Cell biology
Abstract
Chronic liver injury results in activation of quiescent Hepatic Stellate Cells (qHSCs) into Collagen Type I-producing activated HSCs that make liver fibrotic. We identified ETS1/2 (E26 transformation-specific transcription factors 1/2) as lineage-specific transcription factors regulating HSC phenotypes. Here we investigated the role of ETS1/2 in HSCs in liver fibrosis using toxic liver injury models and 3D human liver spheroids. Liver fibrosis was induced in wild-type and HSC-specific Ets1 (Ets1ΔHSC) and Ets2 (Ets2ΔHSC) knockout mice by administration of carbon tetrachloride for 6 weeks, following cessation of liver injury for 2 weeks. Liver fibrosis was more severe in Ets1ΔHSC, and to lesser extent in Ets2ΔHSC, compared to wild-type mice. Regression of liver fibrosis was suppressed only in Ets1ΔHSC, indicating Ets1 as the predominant isoform maintaining quiescent-like phenotype in HSCs. Similar results were obtained in a MASH model using 3D human liver spheroids. Knockdown of ETS1 in human HSCs caused upregulation of fibrogenic genes in MASH human liver spheroids and prevented fibrosis regression. ETS1 regulated the qHSC phenotype via CRTC2/PGC1α/PPARγ pathway. Knockdown of CRTC2 (cAMP response element-binding protein (CREB)-regulated transcription co-activator 2) abrogated PPARγ responses and facilitated HSC activation. These findings suggest that ETS1 may represent a therapeutic target for anti-fibrotic therapy.
Authors
Wonseok Lee, Xiao Liu, Sara Brin Rosenthal, Charlene Miciano, Sadatsugu Sakane, Kanani Hokutan, Debanjan Dhar, Hyun Young Kim, David A. Brenner, Tatiana Kisseleva
Abstract
Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows significant but incomplete benefits. This highlights the need to better understand the interplay between glutamine metabolism and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria in diverse cell types, but not in ccRCC. GLS1 clustering occurs rapidly within 1 to 3 hours, is reversible, is specifically triggered by reduced intracellular glutamate, and is dependent on mitochondrial fission. Clustered GLS1 markedly enhances glutaminase activity and promotes cell death under glutamine-deprived conditions. HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby maintaining low GLS activity and protecting ccRCC cells from glutamine deprivation-induced cell death. Forced clustering of GLS1, using constitutively clustering mutants, restores high GLS activity, promotes apoptosis, and suppresses ccRCC tumor growth in vivo. These findings reveal multiple insights into cellular glutamine handling, including a previously unrecognized process by which HIF2α promotes ccRCC: by suppressing GLS1 clustering and maintaining low GLS activity. This mechanism provides a potential explanation for the lack of clinical efficacy of GLS inhibitors in ccRCC and suggests a therapeutic avenue to combine HIF2α inhibition with strategies that restore GLS1 clustering.
Authors
Wencao Zhao, Sara M. Demczyszyn, Nathan J. Coffey, Yanqing Jiang, Boyoung Kim, Schuyler Bowers, Caitlyn E. Bowman, Michael C. Noji, Cholsoon Jang, M. Celeste Simon, Zoltan Arany, Boa Kim
Abstract
The lung’s mechanosensitive immune response to alveolar overdistension impedes ventilation therapy for hypoxemic respiratory failure. Though mechanistically unclear, the prevailing hypothesis is that the immune response results when alveolar overdistension stretches alveolar macrophages (AMs). Since this hypothesis is untested in live lungs, we optically imaged live mouse alveoli to detect alveolus-adherent, sessile AMs that communicate with the alveolar epithelium through connexin43 (Cx43)-containing gap junctions. Alveolar hyperinflation did not stretch the AMs, but it increased AM Ca2+. AM-specific Cx43 deletion blocked the Ca2+ response, as well lung injury due to mechanical ventilation at high tidal volume (HTV). HTV was also inhibited by AM-targeted delivery of liposomes containing the inhibitor of endosomal Ca2+ release, Xestospongin C. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lung’s mechanosensitive immunity, providing a basis for therapy for ventilator-induced lung injury.
