Cell biology
Abstract
Chronic kidney disease is a global health concern characterized by maladaptive repair processes leading to kidney fibrosis. Following injury, early alterations in the extracellular matrix precede the development of kidney fibrosis and represent potential therapeutic targets to improve kidney repair. In this context, studies from our laboratory and others have shown that the matricellular protein SMOC2 can be targeted to decrease inflammation and tubulointerstitial fibrosis following kidney injury. The tubular epithelial cells (TECs), which are abundant and particularly susceptible to injury, play a central role in maladaptive repair; however, whether SMOC2 affects their functionality after kidney injury has not been explored. In this study, we show that SMOC2 localizes to the basement membrane of injured TECs across three murine models of kidney injury. Our in vitro studies demonstrate that SMOC2 induces a partial epithelial-to-mesenchymal (EMT) transition of TECs. We further demonstrate that its extracellular calcium-binding domain mediates binding to the decellularized extracellular matrix and mediates most of its effects on TECs. Mechanistically, SMOC2 promotes partial EMT effects through an integrin-dependent pathway. Together, these findings provide new mechanistic insight into how SMOC2 drives maladaptive repair by modulating TEC behavior and identify its calcium-binding domain as a key functional mediator.
Authors
Schrodinger Cenatus, Peng Gao, Nathalie Henley, Caroline Lamarche, Xue-Song Liu, Frédérick A. Mallette, Jonatan Barrera-Chimal, Casimiro Gerarduzzi
Abstract
Lineage plasticity, or transdifferentiation, is increasingly recognized as a resistance mechanism to androgen receptor (AR) inhibition in prostate cancer. Lineage plasticity is characterized by loss of AR signaling and epithelial differentiation, along with activation of stemness-associated pathways, epithelial-mesenchymal transition (EMT), or alternative differentiation programs such as neuroendocrine prostate cancer (NEPC). Loss of the tumor suppressors TP53 and RB1 is common in tumors exhibiting lineage plasticity; however, mechanisms by which TP53/RB1 loss promotes this phenotype remain poorly understood, and effective treatments are limited. Using multi-omic profiling of TP53/RB1 loss prostate cancer models, we identified alterations in chromatin accessibility, DNA methylation, and gene expression associated with lineage plasticity. Importantly, many pathways activated upon TP53/RB1 loss could be blocked through BET bromodomain inhibition. TP53/RB1-deficient cells also harbored widespread DNA methylation changes that silenced pathways linked with restraining lineage plasticity. Combined BET bromodomain and DNA methyltransferase (DNMT) inhibition was more effective than single agent treatment in suppressing growth of TP53/RB1 loss models exhibiting a stem-like or NEPC program. This was partly explained by abrogation of discrete lineage plasticity pathways modulated by each agent. Altogether, our work suggests combined BET bromodomain and DNMT inhibition is a promising therapeutic approach for prostate tumors exhibiting lineage plasticity.
Authors
William K. Storck, Diana Flores, Anbarasu Kumaraswamy, Zhi Duan, Shrabastee Chakraborty, Chao Zhang, Eva Rodansky, Dhruv Khokhani, Olivia A. Swaim, Karan Bedi, Raymond G. Cavalcante, Canping Chen, Faming Zhao, Ya-Mei Hu, Zheng Xia, Ryan J. Rebernick, Marcin Cieslik, Rahul Mannan, Somnath Mahapatra, Arul M. Chinnaiyan, Aaron M. Udager, Joshua A. Kuleape, Catherine R. Alumkal, Hannah N. Beck, Peter S. Nelson, Colm Morrissey, Michael C. Haffner, Leigh Ellis, Yuzhuo Wang, Joel A. Yates, Joshi J. Alumkal
Abstract
CFTR in the lung epithelium contributes to the secretion of a surface liquid layer that is essential to lung homeostasis and defense. The understanding of how liquid is secreted in the lung is derived largely from studies of the airway epithelium. Comparatively little is known about liquid secretion mechanisms in the alveolar epithelium, including its cellular source. To define which cell type drives alveolar liquid secretion, we generated transgenic mice that expressed a Cftr null allele in alveolar type 1 (AT1) cells, type 2 (AT2) cells, or both, then viewed liquid secretion in live alveoli using confocal microscopy of isolated, perfused lungs. Our findings show liquid secretion was blocked in alveoli of all three transgenic mice, indicating that both AT1 and AT2 cells contribute to alveolar liquid secretion. Cftr null expression in AT1 cells also blocked the secretion-mediated clearance of small particle and bacterial clusters from alveolar walls, indicating that AT1 cell CFTR contributes to alveolar defense. Together, these findings show alveolar liquid secretion depends on both AT1 and AT2 cell CFTR, and that CFTR in AT1 cells – a cell type not traditionally considered in liquid secretion mechanisms or CFTR-related lung diseases – contributes to lung liquid dynamics and host defense.
