The growth of skeletal muscle relies on a delicate equilibrium between protein synthesis and degradation; however, how proteostasis is managed in the endoplasmic reticulum is largely unknown. Here, we report that the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) complex, the primary molecular machinery that degrades misfolded proteins in the ER is vital to maintain postnatal muscle growth and systemic energy balance. Myocyte-specific SEL1L deletion blunts the hypertrophic phase of muscle growth, resulting in a net zero gain of muscle mass during this developmental period and a 30% reduction in overall body growth. In addition, myocyte-specific SEL1L deletion triggered a systemic reprogramming of metabolism characterized by improved glucose sensitivity, enhanced beiging of adipocytes, and resistance to diet induced obesity. These effects were partially mediated by the upregulation of the myokine FGF21. These findings highlight the pivotal role of SEL1L-HRD1 ERAD activity in skeletal myocytes for postnatal muscle growth, and its physiological integration in maintaining whole-body energy balance.
Benedict Abdon, Yusheng Liang, Débora da Luz Scheffer, Mauricio Torres, Neha Shrestha, Rachel B. Reinert, You Lu, Brent Pederson, Amara Bugarin-Lapuz, Sander Kersten, Ling Qi
Morrbid is a new identified leukocyte-specific long noncoding RNA (lncRNA). However, the expression and biological functions of Morrbid in cardiomyocytes and in heart disease are currently unclear. The study is to determine the roles of cardiac Morrbid in acute myocardial infarction (AMI) and to identify the potential cellular and molecular mechanisms involved. We found that both human and mouse cardiomyocytes could express a significant amount of Morrbid and its expression was increased in cardiomyocytes with hypoxia or oxidative stress, and in mouse hearts with AMI. Overexpression of Morrbid reduced the myocardial infarct size and cardiac dysfunction, whereas the infarct size and cardiac dysfunction were deteriorated in cardiomyocyte-specific Morrbid knockout (Morrbidfl/fl/Myh6-Cre) mice. We identified that Morrbid had a protective effect against hypoxia or H2O2-induce apoptosis, which was also confirmed in vivo in mouse hearts after AMI. We further discovered that serpine1 was a direct target gene of Morrbid, which was involved in Morrbid-mediated protective effect on cardiomyocytes. In summary, we have found for the first time that the cardiac Morrbid is a stress-enhanced lncRNA, which protects hearts from AMI via anti-apoptosis through its target gene serpine1. Morrbid may be a novel promising therapeutic target for ischemic heart diseases such as AMI.
Yang Yu, Haiqiong Yang, Qiuting Li, Nianhui Ding, Jiali Gao, Gan Qiao, Jianguo Feng, Xin Zhang, Jianming Wu, Yajun Yu, Xiangyu Zhou, Xiaobin Wang, Chunxiang Zhang
The mammalian target of rapamycin complex 1 (mTORC1) senses multiple upstream stimuli to orchestrate anabolic and catabolic events that regulate cell growth and metabolism. Hyperactivation of mTORC1 signaling is observed in multiple human diseases; thus, pathways that suppress mTORC1 signaling may help to identify new therapeutic targets. Here, we report that phosphodiesterase 4D (PDE4D) promotes pancreatic cancer tumor growth by increasing mTORC1 signaling. GPCRs paired to Gαs proteins activate adenylyl cyclase, which in turn elevates levels of 3′,5′-cyclic adenosine monophosphate (cAMP), whereas PDEs catalyze the hydrolysis of cAMP to 5′-AMP. PDE4D forms a complex with mTORC1 and is required for mTORC1 lysosomal localization and activation. Inhibition of PDE4D and the elevation of cAMP levels block mTORC1 signaling via Raptor phosphorylation. Moreover, pancreatic cancer exhibits an upregulation of PDE4D expression, and high PDE4D levels predict the poor overall survival of patients with pancreatic cancer. Importantly, FDA-approved PDE4 inhibitors repress pancreatic cancer cell tumor growth in vivo by suppressing mTORC1 signaling. Our results identify PDE4D as an important activator of mTORC1 and suggest that targeting PDE4 with FDA-approved inhibitors may be beneficial for the treatment of human diseases with hyperactivated mTORC1 signaling.
Mi-Hyeon Jeong, Greg Urquhart, Cheryl Lewis, Zhikai Chi, Jenna L. Jewell
Low Density Lipoprotein Receptor-related Protein-1 (LRP1) functions as a receptor for non-pathogenic cellular prion protein (PrPC), which is released from cells by ADAM proteases or in extracellular vesicles. This interaction activates cell-signaling and attenuates inflammatory responses. We screened 14-mer PrPC-derived peptides and identified a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111. A synthetic peptide (P3) corresponding to this region replicated the cell-signaling and biological activities of full-length shed PrPC. P3 blocked lipopolysaccharide (LPS)-elicited cytokine expression in macrophages and microglia and rescued the heightened sensitivity to LPS in mice in which the PrPC gene (Prnp) is deleted. P3 activated ERK1/2 and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA Receptor and was blocked by the PrPC-specific antibody, POM2. P3 has Lys residues, which are typically necessary for LRP1-binding. Converting Lys100 and Lys103 into Ala eliminated the activity of P3, suggesting that these residues are essential in the LRP1 binding motif. A P3 derivative in which Lys105 and Lys109 were converted into Ala retained activity. We conclude that the biological activities of shed PrPC, attributed to interaction with LRP1, are retained in synthetic peptides, which may be templates for therapeutics development.
