Mechanism of Gzma-mediated GEF-H1 activation in intestinal epithelial cells leading to intestinal barrier dysfunction in sepsis

Abstract

Background: Sepsis-induced intestinal injury is a severe complication associated with dysfunction affecting multiple organ systems and a significantly elevated risk of death. Intestinal barrier dysfunction plays a central role, but the underlying molecular pathways remain incompletely understood. The present study sought to explore how the Gzma/GEF-H1/RhoA signalling axis contributes to the disruption of the intestinal epithelial barrier in sepsis.

Methods: Transcriptomic data, clinical samples, and a murine caecal ligation and puncture (CLP) model was used to assess Gzma expression and its correlation with disease severity. We investigated how Gzma—released by activated immune cells—affects epithelial structure and function using in vitro co-culture assays. These experiments assessed key tight junction proteins (occludin, claudin-1, ZO-1, E-cadherin), transepithelial electrical resistance (TEER), and paracellular permeability. GEF-H1 knockout mice and the GEF-H1 activator plinabulin were employed to evaluate the physiological roles of GEF-H1. Mutagenesis revealed how Gzma activates GEF-H1. High-throughput screening identified a GEF-H1 modulator, and its efficacy was validated in septic mice. Gzma expression was significantly elevated during sepsis and correlated with disease severity. Gzma secretion from immune cells impaired the epithelial barrier by downregulating tight junction proteins, increasing permeability, and reducing TEER. Gzma activates GEF-H1 by dephosphorylating Ser886, triggering the RhoA/ROCK pathway and subsequent phosphorylation of MLC2, LIMK, and cofilin—driving cytoskeletal remodelling. GEF-H1 knockout mice showed reduced intestinal injury, higher survival rates, and intact barrier function; conversely, GEF-H1 activation worsened intestinal damage. High-throughput screening identified Epothilone A as a potent GEF-H1 modulator that restores intestinal barrier integrity and improves survival in murine sepsis by suppressing the GEF-H1/librariesRhoA pathway.

Conclusion: This research uncovers the Gzma/GEF-H1/RhoA signalling axis as a pivotal contributor to intestinal barrier dysfunction during sepsis. GEF-H1 represents a promising therapeutic target, and its inhibition by agents such as Epothilone A may offer a novel strategy for treating sepsis.

Key points:

Keywords

Epothilone A / Epithelial integrity / Gzma (granzyme A) / GEF-H1 / Intestinal barrier dysfunction / RhoA/ROCK signalling / Sepsis

References

[1] Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet (London, England). 2020; 395(10219): 200-211.

[2] Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 2016; 315(8): 801-810.

[3] Meyer NJ, Prescott HC. Sepsis and septic shock. N Eng J Med. 2024; 391(22): 2133-2146.

[4] Reintam Blaser A, Preiser JC, Fruhwald S, et al. Gastrointestinal dysfunction in the critically ill: a systematic scoping review and research agenda proposed by the section of metabolism, endocrinology and nutrition of the European Society of Intensive Care Medicine. Crit Care. 2020; 24(1): 224.

[5] Hu B, Sun R, Wu A, et al. Severity of acute gastrointestinal injury grade is a predictor of all-cause mortality in critically ill patients: a multicenter, prospective, observational study. Crit Care. 2017; 21(1): 188.

[6] Tulkens J, Vergauwen G, Van Deun J, et al. Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. Gut. 2020; 69(1): 191-193.

[7] Bros M, Haas K, Moll L, Grabbe S. RhoA as a key regulator of innate and adaptive immunity. Cells. 2019; 8(7): 733.

[8] Li Z, Gao M, Yang B, et al. Naringin attenuates MLC phosphorylation and NF-κB activation to protect sepsis-induced intestinal injury via RhoA/ROCK pathway. Biomed Pharmacother. 2018; 103: 50-58.

[9] Kunimura K, Miki S, Takashima M, Suzuki JI. S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway. Cell Commun Signal. 2021; 19(1): 17.

[10] Lin Z, Ni C, Jiang H, et al. Guanine nucleotide exchange factors and small GTPases: their regulation and functions, diseases, and therapeutic targets. MedComm. 2025; 6(10):e70362.

[11] Birkenfeld J, Nalbant P, Bohl BP, Pertz O, Hahn KM, Bokoch GM. GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases. Dev Cell. 2007; 12(5): 699-712.

