Biology subjects

Shi, H.

Publications and source records attributed to Shi, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Epigenetic Programming of Estrogen Receptor in Adipose Tissue by High Fat Diet Regulates Obesity-Induced Inflammation

Adipose inflammation plays a key role in obesity-induced metabolic abnormalities. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors and complex diseases. Here we found that high fat diet (HFD) feeding induced a dynamic change of DNA methylome in mouse white adipose tissue (WAT) analyzed by reduced representative bisulfite sequencing. Interestingly, DNA methylation at the promoter of estrogen receptor (Esr1) was significantly increased by HFD, concomitant with a down-regulation of Esr1 expression. HFD feeding in mice increased the expression of DNA methyltransferase 1 (Dnmt1) and Dnmt3a, and binding of DNMT1 and DNMT3a to Esr1 promoter in WAT. Mice with adipocyte-specific Dnmt1 deficiency displayed increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity upon HFD challenge; while mice with adipocyte-specific Dnmt3a deficiency showed a mild metabolic phenotype. Using a modified CRISPR/RNA-guided system to specifically target DNA methylation at the Esr1 promoter in WAT, we found that reducing DNA methylation at Esr1 promoter increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity in HFD-challenged mice. Our study demonstrates that DNA methylation at Esr1 promoter plays an important role in regulating adipose inflammation, which may contribute to obesity-induced insulin resistance.

physiology↗

bioRxiv2025

Targeting Regnase-1 unleashes CAR T cell antitumor activity for osteosarcoma and creates a proinflammatory tumor microenvironment

Negative regulators of T cell function represent promising targets to enhance the intrinsic antitumor activity of CAR T cells against solid tumors. However, the endogenous immune ecosystem in solid tumors often represents an immunosuppressive therapeutic barrier to CAR T cell therapy, and it is currently unknown whether deletion of negative regulators in CAR T cells reshapes the endogenous immune landscape. To address this knowledge gap, we developed CAR T cells targeting B7-H3 in immune-competent osteosarcoma models and evaluated the intrinsic and extrinsic effects of deleting a potent negative regulator called Regnase-1 (Reg-1). Deletion of Reg-1 not only improved the effector function of B7-H3-CAR T cells but also endowed them with the ability to create a proinflammatory landscape characterized by an influx of IFN{gamma}-producing endogenous T cells and NK cells and a reduction of inhibitory myeloid cells, including M2 macrophages. Thus, deleting negative regulators in CAR T cells enforces a non-cell-autonomous state by creating a proinflammatory tumor microenvironment.

immunology↗

bioRxiv2025

β-Glucan Modulates Monocyte Plasticity and Differentiation Capacity to Mitigate DSS-Induced Colitis

Trained immunity involves the reprogramming of innate immune cells after an initial exposure, resulting in heightened inflammatory responses to subsequent stimuli and enhanced bactericidal capacity during infection. However, this proinflammatory state could also exacerbate chronic conditions like inflammatory bowel disease (IBD), which is characterized by persistent inflammation and microbial imbalance. It remains unclear how trained immunity influences IBD pathogenesis and whether it can be harnessed therapeutically. In our study, pretreatment with {beta}-glucan reprogrammed bone marrow hematopoietic progenitors and peripheral monocytes, inducing a profound shift in monocyte plasticity and significantly reducing the severity of dextran sulfate sodium (DSS)-induced colitis. Adoptive transfer of bone marrow or peripheral monocytes from {beta}-glucan-trained mice into naive mice conferred robust protection against colitis, demonstrating that this protective effect is transferable. Trained mice also displayed improved clearance of intestinal bacterial infections. Single-cell RNA sequencing revealed an expansion of reparative Cx3cr1 macrophages derived from Ly6Chi monocytes, correlating with accelerated colonic epithelial regeneration. Collectively, these findings reveal how {beta}-glucan-induced trained immunity modulates monocyte differentiation to ameliorate experimental colitis, highlighting the potential of harnessing trained immunity as a therapeutic strategy to recalibrate innate immune responses and restore gut homeostasis in IBD, shedding light for future clinical applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/653036v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1b969c2org.highwire.dtl.DTLVardef@1ce603dorg.highwire.dtl.DTLVardef@10582forg.highwire.dtl.DTLVardef@10d7de6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Metabolic modulation of intratumoral cholesterol with gut microbiota for the treatment of colorectal cancer

