Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing
Using scRNA-Seq and Xenium spatial analysis, Overmiller et al. illustrate that the oral epithelial transcription factor PITX1 transforms epidermal keratinocytes and the skin to promote wound healing. The cover shows various populations of cutaneous keratinocytes (shaded cells) and nuclei (gray) in the skin of a PITX1-expressing mouse accessed by Xenium in situ analysis.
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Physician-Scientist Development
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Abstract
To reduce debt burden and encourage the pursuit of research-focused careers, most MD-PhD programs provide medical school tuition remission and an annual stipend. However, prolonged training compared with MD physicians postpones the time until MD-PhD physicians earn a full salary. We compared lifetime earning potential for MD-PhD physicians in academia with their MD colleagues in the same clinical specialty. We examined the relationship between earning potential based on specialty and the likelihood that MD-PhD physicians reported being engaged predominantly in research. Lifetime earning potential was estimated using 2020–2021 debt and compensation data for 77,701 academic physicians across 47 specialties. Self-reported research effort for 3,025 MD-PhD program alumni in academia was taken from the National MD-PhD Program Outcomes Study. We found that (a) MD-PhD physicians had a lower lifetime earning potential than MD physicians in the same specialty; (b) there was an inverse relationship between earning potential and research effort in different specialties, with MD-PhD physicians in high-earning specialties tending to spend less time on research; and (c) despite this, MD-PhD physicians in academia were more likely to choose clinical fields that allow more time for research.
Authors
Eva Catenaccio, Jonathan Rochlin, Myles H. Akabas, Lawrence F. Brass, Harold K. Simon
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Research Articles
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Abstract
Sphingosine 1-phosphate (S1P) is a lysosphingolipid with antiatherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet–fed LDL receptor–deficient mice with high or low overexpression levels of S1P receptor 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, interferon regulatory factor 8 (IRF8), and liver X receptor (LXR) and were skewed toward an M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5; increased ATP-binding cassette transporter A1– and G1–dependent cholesterol efflux; increased expression of MerTK and efferocytosis; and reduced apoptosis due to elevated B cell lymphoma 6 and Maf bZIP B. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to downregulation of the cAMP-dependent PKA and activation of the signaling cascade encompassing protein kinases AKT and mTOR complex 1 as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1. Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages toward an antiatherogenic functional phenotype and countervails the development of atherosclerosis in mice.
Authors
Francesco Potì, Enrica Scalera, Renata Feuerborn, Josephine Fischer, Lilli Arndt, Georg Varga, Evangelia Pardali, Matthias D. Seidl, Manfred Fobker, Gerhard Liebisch, Bettina Hesse, Alexander H. Lukasz, Jan Rossaint, Beate E. Kehrel, Frank Rosenbauer, Thomas Renné, Christina Christoffersen, Manuela Simoni, Ralph Burkhardt, Jerzy-Roch Nofer
×Abstract
Systemic capillary leak syndrome (SCLS) is a rare life-threatening disorder due to profound vascular leak. The trigger and the cause of the disease are currently unknown and there is no specific treatment. Here, we identified a rare heterozygous splice-site variant in the TLN1 gene in a familial SCLS case, suggestive of autosomal dominant inheritance with incomplete penetrance. Talin1 has a key role in cell adhesion by activating and linking integrins to the actin cytoskeleton. This variant causes in-frame skipping of exon 54 and is predicted to affect talin’s C-terminal actin-binding site (ABS3). Modeling the SCLS-TLN1 variant in TLN1-heterozygous endothelial cells (ECs) disturbed the endothelial barrier function. Similarly, mimicking the predicted actin-binding disruption in TLN1-heterozygous ECs resulted in disorganized endothelial adherens junctions. Mechanistically, we established that the SCLS-TLN1 variant, through the disruption of talin’s ABS3, sequestrates talin’s interacting partner, vinculin, at cell–extracellular matrix adhesions, leading to destabilization of the endothelial barrier. We propose that pathogenic variants in TLN1 underlie SCLS, providing insight into the molecular mechanism of the disease that can be explored for future therapeutic interventions.