Elisabetta Mantuano, Carlotta Zampieri, Pardis Azmoon, Cory B. Gunner, Kyle R. Heye, Steven L. Gonias
Defects in endoplasmic reticulum (ER) proteostasis have been linked to diseases in multiple organ systems. Here we examined the impact of perturbation of ER proteostasis in mice bearing thyrocyte-specific knockout of either HRD1 (to disable ER-associated protein degradation [ERAD]) or ATG7 (to disable autophagy) in the absence or presence of heterozygous expression of misfolded mutant thyroglobulin (the most highly expressed thyroid gene product, synthesized in the ER). Misfolding-inducing thyroglobulin mutations are common in humans but are said to yield only autosomal-recessive disease — perhaps because misfolded thyroglobulin protein might undergo disposal by ERAD or ER macroautophagy. We find that as single defects, neither ERAD, nor autophagy, nor heterozygous thyroglobulin misfolding altered circulating thyroxine levels, and neither defective ERAD nor defective autophagy caused any gross morphological change in an otherwise WT thyroid gland. However, heterozygous expression of misfolded thyroglobulin itself triggered significant ER stress and individual thyrocyte death while maintaining integrity of the surrounding thyroid epithelium. In this context, deficiency of ERAD (but not autophagy) resulted in patchy whole-follicle death with follicular collapse and degeneration, accompanied by infiltration of bone marrow–derived macrophages. Perturbation of thyrocyte ER proteostasis is thus a risk factor for both cell death and follicular demise.
Xiaohan Zhang, Crystal Young, Xiao-Hui Liao, Samuel Refetoff, Mauricio Torres, Yaron Tomer, Mihaela Stefan-Lifshitz, Hao Zhang, Dennis Larkin, Deyu Fang, Ling Qi, Peter Arvan
Epigenetic aberrations, including posttranslational modifications of core histones, are major contributors to cancer. Here, we define the status of histone H2B monoubiquitylation (H2Bub1) in clear cell ovarian carcinoma (CCOC), low-grade serous carcinoma, and endometrioid carcinomas. We report that clear cell carcinomas exhibited profound loss, with nearly all cases showing low or negative H2Bub1 expression. Moreover, we found that H2Bub1 loss occurred in endometriosis and atypical endometriosis, which are established precursors to CCOCs. To examine whether dysregulation of a specific E3 ligase contributes to the loss of H2Bub1, we explored expression of ring finger protein 40 (RNF40), ARID1A, and UBR7 in the same case cohort. Loss of RNF40 was significantly and profoundly correlated with loss of H2Bub1. Using genome-wide DNA methylation profiles of 230 patients with CCOC, we identified hypermethylation of RNF40 in CCOC as a likely mechanism underlying the loss of H2Bub1. Finally, we demonstrated that H2Bub1 depletion promoted cell proliferation and clonogenicity in an endometriosis cell line. Collectively, our results indicate that H2Bub1 plays a tumor-suppressive role in CCOCs and that its loss contributes to disease progression.
Adam J. Ferrari, Priyanka Rawat, Hannah S. Rendulich, Akshaya V. Annapragada, Yasuto Kinose, Xiaoming Zhang, Kyle Devins, Anna Budina, Robert B. Scharpf, Marilyn A. Mitchell, Janos L. Tanyi, Mark A. Morgan, Lauren E. Schwartz, T. Rinda Soong, Victor E. Velculescu, Ronny Drapkin
During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administered E. coli lipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-kB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.
Jennifer M.S. Sucre, Fabian Bock, Nicholas M. Negretti, John T. Benjamin, Peter M. Gulleman, Xinyu Dong, Kimberly T. Ferguson, Christopher S. Jetter, Wei Han, Yang Liu, Seunghyi Kook, Jason J. Gokey, Susan H. Guttentag, Jonathan A. Kropski, Timothy S. Blackwell, Roy Zent, Erin J. Plosa
Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion and blood pressure. Activating P2ry2 in littermate controls but not principal cell specific P2ry2 knockout mice decreases the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolishes increases in sodium excretion in response to stimulation of P2ry2 and compromises the normal ability to excrete a sodium load. Consequently, principal cell specific knockout of P2ry2 prevents decreases in blood pressure in response to P2ry2 stimulation in the DOCA-salt model of hypertension. In wild-type littermate controls, such stimulation decreases blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq-DREADD (Designer Receptors Exclusively Activated by Designer Drugs; GqD) and clozapine N-oxide (CNO) decreases the activity of ENaC in renal tubules promoting a natriuresis that lowers elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation, and that inhibition of ENaC activity in response to P2ry2 mediated Gq signaling lowers blood pressure by increasing renal sodium excretion.
Antonio G. Soares, Jorge Contreras, Elena Mironova, Crystal R. Archer, James D. Stockand, Tarek M. Abd El-Aziz
Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism, which may become overactivated in pathological states resulting intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP, and inhibition of Orai1 could prevent the development of end-stage CP.
Viktória Szabó, Noémi Csákány-Papp, Marietta Görög, Tamara Madacsy, Árpád Varga, Aletta Kiss, Balint Tel, Boldizsár Jójárt, Tim Crul, Krisztina Dudás, Mária Bagyánszki, Nikolett Bódi, Ferhan Ayaydin, Shyam Jee, Laszlo Tiszlavicz, Kenneth A. Stauderman, Sudarshan Hebbar, Petra Pallagi, József Maléth
Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knock-in (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We show that Rbm20ΔRS mice manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized to the sarcoplasm in Rbm20ΔRS mice, which led to the formation of RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif (RRM) show similar mis-splicing of RBM20 target genes, but do not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrate that only DCM-associated mutations in the RS domain facilitate RBM20 nucleocytoplasmic transport and promote granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain. Mutation analysis of phosphorylation sites in the RS domain indicate that this modification is dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.
Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila U. Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo
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