Excess cholesterol is positively correlated with colorectal cancer (CRC). Current therapeutic strategies for modulating cholesterol levels in CRC are limited and often come with complications. Here, we demonstrated that microbiome shunting of intestinal cholesterol to anticancer metabolites is a safe and applicable therapeutic approach for CRC. By screening major microbial metabolic products of cholesterol, we found that 4-cholesten-3-one (4-C-3) was selectively depleted in fecal samples and tumor tissues of patients with CRC. 4-C-3 exhibits strong antitumor effects on human CRC cell lines, patient-derived organoids, and patient-derived xenograft (PDX) models. Mechanistically, 4-C-3 suppresses CRC tumorigenesis by dually targeting the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homologue (KRAS). 4-C-3 directly binds to EGFR, blocking its signal transduction by inhibiting the binding of EGFR ligands. Additionally, 4-C-3 inhibits oncogenic KRAS variants, such as KRASG12D, by suppressing nucleotide exchange activity and effector engagement. By targeting both EGFR and KRAS, 4-C-3 reduces primary resistance to anti-EGFR therapies caused by KRAS mutations in CRC. As a proof-of-concept study, we showed that delivery of 4-C-3 by Oscillibacter ruminantium, a 4-C-3- producing commensal bacterium that is reduced in CRC patients, or a nonpathogenic Escherichia coli strain engineered to specifically convert intestinal cholesterol into 4-C-3, removed intratumoral cholesterol and led to rapid tumor regression in multiple models of CRC. These results suggest that microbial therapies for restoring intestinal cholesterol homeostasis represent a new therapeutic avenue for CRC.

cancer biology↗

bioRxiv2025

Enhance Memory CD8+ T Cell Formation via Downregulation of Hematopoietic Progenitor Kinase 1 and Sustained Mitochondrial Fitness

Memory CD8+ T cells are pivotal for long-term anti-tumor immunity due to their longevity and rapid response upon encountering tumor cells. Hematopoietic progenitor kinase 1 (HPK1), a member of the Ste20 family of kinases, restricts proximal T cell receptor (TCR) signaling and plays critical roles in T cell priming and activation. However, the impact of HPK1 on CD8+ T cell differentiation is not well understood. Here, we demonstrate that chimeric antigen receptor (CAR) T cells derived from patients lacking HPK1 exhibit a stronger memory phenotype. HPK1 deletion promotes the formation of precursor and central memory CD8+ T cells, resulting in superior and long-lasting antitumor activity in murine cancer models compared to WT mice. Additionally, HPK1 knockout induces metabolic reprogramming by enhancing oxidative phosphorylation (OXPHOS) and mitochondrial respiration complex activity. Mechanistically, HPK1 deletion downregulates mTOR signaling by reducing the phosphorylation of mTOR and S6. Increased mitophagy activity is observed in HPK1-depleted cells, with upregulation of key mitophagy regulators, including Pink1 and Bnip3l, maintaining mitochondrial fitness. Our study reveals that HPK1 regulates CD8+ T cell metabolic reprogramming, guiding cell differentiation and antitumor responses.

immunology↗

bioRxiv2025

mcDETECT: Decoding 3D Spatial Synaptic Transcriptomes with Subcellular-Resolution Spatial Transcriptomics