Authors
Naama Elefant, Georgia Rouni, Christina Arapatzi, Danit Oz-Levi, Racheli Sion-Sarid, William J.S. Edwards, Neil J. Ball, Shira Yanovsky-Dagan, Alana R. Cowell, Vardiella Meiner, Vladimir Vainstein, Sofia Grammenoudi, Doron Lancet, Benjamin T. Goult, Tamar Harel, Vassiliki Kostourou
×Abstract
Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MKs) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. Lectin mannose-binding 1 (LMAN1) and multiple coagulation factor deficiency 2 (MCFD2) form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion. In this study, we showed that LMAN1-deficient mice (with complete germline LMAN1 deficiency) exhibited mild thrombocytopenia, whereas the platelet count was entirely normal in mice with approximately 7% Lman1 expression. Surprisingly, mice deleted for Mcfd2 did not exhibit thrombocytopenia. Analysis of peripheral blood from LMAN1-deficient mice demonstrated normal platelet size and normal morphology of dense and alpha granules. LMAN1-deficient mice exhibited a trend toward reduced MK and MK progenitors in the bone marrow. We next showed that hepatocyte-specific but not hematopoietic Lman1 deletion results in thrombocytopenia, with plasma TPO level reduced in LMAN1-deficient mice, despite normal Tpo mRNA levels in LMAN1-deficient livers. TPO and LMAN1 interacted by coimmunoprecipitation in a heterologous cell line, and TPO accumulated intracellularly in LMAN1-deleted cells. Together, these studies verified the hepatocyte as the cell of origin for TPO production in vivo and were consistent with LMAN1 as the endoplasmic reticulum cargo receptor that mediates the efficient secretion of TPO. To our knowledge, TPO is the first example of an LMAN1-dependent cargo that is independent of MCFD2.
Authors
Lesley A. Everett, Zesen Lin, Ann Friedman, Vi T. Tang, Greggory Myers, Ginette Balbin-Cuesta, Richard King, Guojing Zhu, Beth McGee, Rami Khoriaty
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Chemotherapy is often combined with surgery for muscle invasive and nonmuscle invasive bladder cancer (BCa). However, 70% of the patients recur within 5 years. Metabolic reprogramming is an emerging hallmark in cancer chemoresistance. Here, we report a gemcitabine resistance mechanism that promotes cancer reprogramming via the metabolic enzyme OXCT1. This mitochondrial enzyme, responsible for the rate-limiting step in β-hydroxybutyrate (βHB) catabolism, was elevated in muscle invasive disease and in patients with chemoresistant BCa. Resistant orthotopic tumors presented an OXCT1-dependent rise in mitochondrial oxygen consumption rate, ATP, and nucleotide biosynthesis. In resistant BCa, knocking out OXCT1 restored gemcitabine sensitivity, and administering the nonmetabolizable βHB enantiomer (S-βHB) only partially restored gemcitabine sensitivity. Suggesting an extrametabolic role for OXCT1, multi-omics analysis of gemcitabine sensitive and resistant cells revealed an OXCT1-dependent signature with the transcriptional repressor OVOL1 as a master regulator of epithelial differentiation. The elevation of OVOL1 target genes was associated with its cytoplasmic translocation and poor prognosis in a cohort of patients with BCa who have been treated with chemotherapy. The KO of OXCT1 restored OVOL1 transcriptional repressive activity by its nuclear translocation. Orthotopic mouse models of BCa supported OXCT1 as a mediator of gemcitabine sensitivity through ketone metabolism and regulating cancer stem cell differentiation.
Authors
Krizia Rohena-Rivera, Sungyong You, Minhyung Kim, Sandrine Billet, Johanna ten Hoeve, Gabrielle Gonzales, Chengqun Huang, Ashley Heard, Keith Syson Chan, Neil A. Bhowmick
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Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/Staphylococcus aureus Cas9 (CRISPR/SaCas9) system, which can be packaged into a single adeno-associated virus (AAV), holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9–mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single-guide RNAs capable of efficiently editing the mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to codeliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a-deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9–mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.
