Skeletal muscle wasting results from numerous pathological conditions impacting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss of function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 results in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fibre integrity. We further implicate a disruption in atrogin-1 mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We reveal that BiP is not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorates pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicates atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy, and highlights atrogin-1’s essential role in maintaining muscle homeostasis.
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that Caspase 6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) leads to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment can restrain p65 NF-κB transcriptional activity by cytoplasmic sequestration NF-κB signaling component, p65 NF-κB, and attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidney of mice with unilateral ureteral obstruction and lipopolysaccharide-induced acute kidney injury. Collectively, our findings indicate that the C-terminal region of HIPK2 is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential anti-inflammatory agents to treat kidney disease.
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.
Lisa Duvick, W. Michael Southern, Kellie A. Benzow, Zoe N. Burch, Hillary P. Handler, Jason S. Mitchell, Hannah Kuivinen, Udaya Gadiparthi, Praseuth Yang, Alyssa Soles, Carrie A. Sheeler, Orion Rainwater, Shannah Serres, Erin B. Lind, Tessa Nichols-Meade, Brennon O'Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), were dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression in a destabilization of the medial meniscus (DMM) mouse model, while genetic deficiency of plasmin activator inhibitor (PAI-1), or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans, induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, PDK1, AKT, and ERK signaling cascades, and activates matrix metalloproteinases (pro-MMPs) to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
Qian Wang, Guoqiang Shao, Xiaoyi Zhao, Heidi H. Wong, Kate Chin, Mackenzie Zhao, Audrey Bai, Michelle S. Bloom, Zelda Z. Love, Constance R. Chu, Zhen Cheng, William H. Robinson
The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected >5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may impact cardiac contractility.
Colleen M. Kelly, Jody L. Martin, Michael J. Previs
Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the alpha-ketoglutarate dehydrogenase (OGDH) gene, which encodes for an enzyme that is part of the tricarboxylic acid cycle (TCA cycle) as essential for GBM growth. Moreover, by combining a transcriptome and metabolite screening analyses we discovered that loss of function of OGDH by the clinically validated drug compound, CPI-613, was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3-mimetic, ABT263) in patient-derived xenograft as well neurosphere GBM cultures. CPI-613 mediated energy deprivation drove an integrated stress response with an up-regulation of the BH3-only domain protein, Noxa in an ATF4 dependent manner as demonstrated by genetic loss of function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with interference of the TCA-cycle might be a treatment strategy for GBM.
Trang T.T. Nguyen, Consuelo Torrini, Enyuan Shang, Chang Shu, Jeong-Yeon Mun, Qiuqiang Gao, Nelson Humala, Hasan O. Akman, Guoan Zhang, Mike-Andrew Westhoff, Georg Karpel-Massler, Jeffrey N. Bruce, Peter Canoll, Markus D. Siegelin
Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we utilized single-cell RNA sequencing (scRNA-seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment (TME) components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Lastly, murine experiments and scRNA-seq analysis of a treated patient’s tumor demonstrated that Famitinib plus anti-PD-1 antibody could advance TLS in thyroid cancer. Conclusively, we displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.
Pei-Zhen Han, Wei-Dong Ye, Peng-Cheng Yu, Li-Cheng Tan, Xiao Shi, Xu-Feng Chen, Cong He, Jia-Qian Hu, Wen-Jun Wei, Zhong-Wu Lu, Ning Qu, Yu Wang, Qing-Hai Ji, Dong-Mei Ji, Yu-Long Wang
IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator also increased following TCF4 siRNA knockdown and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a, that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together these findings identify a role for TCF4 in the negative regulation of IL-17C, which alone and with TNF-α and IL-17A, feedback to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.
Yanyun Jiang, Dennis Gruszka, Chang Zeng, William R. Swindell, Christa Gaskill, Christian Sorensen, Whitney Brown, Roopesh Singh Gangwar, Lam C. Tsoi, Joshua Webster, Sigrun Laufey Sigurdardottir, Mrinal K. Sarkar, Ranjitha Uppala, Austin Kidder, Xianying Xing, Olesya Plazyo, Enze Xing, Allison C. Billi, Emanual Maverakis, J. Michelle Kahlenberg, Johann Gudjonsson, Nicole L. Ward
IKK2-NF𝜅B pathway mediated-inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2-NF𝜅B pathway in medial calcification remains to be elucidated. In this study, we found that CKD induces inflammatory pathways through the local activation of the IKK2-NF𝜅B pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2-NF𝜅B pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF𝜅B by SMC-specific I𝜅B-α deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2-NF𝜅B pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF𝜅B reduced CKD-dependent vascular cell death. In addition, increased calcifying extracellular vesicles through the inhibition of the IKK2-NF𝜅B pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2-NF𝜅B pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.
Shinobu Miyazaki-Anzai, Masashi Masuda, Audrey L. Keenan, Yuji Shiozaki, Jose G. Miranda, Makoto Miyazaki
Excessive lipolysis in white adipose tissues (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In human, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocytes lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicate that mice lacking P2Y13-R show a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared to their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez
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