Spatial transcriptomics (ST) is a powerful tool for studying the molecular basis of brain diseases. However, most current analyses focus only on nuclear or somatic expression, overlooking distally localized mRNAs, known as the "dark transcriptome." These transcripts, which make up nearly 40% of the brain transcriptome, are packaged into RNA granules and transported to polarized neuronal structures critical for neuronal function. Here, we present mcDETECT, a machine learning framework that leverages in situ ST (iST) data to detect RNA granules and resolve their RNA composition. Applying mcDETECT to simulated and real datasets from multiple iST platforms, we demonstrate robust granule detection and molecular subtyping. Granule-informed downstream analysis further revealed functionally distinct neuronal substates not captured by somatic expression alone. In an Alzheimers disease (AD) mouse model, mcDETECT uncovered alterations in RNA granule distributions and associated neuronal substates prior to measurable neuronal loss, highlighting potential therapeutic targets for early AD pathology.

bioinformatics↗

bioRxiv2025

Multiscale Spatial Mapping of Microbial Communities for Biotherapeutic Development

Live biotherapeutic products (LBPs) are emerging as powerful tools to modulate the microbiome using well-defined microbial communities. Yet, designing, manufacturing, and delivering LBPs remains challenging, in part due to a lack of technologies capable of analyzing LBPs as complete, spatially organized consortia. Conventional sequencing-based methods lack sensitivity and specificity and do not provide critical spatial information. To address this, we present high-phylogenetic-resolution spatial mapping platform (HiPR-Map), a state-of-the-art spectral imaging technology that enables precise enumeration and spatial localization of microbial cells at species-level within complex communities. Through these advantages, HiPR-Map provides unique insights for LBP discovery and development. Applying HiPR-Map to an LBP designed to complement immune checkpoint therapy, we profiled over 1.8 million microbial cells engrafted in the murine gut. Our analysis revealed distinctive microbial spatial organization, underscoring the power of imaging-based microbiome profiling to optimize LBP design and characterization. This work highlights the transformative potential of spatial microbiome analysis for next-generation LBP development.

microbiology↗

bioRxiv2025

Directed evolution expands CRISPR-Cas12a genome editing capacity

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5-TTTV-3 protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12as gene targeting capability to only [~]1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5-TTTV-3 PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5-NYHV-3 PAMs that expand DNA recognition sites to [~]25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

biochemistry↗

bioRxiv2025

Krüppel Regulates Cell Cycle Exit and Limits Adult Neurogenesis of Mushroom Body Neural Progenitors in Drosophila

In many organisms, including Drosophila and humans, neural progenitors exit the cell cycle and are eliminated by the end of development, thereby restricting adult neurogenesis to specific brain regions. Here, we identify the evolutionarily conserved transcription factor Kruppel (Kr) as a lineage-specific regulator of cell cycle exit and elimination of mushroom body neuroblasts (MBNBs), which generate the learning and memory centre of the Drosophila brain, a structure functionally analogous to the mammalian hippocampus. Neuroblast-specific Kr RNAi and the Irregular facet (KrIf-1) mutation prolong MBNB lifespan, enabling continued neurogenesis in the adult brain. Although Kr is expressed only at low levels in postembryonic MBNBs, its pupal stage-specific depletion or misexpression is sufficient to cause MBNB retention, revealing a previously unrecognised postembryonic function distinct from its established role in embryonic neurogenesis. Mechanistically, persistent MBNBs maintain expression of the early temporal factor IGF2 mRNA-binding protein (Imp) and fail to fully induce the late temporal factors Syncrip (Syp) and Eip93F (E93). Co-depletion of Imp suppresses MBNB retention caused by Kr depletion, demonstrating that Imp is a key downstream effector of Kr. In parallel, Kruppel homolog 1 (Kr-h1), another Kr family transcription factor and a well-established mediator of hormone-responsive transcription, functionally antagonises Kr by suppressing E93 expression: Kr-h1 knockdown partially rescues the Kr depletion phenotype, whereas Kr-h1 overexpression drives tumour-like neuroblast overgrowth. Together, our findings establish Kr as an MBNB-specific coordinator that integrates intrinsic temporal programmes with extrinsic signalling pathways to coordinate neural stem cell termination and neuronal fate transitions, with potential parallels in other organisms.

