Cell biology
Abstract
The expression of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium and its functional relevance were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and non-valve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43 and, like Cx43, deletion of Cx45 resulted in shortening and/or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast to Cx43, LEC-specific deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks, or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9 or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.
Authors
Michael J. Davis, Jorge A. Castorena-Gonzalez, Min Li, Scott D. Zawieja, Alexander M. Simon, Xin Geng, R. Sathish Srinivasan
Abstract
The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system (ENS), restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb knock-out (KO) mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colon of recipient Ednrb KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in “neo-ganglia”. Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared to sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.
Authors
Ahmed A. Rahman, Takahiro Ohkura, Sukhada Bhave, Weikang Pan, Kensuke Ohishi, Leah Ott, Christopher Han, Abigail Leavitt, Rhian Stavely, Alan J. Burns, Allan M. Goldstein, Ryo Hotta
Abstract
Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA sequencing, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, healthy individuals, and induced pluripotent stem (iPS) cells generated from a PCD patient. Transcriptomic analysis revealed unique signatures in PCD airway cells compared to their mothers and healthy individuals. Gene expression in heterozygous mothers’ cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases, GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism, Chlamydomonas reinhardtii, demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.
Authors
Jeffrey R. Koenitzer, Deepesh Kumar Gupta, Wang Kyaw Twan, Huihui Xu, Nicholas Hadas, Finn J. Hawkins, Mary Lou Beermann, Gervette M. Penny, Nathan T. Wamsley, Andrew Berical, Michael B. Major, Susan K. Dutcher, Steven L. Brody, Amjad Horani
Abstract
Endoplasmic reticulum (ER) stress and proinsulin misfolding are heralded as contributing factors to β-cell dysfunction in Type 2 diabetes (T2D), yet how ER function becomes compromised is not well understood. Recent data identifies altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β-cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β-cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas Txnip suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β-cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.
Authors
Kristen E. Rohli, Nicole J. Stubbe, Emily M. Walker, Gemma L. Pearson, Scott A. Soleimanpour, Samuel B. Stephens
Abstract
The number of adults living with cystic fibrosis (CF) has already increased significantly due to drastic improvements in life expectancy attributable to advances in treatment including the development of highly effective modulator therapy. Chronic airway inflammation in cystic fibrosis (CF) contributes to morbidity and mortality and aging processes like ‘inflammaging’ and cell senescence impact CF pathology. Our results show that single cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr knockout (Cftr–/–) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1 attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging CF population.
Authors
Molly Easter, Meghan June Hirsch, Elex Harris, Patrick Henry Howze IV, Emma Lea Matthews, Luke I. Jones, Seth Bollenbecker, Shia Vang, Daniel J. Tyrrell, Yan Y. Sanders, Susan E. Birket, Jarrod W. Barnes, Stefanie Krick
Abstract
Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the “hit and run” mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.
Authors
Huw J. Morgan, Carlotta Olivero, Boris Y. Shorning, Alex Gibbs, Alexandra L. Phillips, Lokapriya Ananthan, Annabelle Xiao Hui Lim, Licia Martuscelli, Cinzia Borgogna, Marco De Andrea, Martin Hufbauer, Richard G. Goodwin, Baki Akgül, Marisa Gariglio, Girish K. Patel
Abstract
Acute kidney injury strongly upregulates the transcription factor Foxm1 in proximal tubule in vivo and Foxm1 drives epithelial proliferation in vitro. Here we report that deletion of Foxm1 either with a nephron specific Cre driver or by inducible global deletion reduces proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI-to-CKD transition with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report extracellular signal-regulated kinase (ERK) mediates FOXM1 induction downstream of the epidermal growth factor receptor (EGFR) in primary proximal tubule cells. We defined FOXM1 genomic binding sites by Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned datasets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identify two cis regulatory elements that bind FOXM1 and regulate CCNF expression, demonstrate that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK-FOXM1-CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.
Authors
Megan L. Noonan, Yoshiharu Muto, Yasuhiro Yoshimura, Aidan Leckie-Harre, Haojia Wu, Vladimir V. Kalinichenko, Benjamin D. Humphreys, Monica Chang-Panesso
Abstract
The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of β1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.
Authors
Erin E. Hollander, Rosemary E. Flock, Jayne C. McDevitt, William P. Vostrejs, Sydney L. Campbell, Margo I. Orlen, Samantha B. Kemp, Benjamin M. Kahn, Kathryn E. Wellen, Il-Kyu Kim, Ben Z. Stanger
Abstract
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress–related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins’ physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Authors
Yaqiong Zhang, Aizhen Yang, Zhenzhen Zhao, Fengwu Chen, Xiaofeng Yan, Yue Han, Depei Wu, Yi Wu
Abstract
Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that interleukin 13 (IL-13) activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibits viscoelastic liquid behavior and that mucus secreted by IL-13 activated HAECs exhibits solid-like behavior caused by mucin cross-linking. In addition, IL-13 activated HAECs show increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that is prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), is increased in IL-13 activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings is increased in asthma patients with high airway mucus plug scores. Together, our results show that IL-13 activated HAECs autonomously generate pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.
Authors
Maude A. Liegeois, Margaret Braunreuther, Annabelle R. Charbit, Wilfred W. Raymond, Monica Tang, Prescott G. Woodruff, Stephanie A. Christenson, Mario Castro, Serpil C. Erzurum, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, Wendy C. Moore, Sally E. Wenzel, Gerald G. Fuller, John V. Fahy
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