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TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release.

ABSTRACT: Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)β(5), α(6)β(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.

SUBMITTER: Juric V

PROVIDER: S-EPMC3281933 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.

Project description:Background The metabolite succinate accumulates during cardiac ischemia. Within 5 minutes of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II. The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking MCT1 (monocarboxylate transporter 1) may be beneficial in IR injury, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT1 would retain succinate in cells, exacerbating ROS generation and IR injury. Methods and Results Using the mitochondrial ROS probe mitoSOX and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in situ during the first moments of reperfusion. We found that acute MCT1 inhibition enhanced mitochondrial ROS generation at reperfusion and worsened IR injury (recovery of function and infarct size). Both of these effects were abrogated by tandem inhibition of mitochondrial complex II, suggesting that succinate retention worsens IR because it drives more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator tetramethylrhodamine ethyl ester, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT1 inhibition. Conclusions Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation as key determinants of PT pore opening and IR injury outcomes.

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TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release.

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