developmental biology↗

bioRxiv2025

A novel enterococcus phage endolysin Lys22 with a wide host range against mixed biofilm of Enterococcus faecalis, Staphylococcus aureus and Acinetobacter baumannii

The global surge in multidrug-resistant (MDR) bacterial pathogens has created an urgent imperative for innovative antimicrobial strategies. Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii demonstrate remarkable antibiotic resistance and dominate hospital-acquired infections. These bacteria often form biofilms, a complex community structure that shields them from immune system phagocytosis, resists antibiotic penetration, and enhances their survival in harsh environments. In clinical cases, these bacteria often form mixed biofilms and lead to treatment failures. Phages and their derivatives have emerged as promising candidates in the fight against drug-resistant bacteria. Lys22, an endolysin derived from an enterococcus phage, has been cloned and demonstrated to possess a broad host range, effectively targeting E. faecalis, various Staphylococcus species, and A. baumannii. When applied to the biofilms formed by these bacteria, Lys22 was found to significantly inhibit both simple and complex biofilms in vitro. Virulent genes, including agrA, sarA, and icaA in S. aureus; asa1, cylA, and hyl in E. faecalis; and OmpA and lpsB in A. baumannii were also downregulated by Lys22. Notably, Lys22 also exhibited a robust protective effect against dual or triple infections involving E. faecalis, S. aureus, and A. baumannii in a zebrafish eggs model, highlighting its potential as a therapeutic agent in combatting multi-bacterial infections.

microbiology↗

bioRxiv2025

FOSL2 Directly Regulates FSHR and CYP11A1 Transcription: An Essential Transcription Factor for Gonadotropin-Dependent Folliculogenesis

The meticulous orchestration of gonadotropin-dependent folliculogenesis constitutes the cornerstone of female reproductive cyclicity and fertility, with FSH/FSHR signaling recognized as the master regulator. Achieving the necessary amplification of this signaling is essential for successful GTH-dependent folliculogenesis, yet the mechanisms remain inadequately defined. Our study utilizes single-cell and spatial transcriptomics to identify FOSL2 as an FSH-inducible transcription factor, exhibiting precise spatiotemporal co-expression with FSHR. FOSL2 knockdown in vitro resulted in notable reductions in granulosa cell proliferation, induced apoptosis, and disrupted gonadotropin-dependent folliculogenesis. In vivo studies using conditional FOSL2 deletion in mouse granulosa cells corroborated these results, demonstrating a complete halt in GTH-dependent folliculogenesis and resultant infertility. Mechanistic exploration unveiled that FSH/FSHR initiates FOSL2 expression via the cAMP/PKA/CREB cascade, while FOSL2 in turn enhances FSHR transcription through direct promoter binding, thereby establishing a self-amplifying loop. This loop represents a molecular switch, modeled to the all-or-nothing dynamics of GTH-dependent folliculogenesis. The evolutionary conservation of this mechanism was confirmed through cross-species analyses in sheep, where FOSL2 deficiency similarly attenuated FSH/FSHR signaling and inhibited follicular growth. Our findings advance the understanding of folliculogenesis by revealing a novel FOSL2-centered amplification loop for FSH/FSHR signaling, highlighting the indispensable role of FOSL2 in reproductive biology.

cell biology↗

bioRxiv2025

Dnmt3b deficiency in adipocyte progenitor cells ameliorates obesity in female mice