Authors
Liberty Mthunzi, Mohammad Islam, Galina A Gusarova, Brian Karolewski, Sunita Bhattacharya, Jahar Bhattacharya
Abstract
Apoptosis and necroptosis are 2 distinct destinies of cells stimulated with TNF-α; however, it remains unclear how apoptosis and necroptosis are differentially regulated. This study validates the key regulatory role of speckle-type POZ protein (SPOP) in balancing apoptosis and necroptosis. SPOP promotes the polyubiquitination and degradation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), thereby inhibiting necrosome formation and decreasing cellular sensitivity to necroptosis. Conversely, SPOP interacted with RIPK1 independently of its E3 ubiquitin ligase activity, protecting it from ubiquitination and degradation, thereby enhancing RIPK1 expression and cellular sensitivity to apoptosis. Inhibiting RIPK1 kinase activity with 7-Cl-O-Nec-1 impeded both SPOP-mediated apoptosis and SPOP deficiency–mediated necroptosis. Besides, inhibition or loss of RIPK3 rescued SPOP deficiency–mediated necroptosis. Pancancer analyses indicated that the SPOP/RIPK1/RIPK3 axis is dysfunctional in a variety of tumors. In 3 representative tumor types with high expression of SPOP and RIPK1, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, and breast invasive carcinoma, this regulatory mechanism remains applicable. Based on these findings, a combination therapy using the second mitochondria-derived activator of caspases (Smac) mimetic SM164 and sunitinib was developed, demonstrating a more pronounced efficacy than sunitinib monotherapy, and this sensitizing effect was dependent on the expression level of RIPK1. These results suggest that the combination of Smac mimetics with tyrosine kinase inhibitors holds potential clinical value for tumors with dysregulated SPOP/RIPK1/RIPK3 signaling.
Authors
Yuzhong Ye, Changjie Yue, Zaosong Zheng, Hailong Ruan, Yuanpeng Zhang, Qi Miao, Xiaoping Zhang, Wen Xiao, Lei Liu
Abstract
The gastrointestinal epithelium depends on the apical junctional complex (AJC), composed of tight and adherens junctions, to regulate barrier function. Here, we identify the apical polarity protein Crumbs homolog 3 (CRB3) as an important regulator of AJC assembly and barrier function in intestinal epithelium. Using primary murine colonic epithelial cells (colonoids) from inducible, conditional Crb3-knockout (Crb3ERΔIEC) and control (Crb3fl/fl) mice, we show that CRB3 deficiency compromised barrier function that was associated with a hypercontractile perijunctional actomyosin network and impaired AJC assembly. Loss of CRB3 exacerbated proinflammatory cytokine–induced AJC remodeling, leading to increased intestinal permeability. Crb3ERΔIEC cells exhibited increased RhoA activity and junctional tension, which could be reversed by ROCK-II or myosin II inhibition, restoring junctional architecture. Mechanistically, CRB3A interacts with the actin cytoskeletal linker protein, Merlin (NF2) via its FERM-binding domain, and NF2 knockdown phenocopied CRB3 loss, suggesting their cooperative role in AJC assembly. These findings establish CRB3 and NF2 signaling as key regulators of perijunctional actomyosin contractility and AJC organization during both de novo junctional assembly and inflammation-induced remodeling. This work defines a CRB3- and NF2-dependent pathway by which epithelial cells regulate mechanical tension to coordinate barrier assembly during homeostasis and junctional remodeling under inflammatory stress.