Authors
Sayahi Suthakaran, Sonya Homami, Deebly Chavez, Stephanie Tang, Sarah K.L. Moore, Chaya Sussman, Jimmy Zhang, Clemente J. Britto, Alice Prince, Alison J. May, Jaymin J. Kathiriya, Jaime L. Hook
Abstract
In α-1 antitrypsin (AAT) deficiency (AATD), emphysema is classically linked to protease-antiprotease imbalance caused by decreased antiprotease AAT due levels and function. This decrease is secondary to the impaired release of Z-AAT polymers from hepatocytes carrying Pi*Z, E342K mutation in SERPENA1 gene. Whether the accumulation of Z-AAT polymers in distal lungs contributes directly to emphysema pathogenesis has remained unexplored due to the lack of suitable model systems. We characterized lung injury and airspace enlargement in a Z-AAT–overexpressing murine model. We generated Z-AAT Serpina1Null mice overexpressing human (E342K) SERPENA1 in Serpina1Null mice and analyzed pulmonary phenotypes in young and aged animals, complemented by translational studies using primary cells, bronchoalveolar lavage fluid (BALf), and lung tissue from individuals who have never smoked and individuals with AATD. Young Z-AAT Serpina1Null mice accumulated Z-AAT polymers in hepatocytes, plasma, and BALf, exhibited spontaneous neutrophilic lung inflammation, increased alveolo-capillary permeability, and premature airspace enlargement, which was worse in older Z-AAT Serpina1Null mice. Moreover, Z-AAT polymers accumulated in alveolar type-2 epithelial (AT2) cells and lung macrophages, associated with endoplasmic reticulum (ER) stress, mitochondria dysfunction, and incomplete autophago-lysosomal fusion, which we recapitulated in lung samples from individuals with AATD. These findings support the pathogenic role of Z-AAT polymer accumulation in distal lung epithelium as a driver of epithelial, endothelial, and macrophage dysfunction linked to AATD emphysema.
Authors
Maria Magallón Serrano, Nazli Khodayari, William Bowers, Edward P. Manning, Xinran Liu, Jungnam Lee, Tammy O. Flagg, Regina Oshins, Aidan Griffin, Sahil Patel, Jorge E. Lascano, Divay Chandra, Susan M. Majka, Irina Petrache, Mark L. Brantly, Karina A. Serban
Abstract
To identify therapeutic targets limiting glioblastoma invasion, we applied druggable genome CRISPRi screens and multiomic analysis to patient-derived glioblastoma cells in micro-dissectible biomimetic 3D hydrogels that permitted separation and analysis of core versus invasive fractions. Of 2,550 genes screened, 12 encoded druggable targets whose suppression limited invasion, of which AURKB (encoding aurora kinase B) and ACP1 (encoding low molecular weight protein tyrosine phosphatase, LMW-PTP) were validated in neurosphere assays and in vivo. Proximity labeling identified cortactin as a link between LMW-PTP and aurora B, and we observed that cortactin underwent serine phosphorylation by aurora B and tyrosine dephosphorylation by LMW-PTP. Targeting ACP1 or AURKB via CRISPRi or inhibitors in culture and in vivo shifted the cortactin phosphorylation balance in glioblastoma, reducing levels of cortactin and the actin-related protein 2/3 (Arp2/3) complex that mediates cortactin-induced actin stabilization, thereby reducing actin-cortactin-Arp2/3 colocalization and subsequent actin polymerization. AURKB or ACP1 targeting shifted actin from cytoplasm to the nucleus, reducing mesenchymal gene expression. Biophysical analysis implicated AURKB in glioblastoma cell adhesion and stiffness needed for initial migration and ACP1 in mechanical stress resistance required for later migration. These findings revealed a targetable axis balancing kinase and phosphatase activities to regulate actin polymerization during glioblastoma invasion.