Obesity arises from chronic energy imbalance where energy intake exceeds energy expenditure. Emerging evidence supports a key role of DNA methylation in the regulation of adipose tissue development and metabolism. We recently discovered a key role of DNA methylation, catalyzed by DNA methyltransferase 1 or 3a (Dnmt1 or 3a), in the regulation of adipocyte differentiation and metabolism. Here, we aimed to investigate the role of adipocyte progenitor cell Dnmt3b, an enzyme mediating de novo DNA methylation, in energy metabolism and obesity. We generated a genetic model with Dnmt3b knockout in adipocyte progenitor cells (PD3bKO) by crossing Dnmt3b-floxed mice with platelet-derived growth factor receptor alpha (Pdgfr)-Cre mice. Dnmt3b gene deletion in adipocyte progenitors enhanced thermogenic gene expression in brown adipose tissue, increased overall energy expenditure, and mitigated high-fat diet (HFD)-induced obesity in female mice. PD3bKO mice also displayed a lower respiratory exchange ratio (RER), indicative of a metabolic shift favoring fat utilization as an energy source. Furthermore, female PD3bKO mice exhibited improved insulin sensitivity alongside their lean phenotype. In contrast, male PD3bKO mice showed no changes in body weight but demonstrated decreased insulin sensitivity, revealing a sexually dimorphic metabolic response to Dnmt3b deletion in adipocyte progenitor cells. These findings underscore the critical role of Dnmt3b in regulating energy homeostasis, body weight, and metabolic health, with significant implications for understanding sex-specific mechanisms of obesity and metabolism.

systems biology↗

bioRxiv2025

The metabolic enzyme GYS1 condenses with NONO/p54nrb in the nucleus to spatiotemporally regulate glycogenesis and myogenic differentiation

Accumulating evidence indicates that metabolic enzymes can directly couple metabolic signals to transcriptional adaptation and cell differentiation. Glycogen synthase 1 (GYS1), the key metabolic enzyme for glycogenesis, is a nucleocytoplasmic shuttling protein compartmentalized in the cytosol and nucleus. However, the spatiotemporal regulation and biological function of nuclear GYS1 (nGYS1) microcompartments remain unclear. Here, we show that GYS1 dynamically reorganizes into nuclear condensates under conditions of glycogen depletion or transcription inhibition. nGYS1 complexes with the transcription factor NONO/p54nrb and undergoes liquid-liquid phase separation to form biomolecular condensates, leading to its nuclear retention and inhibition of glycogen biosynthesis. Compared to their wild-type littermates, Nono-deficient mice exhibit exercise intolerance, higher muscle glycogen content, and smaller myofibers. Additionally, Gys1 or Nono deficiency prevents C2C12 differentiation and cardiotoxin-induced muscle regeneration in mice. Mechanistically, nGYS1 and NONO co-condense with the myogenic transcription factor MyoD and preinitiation complex (PIC) proteins to form transcriptional condensates, driving myogenic gene expression during myoblast differentiation. These results reveal the spatiotemporal regulation and subcellular function of nuclear GYS1 condensates in glycogenesis and myogenesis, providing mechanistic insights into glycogenoses and muscular dystrophy.

cell biology↗

bioRxiv2025

Follicular mural granulosa cells stockpile glycogen to fuel corpus luteum pre-vascularization

The corpus luteum (CL) arises from the luteinization of follicular granulosa cells (GCs) and theca cells, marked by rapid progesterone elevation and angiogenesis. Intriguingly, angiogenesis lags behind progesterone elevation, creating an avascular phase during which luteal cells must fuel intensive steroidogenesis without perfusion. How the avascular CL meets this energetic demand remains a mystery. Here, we reveal a novel cellular adaptive mechanism-GC energy storage (GCES)-that resolves this enigma. We demonstrate that upon luteinization initiation, GCs enter a metabolically quiescent state yet enhance glucose uptake via SLC2A1, converting the glucose into glycogen through the hCG (LH)-MAPK-RUNX1-Insulin signaling axis. Catabolism of this glycogen reserve supplies the energy required for the avascular CL, ensuring normal luteogenesis. GCES is evolutionarily conserved across species. Genetic or pharmacological disruption of GCES or glycogenolysis induces luteal insufficiency, whereas timely glucose administration enhances GCES, improving luteal function and optimize reproductive outcome in both mouse and ovine models. In human study, orally intake of glucose post-hCG significantly augments GCES and enhances progesterone production in women. These results advance luteal physiology by uncovering a universal reproductive principle with direct clinical implications.

developmental biology↗

bioRxiv2024

Single-cell transcriptome profiling of the myeloid cells repopulating after chemotherapy identifies a neutrophil-like monocyte subset with pro-tumor activities