Authors
Zhiquan Liu, Siyu Chen, Chien-Hui Lo, Qing Wang, Yang Sun
×Abstract
KRAS mutations are frequent in various human cancers. The development of selective inhibitors targeting KRAS mutations has opened a new era for targeted therapy. However, intrinsic and acquired resistance to these inhibitors remains a major challenge. Here, we found that cancer cells resistant to KRAS G12C inhibitors also display cross-resistance to other targeted therapies, such as inhibitors of RTKs or SHP2. Transcriptomic analyses revealed that the Hippo-YAP/TAZ pathway is activated in intrinsically resistant and acquired-resistance cells. Constitutive activation of YAP/TAZ conferred resistance to KRAS G12C inhibitors, while knockdown of YAP/TAZ or TEADs sensitized resistant cells to these inhibitors. This scenario was also observed in KRAS G12D–mutant cancer cells. Mechanistically, YAP/TAZ protects cells from KRAS inhibitor–induced apoptosis by downregulating the expression of proapoptotic genes such as BMF, BCL2L11, and PUMA, and YAP/TAZ reverses KRAS inhibitor–induced proliferation retardation by activating the SLC7A5/mTORC1 axis. We further demonstrated that dasatinib and MYF-03-176 notably enhance the efficacy of KRAS inhibitors by reducing SRC kinase activity and TEAD activity. Overall, targeting the Hippo-YAP/TAZ pathway has the potential to overcome resistance to KRAS inhibitors.
Authors
Wang Yang, Ming Zhang, Tian-Xing Zhang, Jia-Hui Liu, Man-Wei Hao, Xu Yan, Haicheng Gao, Qun-Ying Lei, Jiuwei Cui, Xin Zhou
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Patients with immune-mediated inflammatory diseases (IMIDs) like rheumatoid arthritis (RA) are at higher risk for severe COVID-19 and long-term complications in bone health. Emerging clinical evidence demonstrated that SARS-CoV-2 infection reduces bone turnover and promotes bone loss, but the mechanism underlying worsened bone health remains elusive. This study sought to identify specific immune mediators that exacerbated preexisting IMIDs after SARS-CoV-2 exposure. Plasma samples from 4 groups were analyzed: healthy, IMID only, COVID-19 only, and COVID-19 + IMID. Using high-throughput multiplexed proteomics, we profiled 1,500 protein biomarkers and identified 148 unique biomarkers in COVID-19 patients with IMIDs, including elevated inflammatory cytokines (e.g., IL-17F) and bone resorption markers. Long-term circulating SARS-CoV-2 ORF8, a virulence factor for COVID-19, was detected in the COVID + IMID group. RA was one of the most common IMIDs in our study. ORF8 treatment of RA-derived human osteoblasts (RA-hOBs) increased levels of inflammatory (TNF, IL6, CCL2) and bone resorption (RANKL/osteoprotegerin ratio) markers compared with healthy controls. Supernatants from ORF8-treated RA-hOBs drove the differentiation of macrophages into osteoclast-like cells. These findings suggest that SARS-CoV-2 exposure can exacerbate IMIDs through ORF8-driven inflammation and osteoclastogenesis, highlighting potential therapeutic targets for managing COVID-19–induced bone pathologies.