Authors
Mufeng Hu, Anna Weldy, Isabella M. Lovalvo, Erin A. Akins, Saket Jain, Alexander Chang, Ankita Sati, Meeki Lad, Austin Lui, Akhil Rajidi, Ameya Kothekar, Erika A. Ding, Juan A. Oses Prieto, Pablo Estevez, Alma L. Burlingame, Sanjay Kumar, Manish K. Aghi
Abstract
Women with PMOS (formally termed PCOS) have an overall increased prevalence of metabolic syndrome (MetS) and central obesity. To help determine whether there might be changes in s.c. adipose tissue (SAT) associated with these abnormalities, we performed single-nuclei and scRNA-seq on SAT biopsies from 15 premenopausal PMOS women with signs of insulin resistance and 17 healthy BMI-matched controls. In SAT from PMOS versus control we observed a higher ratio of fibrotic versus insulin sensitive adipocytes and a higher ratio of mesenchymal stem cells (MSCs) to preadipocytes. Further in silico analysis suggested that preadipocytes in PMOS are more inflammatory and have a reduced capacity for differentiation. Slit homolog 2 (SLIT2), which is expressed at higher levels in MSC from PMOS, decreased adipogenesis in cell culture assays likely through its interaction with the Roundabout homolog 1 and homolog 2 (ROBO1/2) receptor expressed on the surface of preadipocytes. These new observations are consistent with higher SLIT/ROBO signaling, leading to reduced differentiation in the SAT of PMOS as an underlying mechanism for the aberrant ectopic fat accumulation and the development of MetS in PMOS.
Authors
Adeline Divoux, Edina Erdos, Katie L. Whytock, Timothy F. Osborne, Steven R. Smith
Abstract
Natural killer (NK) cells undergo stepwise differentiation from multipotent progenitors within secondary lymphoid tissues. Despite the central importance of the tissue microenvironment in their development, little is known about cell-cell interactions that regulate human NK cell trafficking and maturation. Here, we identify the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of stromal-NK cell interactions required for NK cell maturation. We demonstrate that CXCR4 is expressed throughout human NK cell development in peripheral blood and tonsil, and CXCL12 is enriched in stromal niches containing developing NK cells. Pharmacologic blockade or genetic disruption of CXCR4 resulted in diminished adhesion to integrin ligands, and high-resolution imaging demonstrated crosstalk between CXCR4 and integrins, providing a mechanistic basis for chemokine-dependent modulation of adhesion. Further, CXCR4 blockade resulted in altered contact-dependent motility on stromal cells and integrin ligands, with decreased stable stromal engagement and increased cell speed. Consistent with a requirement for these interactions, treatment with the CXCR4 antagonist plerixafor (AMD3100) impaired NK cell generation from CD34+ precursors. Analysis of NK cells from WHIM syndrome patients with CXCR4 gain-of-function mutations treated with plerixafor revealed similar defects in migration and adhesion, supporting the in-vivo relevance of CXCR4-dependent regulation of NK cell adhesion and motility.
Authors
Shira E. Eisman, Francesca E. Grossberg, Batya S. Koenigsberg, David H. McDermott, Frédérique van den Haak, Luis A. Pedroza, Everardo Hegewisch-Solloa, Philip M. Murphy, Emily M. Mace
Abstract
Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2, expressed in renal epithelial cells, leading to progressive kidney dysfunction and end-stage kidney disease (ESKD). PKD patients exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell RNA sequencing atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Across models, single cell RNA sequencing (scRNAseq) data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters. Global deletion of Spp1 in Pkd1RC/RC mice resulted in a modest reduction in cyst severity and improved kidney function. From these studies, we created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.
