Cell biology
Abstract
Lineage plasticity mediates resistance to androgen receptor pathway inhibitors (ARPIs) and progression from adenocarcinoma to neuroendocrine prostate cancer (NEPC), a highly aggressive and poorly understood subtype. ASCL1 has emerged as a central regulator of the lineage plasticity driving neuroendocrine differentiation. Here, we showed that ASCL1 was reprogrammed in ARPI-induced transition to the terminal NEPC and identified that the ASCL1 binding pattern tailored the expression of lineage-determinant transcription factor combinations that underlying discrete terminal NEPC identity. Notably, we identified FOXA2 as a major co-factor of ASCL1 in terminal NEPC, which is highly expressed in ASCL1-driven NEPC. Mechanistically, FOXA2 and ASCL1 interacted and worked in concert to orchestrate terminal neuronal differentiation. We identified that Prospero-Related Homeobox 1 was a target of ASCL1 and FOXA2. Targeting prospero-related homeobox 1 abrogated neuroendocrine characteristics and led to a decrease in cell proliferation in vitro and tumor growth in vivo. Our findings provide insights into the molecular conduit underlying the interplay between different lineage-determinant transcription factors to support the neuroendocrine identity and nominate prospero-related homeobox 1 as a potential target in ASCL1 high NEPC.
Authors
Shaghayegh Nouruzi, Takeshi Namekawa, Nakisa Tabrizian, Maxim Kobelev, Olena Sivak, Joshua M. Scurll, Cassandra Jingjing Cui, Dwaipayan Ganguli, Amina Zoubeidi
Abstract
Sjögren’s disease is a chronic autoimmune disease characterized by symptoms of oral and ocular dryness and extra-glandular manifestations. Mouth dryness is not only due to reduced saliva volume but also to alterations in the quality of salivary mucins in these patients. Mucins play a leading role in mucosa hydration and protection, where sulfated and sialylated oligosaccharides retain water molecules at the epithelial surface. The correct localization of glycosyltransferases and sulfotransferases within the Golgi apparatus determines adequate O-glycosylation and sulfation of mucins, which depends on specific golgins that tether enzyme-bearing vesicles. Here, we show that a golgin called Giantin is mislocalized in salivary glands from patients with Sjögren’s disease and forms protein complexes with Gal3-O-sulfotransferases (Gal3STs), which change their localization in Giantin knockout and knockdown cells. Our results suggest that Giantin could tether Gal3ST-bearing vesicles and that its altered localization could affect Gal3ST activity, explaining the decreased sulfation of MUC5B observed in salivary glands from patients with Sjögren’s disease.
Authors
Matilde Nuñez, Patricia Carvajal, Sergio Aguilera, María-José Barrera, Soledad Matus, Alicia Couto, Malena Landoni, Gaelle Boncompain, Sergio González, Claudio Molina, Karina Pino, Sebastián Indo, Lourdes Figueroa, María-Julieta González, Isabel Castro
Abstract
Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin-2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.
Authors
Joshua S. Carty, Ryoichi Bessho, Yvonne Zuchowski, Jonathan B. Trapani, Olena Davidoff, Hanako Kobayashi, Joseph T. Roland, Jason A. Watts, Andrew S. Terker, Fabian Bock, Juan Pablo Arroyo, Volker H. Haase
Abstract
Pneumonia is a worldwide threat to public health, demanding novel preventative and therapeutic strategies. The lung epithelium is a critical environmental interface that functions as a physical barrier to pathogen invasion while also actively sensing and responding to pathogens. We have reported that stimulating lung epithelial cells with a combination therapeutic consisting of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) induces synergistic pneumonia protection against a wide range of pathogens. We report here that mice deficient in Toll-like receptor 9 (TLR9), the previously described receptor for ODN, still displayed partial ODN-induced protection. This prompted us to seek an alternate ODN receptor, and we discovered by mass spectroscopy that the RNA sensor RIG-I could also bind DNA-like ODN. ODN binding by RIG-I resulted in MAVS-dependent pneumonia-protective signaling events. While RIG-I is essential to native defenses against viral infections, we report that therapeutic RIG-I activation with ODN promoted pathogen killing and host survival following both viral and bacterial challenges. These data indicate that maximal ODN-induced pneumonia protection requires activation of both TLR9/MyD88 and RIG-I/MAVS signaling pathways. These findings not only identify what we believe to be a novel pattern recognition receptor for DNA-like molecules, but reveal a potential therapeutic strategy to protect susceptible individuals against lethal pneumonias during periods of peak vulnerability.