Authors
Ivonne Melano, Tamiris Azamor, Camila C.S. Caetano, Nikki M. Meyer, Chineme Onwubueke, Anabelle Visperas, Débora Familiar-Macedo, Gielenny M. Salem, Brandy-Lee Soos, Cassandra M. Calabrese, Youn Jung Choi, Shuyang Chen, Younho Choi, Xianfang Wu, Zilton Vasconcelos, Suzy A.A. Comhair, Karin Nielsen-Saines, Leonard H. Calabrese, M. Elaine Husni, Jae U. Jung, Nicolas S. Piuzzi, Suan-Sin Foo, Weiqiang Chen
×Abstract
Metabolic reprogramming is a common feature in tumor progression and metastasis. Like proteins, lipids can transduce signals through lipid-protein interactions. During tumor initiation and metastasis, dysregulation of the Hippo pathway plays a critical role. Specifically, the inhibition of YAP1 phosphorylation leads to the relocation of YAP1 to the nucleus to activate transcription of genes involved in metastasis. Although recent studies reveal the involvement of phosphatidylethanolamine (PE) synthesis enzyme phosphoethanolamine cytidylyltransferase 2 (PCYT2) in tumor chemoresistance, the effect of PCYT2 on tumor metastasis remains elusive. Here, we show that PCYT2 was significantly downregulated in metastatic colorectal cancer (CRC) and acted as a tumor metastasis suppressor. Mechanistically, PCYT2 increased the interaction between PEBP1 and YAP1–phosphatase PPP2R1A, thus disrupting PPP2R1A-YAP1 association. As a result, phosphorylated YAP1 levels were increased, leading to YAP1 degradation through the ubiquitin protease pathway. YAP1 reduction in the nucleus repressed the transcription of ZEB1 and SNAIL2, eventually resulting in metastasis suppression. Our work provides insight into the role of PE synthesis in regulating metastasis and presents PCYT2 as a potential therapeutic target for CRC.
Authors
Lian Zhou, Su Zhang, Lingli Wang, Xueqin Liu, Xuyang Yang, Lei Qiu, Ying Zhou, Qing Huang, Yang Meng, Xue Lei, Linda Wen, Junhong Han
×Abstract
BACKGROUND Sodium-glucose cotransporter 2 inhibitors slow down progression of chronic kidney disease (CKD). We tested whether the circulating substrate mix is related to CKD progression and cardiovascular outcomes in patients with type 2 diabetes (T2D) and albuminuric CKD in the CREDENCE trial.METHODS We measured fasting substrates in 2,543 plasma samples at baseline and 1 year after randomization to either 100 mg canagliflozin or placebo and used multivariate Cox models to explore their association with CKD progression, heart failure hospitalization/cardiovascular death (hHF/CVD), and mortality.RESULTS Higher baseline lactate and free fatty acids (FFAs) were independently associated with a lower risk of CKD progression (HR = 0.73 [95% CI: 0.54–0.98] and HR = 0.67 [95% CI: 0.48–0.95], respectively) and hHF/CVD HR = 0.70 [95% CI: 0.50–0.99] and HR = 0.63 [95% CI: 0.42–0.94]). Canagliflozin led to a rise in plasma FFAs, glycerol, β-hydroxybutyrate, and acetoacetate. Changes in substrate between baseline and year 1 predicted an approximately 30% reduction in relative risk of both CKD progression and hHF/CVD independently of treatment. More patients who did not respond to canagliflozin treatment in terms of CKD progression belonged to the bottom lactate and FFA distribution tertiles.CONCLUSION In T2D patients with albuminuric CKD, basic energy substrates selectively influenced major long-term endpoints; canagliflozin treatment amplified their effects by chronically raising their circulating levels.
Authors
Ele Ferrannini, Simona Baldi, Maria Tiziana Scozzaro, Giulia Ferrannini, Michael K. Hansen
×Abstract
Deficits in IL-2 signaling can precipitate autoimmunity by altering the function and survival of FoxP3+ regulatory T cells (Tregs) while high concentrations of IL-2 fuel inflammatory responses. Recently, we showed that the non-beta IL-2 SYNTHORIN molecule SAR444336 (SAR’336) can bypass the induction of autoimmune and inflammatory responses by increasing its reliance on IL-2 receptor α chain subunit (CD25) to provide a bona fide IL-2 signal selectively to Tregs, making it an attractive approach for the control of autoimmunity. In this report, we further demonstrate that SAR’336 can support non-beta IL-2 signaling in murine Tregs and limit NK and CD8+ T cells’ proliferation and function. Using a murine model of spontaneous type 1 diabetes, we showed that the administration of SAR’336 slows the development of disease in mice by decreasing the degree of insulitis through the expansion of antigen-specific Tregs over Th1 cells in pancreatic islets. Specifically, SAR’336 promoted the differentiation of IL-33–responsive (ST2+), IL-10–producing GATA3+ Tregs over other Treg subsets in the pancreas, demonstrating the ability of this molecule to further orchestrate Treg adaptation. These results offer insight into the capacity of SAR’336 to generate highly specialized, tissue-localized Tregs that promote restoration of homeostasis during ongoing autoimmune disease.