Patients with cancer often receive chemotherapy to control tumor progression and reduce disease symptoms. Cytotoxic chemotherapeutic agents not only kill rapidly growing cancer cells but also reduce normal cells including myeloid cells, the main innate immune population involved in fighting infections and repairing tissue damages. Rapid loss of myeloid cells caused by chemotherapy triggers myelopoiesis, a process in which the hematopoietic stem and progenitor cells in the bone marrow regenerate myeloid cells, including monocytes, neutrophils, dendritic cells and macrophages, to reconstitute the myeloid cell compartment. We previously reported that chemotherapy with an alkylating agent cyclophosphamide (CTX) in mice leads to repopulation of myeloid cells that acquire immunosuppressive activities within the monocyte subset. However, detailed information on the cellular composition and molecular identity of these chemotherapy- induced immunosuppressive monocytes is lacking. Here, we investigated how the various myeloid cell subsets in the bone marrow of mice respond to CTX chemotherapy through single-cell RNA sequencing analysis (scRNAseq). We found that myeloid progenitor cells and monocytes were reduced 2 days after chemotherapy but rebounded and surpassed their pretreatment levels by day 7. Further scRNAseq analysis of pre-enriched monocytes revealed that the monocyte population was heterogenous, and that chemotherapy tilted myelopoiesis towards the production of neutrophil-like monocytes (NeuMo). We identified Cxcr4 and Cx3cr1 as suitable markers for isolation of chemotherapy-induced NeuMo and demonstrated that these cells were suppressive to T cells. Together with the evidence that CTX-induced monocytes can promote breast cancer metastasis in mice, our data reveal the heterogeneity of the monocytes reemerging after chemotherapy and identify the NeuMo subset as a potential therapeutic target for enhancing the efficacy of chemotherapy in cancer.

immunology↗

bioRxiv2024

MOZ and HBO1 Histone Acetyltransferase Complexes Are Molecular Dependencies and Therapeutic Targets in NUP98-Rearranged Acute Myeloid Leukemia

NUP98 fusion oncoproteins (FOs) are a hallmark of childhood acute myeloid leukemia (AML) and drive leukemogenesis through liquid-liquid phase separation-mediated nuclear condensate formation. However, the composition and consequences of NUP98 FO-associated condensates are incompletely understood. Here we show that MYST family histone acetyltransferase (HAT) complex proteins including MOZ/KAT6A, HBO1/KAT7, and the common MOZ/HBO1 complex subunit BRPF1 associate with NUP98 FOs on chromatin and within condensates. MYST HATs are molecular dependencies in NUP98-rearranged (NUP98-r) leukemia, and genetic inactivation or pharmacologic inhibition of Moz and Hbo1 impairs NUP98-r cell fitness. MOZ/HBO1 inhibition decreased global H3K23ac levels, displaced NUP98::HOXA9 from chromatin at the Meis1 locus, and led to myeloid cell differentiation. Additionally, MOZ/HBO1 inhibition decreased leukemic burden in multiple NUP98-r leukemia xenograft mouse models, synergized with Menin inhibitor treatment, and was efficacious in Menin inhibitor-resistant cells. In summary, we show that MYST family HATs are therapeutically actionable dependencies in NUP98-r AML. SIGNIFICANCE STATEMENTMOZ and HBO1 associate with NUP98 fusion oncoprotein condensates to drive leukemogenesis. Inhibition of their histone acetyltransferase activity is an effective therapeutic strategy in NUP98-rearranged leukemias, including those resistant to Menin inhibition. Moreover, combined MOZ/HBO1 and Menin inhibition is synergistic, supporting clinical translation to improve outcomes of NUP98 FO-driven leukemias.

cancer biology↗

bioRxiv2024

Shroom3-Rock interaction and profibrotic function: Resolving mechanism of an intronic CKD risk allele.