Authors
Yongxing Wang, Vikram V. Kulkarni, Jezreel Pantaleón García, Michael K. Longmire, Mathilde Lethier, Stephen Cusack, Scott E. Evans
Abstract
Transcriptomic analyses have advanced the understanding of complex disease pathophysiology including chronic obstructive pulmonary disease (COPD). However, identifying relevant biologic causative factors has been limited by the integration of high dimensionality data. COPD is characterized by lung destruction and inflammation with smoke exposure being a major risk factor. To define novel biological mechanisms in COPD, we utilized unsupervised and supervised interpretable machine learning analyses of single cell-RNA sequencing data from the gold standard mouse smoke exposure model to identify significant latent factors (context-specific co-expression modules) impacting pathophysiology. The machine learning transcriptomic signatures coupled to protein networks uncovered a reduction in network complexity and novel biological alterations in actin-associated gelsolin (GSN), which was transcriptionally linked to disease state. GSN was altered in airway epithelial cells in the mouse model and in human COPD. GSN was increased in plasma from COPD patients, and smoke exposure resulted in enhanced GSN release from airway cells from COPD patients. This method provides insights into rewiring of transcriptional networks that are associated with COPD pathogenesis and provide a novel analytical platform for other diseases.
Authors
Justin Sui, Hanxi Xiao, Ugonna Mbaekwe, Nai-Chun Ting, Kaley Murday, Qianjiang Hu, Alyssa D. Gregory, Theodore S. Kapellos, Ali Öender Yildirim, Melanie Königshoff, Yingze Zhang, Frank C. Sciurba, Jishnu Das, Corrine R. Kliment
Abstract
Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3′sialyllactose (3′SL) as a natural inhibitor of Toll-Like Receptor (TLR) 4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3′SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of Liver X Receptor (LXR) and Sterol Regulatory Element-binding Protein-1 (SREBP). These acute anti-inflammatory effects of 3′SL were associated with reduced histone H3K27 acetylation at a subset of lipopolysaccharide (LPS)-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), Interferon Regulatory Factor 2 (IRF2), B-cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3′SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3′SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.
Authors
Ariane R. Pessentheiner, Nathanael J. Spann, Chloe A. Autran, Tae Gyu Oh, Kaare V. Grunddal, Joanna K.C. Coker, Chelsea D. Painter, Bastian Ramms, Austin W.T. Chiang, Chen-Yi Wang, Jason Hsiao, Yiwen Wang, Anthony Quach, Laela M. Booshehri, Alexandra Hammond, Chiara Tognaccini, Joanna Latasiewicz, Lisa Willemsen, Karsten Zengler, Menno P.J. de Winther, Hal M. Hoffman, Martin Philpott, Adam P. Cribbs, Udo Oppermann, Nathan E. Lewis, Joseph L. Witztum, Ruth Yu, Annette R. Atkins, Michael Downes, Ron M. Evans, Christopher K. Glass, Lars Bode, Philip L.S.M. Gordts
Abstract
Grover disease is an acquired epidermal blistering disorder in which keratinocytes lose intercellular connections. While its pathologic features are well-defined, its etiology remains unclear and it lacks any FDA-approved therapy. Interestingly, Grover disease was a common adverse event in clinical trials for cancer using B-RAF inhibitors, but it remained unknown how B-RAF blockade compromised skin integrity. Here we identified ERK hyperactivation as a key driver of Grover disease pathology. We leveraged a fluorescent biosensor to confirm that B-RAF inhibitors, dabrafenib and vemurafenib, paradoxically activated ERK in human keratinocytes and organotypic epidermis, disrupting cell-cell junctions and weakening epithelial integrity. Consistent with clinical data showing that concomitant MEK blockade prevents Grover disease in patients receiving B-RAF inhibitors, we found that MEK inhibition suppressed ERK and rescued cohesion of B-RAF-inhibited keratinocytes. Validating these results, we demonstrated ERK hyperactivation in patient biopsies from vemurafenib-induced Grover disease, but also from spontaneous Grover disease, revealing a common etiology for both. Finally, in line with our recent identification of ERK hyperactivation in Darier disease, a genetic disorder with identical pathology to Grover disease, our studies uncovered that the pathogenic mechanisms of these two diseases converge on ERK signaling and support MEK inhibition as a therapeutic strategy.