Authors
Fernando Alvarez, Nicole V. Acuff, Glenn M. La Muraglia II, Nazila Sabri, Marcos E. Milla, Jill M. Mooney, Matthew F. Mackey, Mark Peakman, Ciriaco A. Piccirillo
×Abstract
T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function in response to weak TCR stimulation. T cell–specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased Th1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and ERK1/2 upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single-cell RNA-Seq suggested that Flot2-null CD4+ T cells follow a similar route of activation as WT CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naive CD4+ T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.
Authors
Sookjin Moon, Fei Zhao, Mohammad N. Uddin, Charles J. Tucker, Peer W.F. Karmaus, Michael B. Fessler
×Abstract
BACKGROUND Studies have demonstrated the role of ghrelin in alcohol-related behaviors and consumption. Blockade of the growth hormone secretagogue receptor (GHSR), which is the ghrelin receptor, has been shown to decrease alcohol drinking and reward-related behaviors across several animal models. We previously conducted a human study testing a GHSR inverse agonist/competitive antagonist, PF-5190457, in individuals who are heavy drinkers and showed its safety when coadministered with alcohol. Here, we conducted a phase IIa experimental medicine study in patients with alcohol use disorder (AUD) to investigate the effects of PF-5190457 on alcohol- and food-related outcomes.METHODS Forty-two individuals with AUD (n = 29 completers) participated in a randomized, double-blind, placebo-controlled study where they received PF-5190457 100mg b.i.d. (or placebo) in 2 counterbalanced, within-subject stages. Participants completed an alcohol cue-reactivity (CR) experiment in a bar-like laboratory and a virtual food choice experiment in a cafeteria-like virtual reality (VR) environment. A subset of participants (n = 12) performed a CR task during a brain functional MRI (fMRI) experiment.RESULTS PF-5190457 did not reduce cue-elicited alcohol craving. PF-5190457 reduced virtual calories selected (P = 0.04) in the VR environment. PF-5190457 did not influence neural activation during CR task in the fMRI experiment.CONCLUSION This study provides human evidence of the role of GHSR blockade in behaviors related to food selection and highlights the need for future investigations into targeting the ghrelin system in AUD.TRIAL REGISTRATION ClinicalTrials.gov (accession no. NCT02707055).FUNDING NIDA and NIAAA ZIA-DA000635; National Center for Advancing Translational Sciences UH2/UH3-TR000963.
Authors
Monica L. Faulkner, Mehdi Farokhnia, Mary R. Lee, Lisa Farinelli, Brittney D. Browning, Kelly Abshire, Allison M. Daurio, Vikas Munjal, Sara L. Deschaine, Selim R. Boukabara, Christopher Fortney, Garrick Sherman, Melanie Schwandt, Fatemeh Akhlaghi, Reza Momenan, Thomas J. Ross, Susan Persky, Lorenzo Leggio
×Abstract
Despite recent advances in the treatment of thyroid eye disease thyroid-related eye disease (TED), marked gaps remain in our understanding of the underlying molecular mechanisms, particularly concerning the insulin-like growth factor-1 receptor (IGF-1R) pathway. To dissect the pathophysiology of TED, we used single-nucleus RNA-Seq to analyze orbital fat specimens from both patients with TED and matched individuals acting as controls. The analysis demonstrated a marked increase in the proportion of fibroblasts transitioning to adipogenesis in the orbital fat of patients with TED compared with that in control patients. This was associated with diverse alterations in immune cell composition. Significant alterations in the IGF-1R signaling pathway were noted between TED specimens and those from control patients, indicating a potential pathological mechanism driven by IGF-1R signaling abnormalities. Additionally, our data showed that linsitinib, a small-molecule inhibitor of IGF-1R, effectively reduced adipogenesis in TED orbital fibroblasts in vitro, suggesting its potential utility as a therapeutic agent. Our findings reveal that, beyond immune dysfunction, abnormal IGF-1R signaling leading to enhanced adipogenesis is a crucial pathogenic mechanism in TED.