Authors
Shuling Fan, Saranyaraajan Varadarajan, Vicky Garcia-Hernandez, Sven Flemming, Arturo Raya-Sandino, Ben Margolis, Charles A. Parkos, Asma Nusrat
Abstract
Laminin-α2-related Congenital Muscular Dystrophy (LAMA2-CMD) is a severe neuromuscular disorder caused by mutations in the LAMA2 gene, leading to loss of heterotrimers laminin-211/221, key components of the skeletal muscle extracellular matrix. Their absence disrupts adhesion between the cytoskeleton and extracellular matrix, resulting in progressive muscle wasting. Laminin-211/221 interacts with adhesion complexes such as the dystrophin/Utrophin glycoprotein complex and the α7β1-integrin. However, the regulatory mechanisms of these laminin-binding complexes and the broader role of laminin’s influence on the formation of the macromolecular network in skeletal muscle remain unclear. We previously demonstrated that mouse laminin-111 delivered in the dyW⁻/⁻ mouse model of LAMA2-CMD prevented disease progression, improved strength, and extended survival. We hypothesize that laminin-111, the embryonic laminin isoform, restores key adhesion-signaling networks. Using spatial-proteomics on patient and mouse muscle, we identified loss of essential signaling components: heat shock proteins 27 and 70, c-Jun N-terminal kinase, and glucose transporter 1 in laminin-α2 deficient muscle. Treatment with recombinant human laminin-111 (rhLAM-111) restored protein localization, reduced ROS, and promoted glycolytic, pro-survival signaling. These findings highlight laminin’s role in maintaining muscle homeostasis and metabolism and support the therapeutic potential of rhLAM-111 for treating LAMA2-CMD by restoring adhesion and intracellular signaling in dystrophic muscle.
Authors
Hailey J. Hermann, Ryan D. Wuebbles, Marisela Dagda, Axel Munoz, Lauren L. Parker, Paula C. C Guzman, Lola T. Byrne, Steven A. Moore, Dean J. Burkin
Abstract
Impaired wound healing poses a major and increasingly frequent health problem. Among the key players in the healing process are fibroblasts, but their metabolic profile in healing wounds is largely unknown. Using a combination of transcriptomics, targeted proteomics and metabolomics, we identified retinol metabolism as a top regulated pathway in wound fibroblasts. This is functionally relevant, since even a mild retinol deficiency caused a delay in wound closure and re-epithelialization, which mainly resulted from misdirected keratinocyte migration on the new granulation tissue. Quantitative proteomics identified integrin alpha 11 (Itga11) as a less abundant protein in wounds of mice subjected to a retinol-deficient diet. Reduced levels of this fibroblast-specific protein likely altered the granulation tissue matrix, which in turn affected re-epithelialization. These results provide a comprehensive overview on the transcriptome, proteome and metabolome of wound fibroblasts and identify retinol metabolism in fibroblasts as a key regulator of tissue repair.
Authors
Till Wüstemann, Elizabeta Madzharova, Mateusz S. Wietecha, Norbert B. Ghyselinck, Marcus Höring, Gerhard Liebisch, Nicola Zamboni, Ulrich auf dem Keller, Sabine Werner
Abstract
IDH1/2 mutations (IDHmut) increase methylation of DNA and histones in gliomas. IDHmut inhibitors are effective against low-grade IDHmut gliomas, but new strategies against high grade IDHmut gliomas are needed. Although histone deacetylase inhibitors (HDACi) are ineffective against IDHwt glioblastoma (GBM), their potential in IDHmut gliomas has not been extensively studied. We previously established that IDHmut gliomas are more sensitive to HDACi than IDHwt GBM. Here we show that IDHmut is associated with greater sensitivity to HDACi only in glioma, not in IDHmut chondrosarcoma or cholangiocarcinoma. While HDACi induced more histone acetylation and gene regulation in IDHmut glioma than in IDHwt GBM, such acetylation was mostly within gene deserts, whereas IDHmut glioma promoters paradoxically lost histone acetylation. Two mediators of HDACi resistance, YAP and TAZ, were methylated and suppressed in IDHmut gliomas, but not in other IDHmut cancers. Inducing YAP or TAZ expression in IDHmut gliomas conferred resistance to HDACi. Finally, belinostat extended in vivo survival only in IDHmut glioma models, not in IDHmut GBM models. Our findings provide a mechanistic rationale for further studies of HDACi in IDHmut glioma patients, as well as the potential use of YAP/TAZ as a biomarker of HDACi sensitivity in cancers.