Authors
Cory L. Simpson, Afua Tiwaa, Shivam A. Zaver, Christopher J. Johnson, Emily Y. Chu, Paul W. Harms, Johann E. Gudjonsson
Abstract
Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor–β1 (TGF-β1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β1–induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β1–induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-β1–induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.
Authors
Greg Contento, Jo-Anne A.M. Wilson, Brintha Selvarajah, Manuela Platé, Delphine Guillotin, Valle Morales, Marcello Trevisani, Vanessa Pitozzi, Katiuscia Bianchi, Rachel C. Chambers
Abstract
Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/β-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.
Authors
Olivier Burgy, Christoph H. Mayr, Déborah Schenesse, Efthymios Fousekis Papakonstantinou, Beatriz Ballester, Arunima Sengupta, Yixin She, Qianjiang Hu, Maria Camila Melo-Narvaéz, Eshita Jain, Jeanine C. Pestoni, Molly Mozurak, Adriana Estrada-Bernal, Ugochi Onwuka, Christina Coughlan, Tanyalak Parimon, Peter Chen, Thomas Heimerl, Gert Bange, Bernd T. Schmeck, Michael Lindner, Anne Hilgendorff, Clemens Ruppert, Andreas Güenther, Matthias Mann, Ali Önder Yildirim, Oliver Eickelberg, Anna Lena Jung, Herbert B. Schiller, Mareike Lehmann, Gerald Burgstaller, Melanie Königshoff
Abstract
Emerging studies suggest that various parental exposures affect offspring cardiovascular health, yet the specific mechanisms, particularly the influence of paternal cardiovascular disease (CVD) risk factors on offspring cardiovascular health, remain elusive. The present study explores how paternal hypercholesterolemia affects offspring atherosclerosis development using the LDL receptor-deficient (LDLR–/–) mouse model. We found that paternal high-cholesterol diet feeding led to significantly increased atherosclerosis in F1 female, but not male, LDLR–/– offspring. Transcriptomic analysis highlighted that paternal hypercholesterolemia stimulated proatherogenic genes, including Ccn1 and Ccn2, in the intima of female offspring. Sperm small noncoding RNAs (sncRNAs), particularly transfer RNA–derived (tRNA-derived) small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), contribute to the intergenerational transmission of paternally acquired metabolic phenotypes. Using a newly developed PANDORA-Seq method, we identified that high-cholesterol feeding elicited changes in sperm tsRNA/rsRNA profiles that were undetectable by traditional RNA-Seq, and these altered sperm sncRNAs were potentially key factors mediating paternal hypercholesterolemia-elicited atherogenesis in offspring. Interestingly, high-cholesterol feeding altered sncRNA biogenesis–related gene expression in the epididymis but not testis of LDLR–/– sires; this may have led to the modified sperm sncRNA landscape. Our results underscore the sex-specific intergenerational effect of paternal hypercholesterolemia on offspring cardiovascular health and contribute to the understanding of chronic disease etiology originating from parental exposures.
Authors
Rebecca Hernandez, Xiuchun Li, Junchao Shi, Tejasvi R. Dave, Tong Zhou, Qi Chen, Changcheng Zhou
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