Authors
Dong Won Kim, Soohyun Kim, Jeong Han, Karan Belday, Emily Li, Nicholas Mahoney, Seth Blackshaw, Fatemeh Rajaii
×Abstract
Macrophages are required for healthy repair of the lungs following injury, but they are also implicated in driving dysregulated repair with fibrosis. How these 2 distinct outcomes of lung injury are mediated by different macrophage subsets is unknown. To assess this, single-cell RNA-Seq was performed on lung macrophages isolated from mice treated with LPS or bleomycin. Macrophages were categorized based on anatomic location (airspace versus interstitium), developmental origin (embryonic versus recruited monocyte derived), time after inflammatory challenge, and injury model. Analysis of the integrated dataset revealed that macrophage subset clustering was driven by macrophage origin and tissue compartment rather than injury model. Gpnmb-expressing recruited macrophages that were enriched for genes typically associated with fibrosis were present in both injury models. Analogous GPNMB-expressing macrophages were identified in datasets from both fibrotic and nonfibrotic lung disease in humans. We conclude that this subset represents a conserved response to tissue injury and is not sufficient to drive fibrosis. Beyond this conserved response, we identified that recruited macrophages failed to gain resident-like programming during fibrotic repair. Overall, fibrotic versus nonfibrotic tissue repair is dictated by dynamic shifts in macrophage subset programming and persistence of recruited macrophages.
Authors
Emily M. King, Yifan Zhao, Camille M. Moore, Benjamin Steinhart, Kelsey C. Anderson, Brian Vestal, Peter K. Moore, Shannon A. McManus, Christopher M. Evans, Kara J. Mould, Elizabeth F. Redente, Alexandra L. McCubbrey, William J. Janssen
×Abstract
Cutaneous wound healing is a slow process that often terminates with permanent scarring while oral wounds, in contrast, regenerate after damage faster. Unique molecular networks in epidermal and oral epithelial keratinocytes contribute to the tissue-specific response to wounding, but key factors that establish those networks and how the keratinocytes interact with their cellular environment remain to be elucidated. The transcription factor PITX1 is highly expressed in the oral epithelium but is undetectable in cutaneous keratinocytes. To delineate if PITX1 contributes to oral keratinocyte identity, cell-cell interactions, and the improved wound healing capabilities, we ectopically expressed PITX1 in the epidermis of murine skin. Using comparative analysis of murine skin and oral (buccal) mucosa with single-cell RNA-Seq and spatial transcriptomics, we found that PITX1 expression enhances epidermal keratinocyte migration and proliferation and alters differentiation to a quasi–oral keratinocyte state. PITX1+ keratinocytes reprogrammed intercellular communication between skin-resident cells to mirror buccal tissue while stimulating the influx of neutrophils that establish a pro-inflammatory environment. Furthermore, PITX1+ skin healed significantly faster than control skin via increased keratinocyte activation and migration and a tunable inflammatory environment. These results illustrate that PITX1 programs oral keratinocyte identity and cellular interactions while revealing critical downstream networks that promote wound closure.
