Cell biology
Abstract
The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we follow heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.
Authors
Alex Agelidis, Benjamin A. Turturice, Rahul K. Suryawanshi, Tejabhiram Yadavalli, Dinesh Jaishankar, Joshua Ames, James Hopkins, Lulia Koujah, Chandrashekhar D. Patil, Satvik R. Hadigal, Evan J. Kyzar, Anaamika Campeau, Jacob M. Wozniak, David J. Gonzalez, Israel Vlodavsky, Jin-ping Li, David L. Perkins, Patricia W. Finn, Deepak Shukla
Abstract
Myofibroblasts are the major cellular source of collagen, and their accumulation – via differentiation from fibroblasts and resistance to apoptosis – is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast de-differentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.
Authors
Sean M. Fortier, Loka R. Penke, Dana M. King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden
Abstract
Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with Primary Open Angle Glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic cause of POAG. We have previously shown that mutant myocilin accumulates in the endoplasmic reticulum (ER) and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activates autophagy but it is functionally impairecd in cultured human trabecular meshwork (TM) cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsens mutant myocilin accumulation and exacerbates IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy is associated with chronic ER stress-induced transcriptional factor, CHOP. Deletion of CHOP corrects impaired autophagy, enhances recognition and degradation of mutant myocilin by autophagy,and reduces glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via Tat-beclin 1 peptide or torin 2, promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOCY437H mice. Together, our studies provide a novel treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.
Authors
Ramesh B. Kasetti, Prabhavathi Maddineni, Charles C. Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode
Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size
Abstract
Limitations in cell proliferation are important for normal function of differentiated tissues, and essential for the safty of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion and determined growth potential, differentiation and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S-phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.
Authors
Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli
Abstract
An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well-understood. Here, we showed that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport-independent mechanism. Using immunoprecipitation and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase-related cdc42-binding kinase α (MRCKα), a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with Acute Respiratory Distress Syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a novel mechanism by which NKA regulates epithelial tight junctions and identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.
Authors
Haiqing Bai, Rui Zhou, Michael Barravecchia, Rosemary Norman, Alan E. Friedman, Deborah Yu, Xin Lin, Jennifer L. Young, David A. Dean
Abstract
Myotonic dystrophy type 1 (DM1) is caused by a CTG-repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG-repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-seq results, calcium handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.
Authors
Ashish N. Rao, Hannah M. Campbell, Xiangnan Guan, Tarah A. Word, Xander H.T. Wehrens, Zheng Xia, Thomas A. Cooper
Abstract
Triple negative breast cancers (TNBC) lack effective targeted therapies and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, PARP inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohorts of patients do respond. Moreover, recent studies suggest that an increase in levels of reactive oxygen species (ROS) may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that in response to therapy-induced stress, p66ShcA stimulates ROS production, which, in turn, potentiates synergy between doxorubicin/PARPi combination therapy in TNBCs. This p66ShcA-induced sensitivity relies on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.
Authors
Eduardo Cepeda Cañedo, Stephanie Totten, Ryuhjin Ahn, Paul Savage, Deanna MacNeil, Jesse Hudson, Chantal Autexier, Genevieve Deblois, Morag Park, Michael Witcher, Josie Ursini-Siegel
Abstract
Rewiring tumor cells to undergo drug-induced apoptosis could be a promising way to overcome chemoresistance, therefore identifying causative factors for chemoresistance is of high importance. Global proteome-profiling of sensitive, early and late cisplatin resistant OSCC lines identified CMTM6 as a top ranked up-regulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy-non-responders as compared to responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in ESCC, HNSCC was monitored from Kaplan-Meier-Plot analysis. Stable knockdown of CMTM6 restores cisplatin-mediated cell death in chemoresistant OSCC cell lines. Similarly, upon CMTM6 overexpression in CMTM6KD lines, the cisplatin resistant phenotype was efficiently rescued. The patient-derived cell xenograft model of chemoresistant OSCC displayed CMTM6 depletion restored the cisplatin-induced cell death and tumor burden significantly. The transcriptome analysis of CMTM6KD and control chemoresistant cells depicted enrichment of Wnt-signaling pathway. Mechanistically, we demonstrated that CMTM6 interaction with membrane bound Enolase-1 stabilized its expression, leading to AKT-GSK3β mediated activation of Wnt-signaling. CMTM6 has been identified as a stabilizer of PD-L1 thereby facilitates immune evasion by tumor cells. As CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it can be an important therapeutic target for therapy resistant OSCC.
Authors
Pallavi Mohapatra, Omprakash Shriwas, Sibasish Mohanty, Arup Ghosh, Shuchi Smita, Sandeep Rai Kaushik, Rakesh Arya, Rachna Rath, Saroj Das Majumdar, Dillip Kumar Muduly, Sunil Raghav, Ranjan K. Nanda, Rupesh Dash
Abstract
Background: Loss-of-function variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including epileptic encephalopathy and cardiac arrhythmia. β1 subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming a subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Methods: We investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase.Results: We show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA-seq, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null cardiac tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. Conclusion: We propose that human disease variants resulting in SCN1B loss-of-function cause transcriptional dysregulation that contributes to altered excitability. These results provide important new insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multi-functionality of sodium channel β1 subunits.
Authors
Alexandra A. Bouza, Nnamdi Edokobi, Samantha L. Hodges, Alexa M. Pinsky, James Offord, Lin Piao, Yan-Ting Zhao, Anatoli N. Lopatin, Luis F. Lopez-Santiago, Lori L. Isom
Abstract
Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2 and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK up-regulated the expression of genes involved in OS cell metabolism and down-regulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux towards lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of peroxisome proliferator-activated receptor-γ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.
Authors
Toru Hirozane, Mari Masuda, Teppei Sugano, Tetsuya Sekita, Naoko Goto, Toru Aoyama, Takato Sakagami, Yuko Uno, Hideki Moriyama, Masaaki Sawa, Naofumi Asano, Masaya Nakamura, Morio Matsumoto, Robert Nakayama, Tadashi Kondo, Akira Kawai, Eisuke Kobayashi, Tesshi Yamada
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