Authors
Thomas K. Sears, Matthew McCord, Wenxia Wang, Alicia Steffens, Kathleen McCortney, Rahul Chaliparambil, Jann N. Sarkaria, Craig M. Horbinski
Abstract
Coronary artery disease (CAD) is the leading cause of mortality worldwide, with macrophages playing a central role in shaping the inflammatory environment through cytokines, chemokines, and other mediators. Long noncoding RNAs (lncRNAs) are emerging as key regulators of cellular processes due to their interactions with DNA, RNA, microRNAs, and proteins, positioning them as promising therapeutic targets. Through integrative transcriptomic analysis, we identified SPANXA2-OT1 as a primate-specific lncRNA with a potential role in macrophage-mediated inflammation in CAD. Functional studies in primary human macrophages demonstrated that SPANXA2-OT1 is induced by inflammatory stimulation, localized to the cytoplasm, and exerts regulatory effects on chemokine expression and macrophage chemotaxis. Mechanistically, SPANXA2-OT1 acts as a molecular sponge for microRNA-338, thereby influencing the expression of interleukin-8 (IL-8), a critical mediator of monocyte recruitment and inflammatory signaling. Collectively, these findings establish SPANXA2-OT1 as a human-specific regulator of inflammatory pathways in CAD and highlight its translational potential as both a biomarker and therapeutic target.
Authors
Prabhash Kumar Jha, Sarvesh Chelvanambi, Yuto Nakamura, Lucas Yuji Umesaki Itto, Aatira Vijay, Adrien Lupieri, Miguel Cantadori Barbeiro, Thanh-Dat Le, Caio Borges Nascimento, Taku Kasai, Mary C. Whelan, Daiki Hosokawa, Dakota Becker-Greene, Sasha A. Singh, Elena Aikawa, Shizuka Uchida, Masanori Aikawa
Abstract
The unfolded protein response (UPR), triggered by endoplasmic reticulum (ER) stress, comprises distinct pathways orchestrated by conserved molecular sensors. Although several of these components have been suggested to protect cardiomyocytes from ischemic injury, their precise functions and mechanisms remain elusive. In this study, we observed a marked increase in glucose-regulated protein 94 (GRP94) expression at the border zone of cardiac infarct in a mouse model. GRP94 overexpression ameliorated post-infarction myocardial damage and reduced infarct size. Conversely, GRP94 deficiency exacerbated myocardial dysfunction and infarct size. Mechanistically, GRP94 alleviated hypoxia-induced mitochondrial fragmentation, whereas its depletion exacerbated this fragmentation. Molecular investigations revealed that GRP94 specifically facilitated the cleavage of Opa1 into L-Opa1, but not S-Opa1. The study further elucidated that under hypoxic conditions, the binding shift of Yy1 from lncRNA Oip5os1 to AI662270 promoted Yy1’s binding on the GRP94 promoter, thereby enhancing GRP94 expression. AI662270 attenuated mitochondrial over-fragmentation and ischemic injury after myocardial infarction similarly to GRP94. Moreover, coimmunoprecipitation coupled with LC-MS/MS identified the interaction of GRP94 with Anxa2, which regulates Akt1 signaling to maintain L-Opa1 levels. Overall, these findings unveiled what we believe is a novel role for the AI662270/GRP94 axis in linking ER stress to mitochondrial dynamics regulation, proposing new therapeutic avenues for managing cardiovascular conditions through ER stress modulation.
Authors
Suling Ding, Wen Liu, Zhiwei Zhang, Xiyang Yang, Dili Sun, Jianfu Zhu, Xiaowei Zhu, Shijun Wang, Mengshi Xie, Hongyu Shi, Junbo Ge, Xiangdong Yang
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