Authors
Andrew M. Overmiller, Akihiko Uchiyama, Emma D Hope, Subhashree Nayak, Christopher G. O’Neill, Kowser Hasneen, Yi-Wen Chen, Faiza Naz, Stefania Dell’Orso, Stephen R. Brooks, Kan Jiang, Maria I. Morasso
×Abstract
MYB fusions are recurrently found in select cancers, including blastic plasmacytoid DC neoplasm (BPDCN), an acute leukemia with poor prognosis. They are markedly enriched in BPDCN compared with other blood cancers and, in some patients, are the only obvious somatic mutation detected. This suggests that they may alone be sufficient to drive DC transformation. MYB fusions are hypothesized to alter the normal transcription factor activity of MYB, but, mechanistically, how they promote leukemogenesis is poorly understood. Using CUT&RUN chromatin profiling, we found that, in BPDCN leukemogenesis, MYB switches from being a regulator of DC lineage genes to aberrantly regulating G2/M cell cycle control genes. MYB fusions found in patients with BPDCN increased the magnitude of DNA binding at these locations, and this was linked to BPDCN-associated gene expression changes. Furthermore, expression of MYB fusions in vivo impaired DC differentiation and induced transformation to generate a mouse model of myeloid-dendritic acute leukemia. Therapeutically, we present evidence that all-trans retinoic acid (ATRA) may cause loss of MYB protein and cell death in BPDCN.
Authors
Christopher A.G. Booth, Juliette M. Bouyssou, Katsuhiro Togami, Olivier Armand, Hembly G. Rivas, Kezhi Yan, Siobhan Rice, Shuyuan Cheng, Emily M. Lachtara, Jean-Pierre Bourquin, Alex Kentsis, Esther Rheinbay, James A. DeCaprio, Andrew A. Lane
×Abstract
Osteosarcoma (OS) is the most common malignant bone tumor, characterized by a high propensity for metastasis. Recent studies have highlighted the role of alternative splicing in cancer metastasis, although the precise mechanisms underlying aberrant splicing in OS invasion and metastasis remain unclear. Here, we analyzed consistently differentially expressed genes and differentially alternative splicing events between primary and metastatic OS to identify potential genes associated with OS progression. U1 small nuclear ribonucleoprotein 70K (SNRNP70) emerged as both differentially expressed and spliced, with elevated SNRNP70 levels correlating with poor prognosis in pateints with OS. Functional experiments demonstrated that SNRNP70 overexpression enhanced the proliferation and metastasis of OS cells in vitro, while its depletion reduced these capabilities in vivo. Mechanistically, SNRNP70 directly interacted with CD55, modulating its alternative splicing and promoting tumor progression in OS. Additionally, metastatic OS samples exhibited increased infiltration of resting immune cells, and single-cell RNA sequencing revealed communication between SNRNP70-expressing osteoblastic cells and macrophages via the ADGRE5/CD55 signaling pathway. Overall, our results showed that SNRNP70 knockdown inhibited OS progression, which was associated with the splicing of CD55, indicating SNRNP70 as a promising target for OS treatment.
Authors
Wenyue Li, Linzhu Wang, Wen Tian, Weihang Ji, Danyang Bing, Yan Wang, Bingqian Xu, Jiayue Feng, Peng Zhang, Haihai Liang, Yunyan Gu, Baofeng Yang
×Abstract
Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells and involves an interplay between β cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in β cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages, with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift toward an immunosuppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in IFN response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.
Authors
Titli Nargis, Charanya Muralidharan, Jacob R. Enriquez, Jiayi E. Wang, Kerim B. Kaylan, Advaita Chakraborty, Sarida Pratuangtham, Kayla Figatner, Jennifer B. Nelson, Sarah C. May, Jerry L. Nadler, Matthew B. Boxer, David J. Maloney, Sarah A. Tersey, Raghavendra G. Mirmira
×Abstract
Diabetic patients have increased susceptibility to acute kidney injury (AKI), and AKI could progress to chronic tubulointerstitial injury and fibrosis, referred to as AKI-to–chronic kidney disease (AKI-to-CKD) transition. However, whether diabetes directly promotes AKI-to-CKD transition is not known. We previously showed that reticulon-1A (RTN1A), a gene highly upregulated in injured renal tubular epithelial cells (RTECs), promotes AKI-to-CKD transition in nondiabetic settings. Therefore, we also examined whether reducing RTN1A expression could attenuate diabetes-induced AKI-to-CKD transition. Diabetes was induced by a high-fat diet and streptozotocin injections, and unilateral ischemic reperfusion injury was created as an AKI model in control, diabetic, and RTEC-specific Rtn1a-knockdown diabetic mice. AKI induced greater renal function decline, tubulointerstitial injury, and fibrosis in diabetic mice than in nondiabetic mice. Reduction of RTN1A markedly reduced the CKD development following AKI in diabetic mice, which was associated with reduced ER stress and mitochondrial dysfunction in RTECs. These findings indicate that diabetes markedly accelerates AKI-to-CKD transition and that RTN1A is a crucial mediator of diabetes-induced AKI-to-CKD transition. The development of RTN1A inhibitors could potentially attenuate AKI-to-CKD transition in diabetic patients.