Common intronic enhancer SNPs in Shroom3 associate with CKD in GWAS, although there is paucity of detailed mechanism. Previously, we reported a role for Shroom3 in mediating crosstalk between TGF{beta}1- & Wnt/Ctnnb1 pathways promoting renal fibrosis (TIF). However, beneficial roles for Shroom3 in proteinuria have also been reported suggesting pleiotropic effects. Here we focused on identifying the specific profibrotic Shroom3 motif. Given known therapeutic roles for Rho-kinase inhibitors in experimental CKD, and the established interaction between Shroom3 and Rock via its ASD2 domain, we hypothesized that Shroom3-mediated ROCK activation played a crucial role in its profibrotic function in high expressors. To test this hypothesis, we developed transgenic mice and cell lines that inducibly overexpressed wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2{Delta}-Sh3). Prior scRNAseq data showed that during TIF, Shroom3 and Rock co-expression occurred in injured tubular cells and fibroblasts, highlighting cell-types where this mechanism could be involved. Using HEK293T cells, we first confirmed absent ROCK binding and inhibited TGF{beta}1-signaling with ASD2{Delta}-Sh3-overexpression vs WT-Sh3. In mIMCD cells, ASD2{Delta}-Sh3 overexpression, reduced Rock activation (phospho-MYPT1), pro-fibrotic and pro-inflammatory transcripts vs WT-Sh3. Fibroblast proliferation (3T3) was also reduced with ASD2{Delta}-Sh3. In vivo, we studied ureteric obstruction (UUO) and Aristolochic nephropathy (AAN) as TIF models. In AAN, inducible global-, or Pan-tubular specific-, WTSh3-overexpression showed increased azotemia, and TIF vs ASD2{Delta}-Sh3 mice. WT-Sh3 mice consistently showed significant enrichment of Rho-GTPase, TGF{beta}1- and Wnt/CtnnB1- signaling in kidney transcriptome, paralleling Shroom3-coexpressed genes in tubulo-interstitial transcriptomes from human CKD. In UUO, again WT-Sh3 mice recapitulated increased fibrosis vs ASD2{Delta}-Sh3. Importantly, ASD2{Delta}-Sh3 did not develop albuminuria vs WT-Sh3, while mutating a disparate Fyn-binding Shroom3 motif induced albuminuria in mice, suggesting motif-specific roles for Shroom3 in the kidney. Hence, our data show a critical role for the Rock-binding, ASD2-domain in mediating TIF in milieu of Shroom3 excess, with relevance to human CKD.

systems biology↗

bioRxiv2024

An immunomechanical checkpoint PYK2 governs monocyte-to-macrophage differentiation in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic, stiff tumor microenvironment (TME), where tumor-associated macrophages (TAMs) drive ECM remodeling, progression, and immune evasion. The contribution of mechanical cues to monocyte differentiation into TAMs remains largely unexplored. Here we show that mechanical force is required for monocyte-to-macrophage differentiation. PYK2, as an innovative immunomechanical checkpoint, de facto governs this differentiation process. We demonstrated that PYK2 senses mechanical signals via Piezo1 and integrins, triggering F-actin polymerization and translocating to the nucleus to regulate mechanotransduction and differentiation genes (e.g., ACTR3, RELA). Targeted deletion of PYK2 impairs the differentiation and polarization of monocyte-derived macrophages, reshapes the PDAC microenvironment, and enhances the efficacy of anti-PD-1 immunotherapy. These findings underscore the critical role of mechanical cues in monocyte differentiation and suggest that targeting PYK2 is a promising strategy to modulate TAM function and improve immunotherapy outcomes in patients with PDAC. Statement of significanceThis study identifies PYK2 as an immunomechanical checkpoint that drives monocyte-to-macrophage differentiation in PDAC via Piezo1/integrin-mediated mechanical cues. Targeted deletion of PYK2 reshapes the PDAC microenvironment, and enhances the efficacy of anti-PD-1 immunotherapy, suggesting PYK2 as a promising therapeutic target to overcome immunotherapy resistance.

immunology↗