Authors
Sarah J. Miller, Hua Zhong, Weidong Wu, Audrey M. Cordova, Morgan E. Yashchenko, Alex Yashchenko, Zhang Li, Daniyal J. Jafree, Chelsea N. Zimmerman, Christa I. DeVette, Vicki Do, Maya E. Hignite, Yohan Park, Fariha Nusrat, Bibi Maryam, Sizhao Lu, Xiaoyan Li, Jenny R. Gipson, Xiaogang Li, David A. Long, Mary C.M. Weiser-Evans, Bradley K. Yoder, Benjamin D. Cowley, Jr., Katharina Hopp, Jason R. Stubbs, Qin Ma, Anjun Ma, Kurt A. Zimmerman
Abstract
Malattia Leventinese (MAL) is an inherited macular degeneration disorder characterized by retinal drusen formation in adolescence, leading to vision loss. A mutation in the fibulin-3 gene (EFEMP1) causes MAL; however, the mechanisms underlying disease onset and drusen formation remain unclear. In this study, we generated induced pluripotent stem cell–derived retinal pigment epithelial (iPSC-RPE) cells from a patient with MAL to investigate disease mechanisms and potential therapies. MAL iPSC-RPE exhibited fibulin-3 and apolipoprotein E (ApoE) aggregation, increased endoplasmic reticulum stress, and enhanced apoptosis. Long-term culture with photoreceptor outer segments led to drusen-like deposits containing ApoE, complement components, and collagen IV accumulation, and it showed activation of matrix metalloproteinase-2 (MMP2). Untargeted lipid analysis revealed increased hexosylceramide and bis-monoacylglycerophosphate levels in MAL iPSC-RPE cells. A key pathological feature was lysosomal dysfunction associated with altered regulation of lysosomal gene programs, including reduced transcription factor EB transcript levels. Treatment with trehalose, a lysosome-modulating compound, increased lysosomal content and function, reducing drusen-like deposit formation, inhibiting MMP2 activation, and suppressing apoptosis. This study highlighted lysosomal dysfunction as a contributor to RPE damage, drusen-like deposit accumulation, and extracellular matrix degradation. Pharmacological restoration of lysosomal function alleviated these defects, suggesting therapeutic potential for MAL and other drusen-related diseases, including age-related macular degeneration.
Authors
Yumi Inoue, Hanako O. Ikeda, Masayuki Hata, Yuto Iida, Keiko Okamoto-Furuta, Isao Asaka, Makoto Arita, Akitaka Tsujikawa
Abstract
Mitochondrial gene expression is essential for oxidative phosphorylation that generates the bulk of the cellular ATP, and mitochondrial dysfunction is a common cause of human metabolic diseases. Recently, the first pathogenic variants in the only known mitochondrial RNA polymerase (POLRMT) were described in patients presenting with a wide variety of clinical manifestations, including hypotonia, short stature, and developmental delay. Here, we modeled two human pathogenic POLRMT variants by creating the corresponding substitutions in mice: the dominant S582F and the recessive R984C variant. Mice homozygous for the R984C variant showed perinatal lethality without apparent embryonic developmental defects, a finding consistent with a failure to adapt to the metabolic transition to oxidative metabolism at birth. Mice carrying the S582F variant were viable and exhibited decreased mitochondrial transcript levels due to impaired de novo transcription. However, mtDNA levels and in organello mtDNA replication remained normal, which recapitulates the molecular phenotypes observed in patients. Altogether, our findings indicate that the conserved arginine near the active site is essential for POLRMT function, while the serine in the intercalating hairpin of the N-terminal domain is required for near-genome length transcription but not primase activity. This study highlights genotype-phenotype differences and provides new insights into POLRMT function.
Authors
David Alsina, Diana Rubalcava-Gracia, Kristina Bubb, Rodolfo Garcia-Villegas, Akos Vegvari, Roberta Filograna, Florian A. Rosenberger, Camilla Koolmeister, Nils-Göran Larsson
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