and Nox-2 expression in IL-1␤-treated ATDC5 cells
We found that IL-1␤ induced biphasic activation of the canonical nuclear factor ␬B (NF-␬B) pathway in chondrocytic ATDC5 cells

Summary

Introduction

Introduction of siRNAStealthTM siRNAs for mouse Mct-1, Mct-6, Mct-13, and Nox-2 and their control non-silencing siRNAs were purchased from Invitrogen. We investigated the role of MCT-1 in IL-1␤-induced gene expression and cell death in ATDC5 cells by introducing siRNA for Mct-1, which nearly completely suppressed the expression of Mct-1 mRNA both in the presence and absence of IL-1␤ (Fig. 2D). Because undifferentiated ATDC5 cells at higher density tended to die following IL-1␤ treatment (supplemental Fig. S2), the speculation that NOX-2 plays an important role in IL-1␤-induced cell death was reinforced.

Results

Conclusion