Authors
Lulin Min, Ya Chen, Yixin Chen, Fang Zhong, Zhaohui Ni, Leyi Gu, Kyung Lee, John Cijiang He
×Abstract
Pseudohypoparathyroidism type 1B (PHP1B) is associated with epigenetic changes in the maternal allele of the imprinted GNAS gene that inhibit expression of the α subunit of Gs (Gsα), thereby leading to parathyroid hormone resistance in renal proximal tubule cells where expression of Gsα from the paternal GNAS allele is normally silent. Although all patients with PHP1B show loss of methylation for the exon A/B differentially methylated region (DMR), some patients with autosomal dominant PHP1B (AD-PHP1B) and most patients with sporadic PHP1B have additional methylation defects that affect the DMRs corresponding to exons XL, AS1, and NESP. Because the genetic defect is unknown in most of these patients, we sought to identify the underlying genetic basis for AD-PHP1B in 2 multigenerational families with broad GNAS methylation defects and negative clinical exomes. Genome sequencing identified small GNAS variants in each family that were also present in unrelated individuals with PHP1B in a replication cohort. Maternal transmission of one GNAS microdeletion showed reduced penetrance in some unaffected patients. Expression of AS transcripts was increased, and NESP was decreased, in cells from affected patients. These results suggest that the small deletion activated AS transcription, leading to methylation of the NESP DMR with consequent inhibition of NESP transcription, and thereby provide a potential mechanism for PHP1B.
Authors
Dong Li, Suzanne Jan de Beur, Cuiping Hou, Maura R.Z. Ruzhnikov, Hilary Seeley, Garry R. Cutting, Molly B. Sheridan, Michael A. Levine
×Abstract
T cells are involved in protective immunity against numerous viral infections. Data regarding functional roles of human T cells in SARS-CoV-2 (SARS2) viral clearance in primary COVID-19 are limited. To address this knowledge gap, we assessed samples for associations between SARS2 upper respiratory tract viral RNA levels and early virus-specific adaptive immune responses for 95 unvaccinated clinical trial participants with acute primary COVID-19 aged 18–86 years old, approximately half of whom were considered at high risk for progression to severe COVID-19. Functionality and magnitude of acute SARS2-specific CD4+ and CD8+ T cell responses were evaluated, in addition to antibody responses. Most individuals with acute COVID-19 developed SARS2-specific T cell responses within 6 days of COVID-19 symptom onset. Early CD4+ T cell and CD8+ T cell responses were polyfunctional, and both strongly associated with reduced upper respiratory tract SARS2 viral RNA, independent of neutralizing antibody titers. Overall, these findings provide evidence for protective roles for circulating SARS2-specific CD4+ and CD8+ T cells during acute COVID-19.
Authors
Sydney I. Ramirez, Paul G. Lopez, Farhoud Faraji, Urvi M. Parikh, Amy Heaps, Justin Ritz, Carlee Moser, Joseph J. Eron, David Wohl, Judith Currier, Eric S. Daar, Alex Greninger, Paul Klekotka, Alba Grifoni, Daniela Weiskopf, Alessandro Sette, Bjoern Peters, Michael D. Hughes, Kara W. Chew, Davey M. Smith, Shane Crotty, for the Accelerating COVID-19 Therapeutic Interventions and Vaccines-2 (ACTIV-2)/A5401 Study Team