[12] Yamahashi Y, Saito Y, Murata-Kamiya N, Hatakeyama M. Polarity-regulating kinase partitioning-defective 1b (PAR1b) phosphorylates guanine nucleotide exchange factor H1 (GEF-H1) to regulate RhoA-dependent actin cytoskeletal reorganization. J Biol Chem. 2011; 286(52): 44576-44584.

[13] Zenke FT, Krendel M, DerMardirossian C, King CC, Bohl BP, Bokoch GM. p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor. J Biol Chem. 2004; 279(18): 18392-18400.

[14] Chang YC, Nalbant P, Birkenfeld J, Chang ZF, Bokoch GM. GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA. Mol Biol Cell. 2008; 19(5): 2147-2153.

[15] Krendel M, Zenke FT, Bokoch GM. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat Cell Biol. 2002; 4(4): 294-301.

[16] Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004; 4(4): 253-265.

[17] Nogales E, Whittaker M, Milligan RA, Downing KH. High-resolution model of the microtubule. Cell. 1999; 96(1): 79-88.

[18] Ravelli RB, Gigant B, Curmi PA, et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature. 2004; 428(6979): 198-202.

[19] Birkenfeld J, Nalbant P, Yoon SH, Bokoch GM. Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis? Trends Cell Biol. 2008; 18(5): 210-219.

[20] Cheng IK, Tsang BC, Lai KP, et al. GEF-H1 over-expression in hepatocellular carcinoma promotes cell motility via activation of RhoA signalling. J Pathol. 2012; 228(4): 575-585.

[21] Pan W, Nagpal K, Suárez-Fueyo A, et al. The regulatory subunit PPP2R2A of PP2A enhances Th1 and Th17 differentiation through activation of the GEF-H1/RhoA/ROCK signaling pathway. J Immunol (Baltimore, MD: 1950). 2021; 206(8): 1719-1728.

[22] Martinez J, Rodriguez Hovnanian KM, Martinez EE. Biomarkers and functional assays of epithelial barrier disruption and gastrointestinal dysmotility in critical illness: a narrative review. Nutrients. 2023; 15(18): 4052.

[23] Deruelle V, Bouillot S, Job V, et al. The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis. Nat Commun. 2021; 12(1): 4024.

[24] Villalba N, Ma Y, Gahan SA, et al. Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice. J Neuroinflam. 2023; 20(1): 127.

[25] Garzón-Tituaña M, Sierra-Monzón JL, Comas L, et al. Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis. Theranostics. 2021; 11(8): 3781-3795.

[26] García-Laorden MI, Stroo I, Terpstra S, et al. Expression and function of granzymes A and B in Escherichia coli peritonitis and sepsis. Mediators Inflamm. 2017; 2017:4137563.

[27] Mills C, Hemkemeyer SA, Alimajstorovic Z, et al. Therapeutic validation of GEF-H1 using a de novo designed inhibitor in models of retinal disease. Cells. 2022; 11(11): 1733.

[28] Baghela A, Pena OM, Lee AH, et al. Predicting sepsis severity at first clinical presentation: the role of endotypes and mechanistic signatures. EBioMedicine. 2022; 75:103776.

[29] Zhou Z, He H, Wang K, et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science. 2020; 368(6494):eaaz7548.

[30] Azoitei ML, Noh J, Marston DJ, et al. Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways. J Cell Biol. 2019; 218(9): 3077-3097.

[31] Chen RH, Corbalan-Garcia S, Bar-Sagi D. The role of the PH domain in the signal-dependent membrane targeting of Sos. EMBO J. 1997; 16(6): 1351-1359.

[32] Comer S, Nagy Z, Bolado A, et al. The RhoA regulators Myo9b and GEF-H1 are targets of cyclic nucleotide-dependent kinases in platelets. J Thromb Haemost 2020; 18(11): 3002-3012.

[33] Leguay K, Kent OA. Dynamic coupling of MAPK signaling to the guanine nucleotide exchange factor GEF-H1. OncoTargets Ther. 2025; 18: 147-159.

[34] Fujishiro SH, Tanimura S, Mure S, Kashimoto Y, Watanabe K, Kohno M. ERK1/2 phosphorylate GEF-H1 to enhance its guanine nucleotide exchange activity toward RhoA. Biochem Biophys Res Commun. 2008; 368(1): 162-167.

[35] Kratzer E, Tian Y, Sarich N, et al. Oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization. Am J Respir Cell Mol Biol. 2012; 47(5): 688-697.

[36] Niu R, Lan J, Liang D, et al. GZMA suppressed GPX4-mediated ferroptosis to improve intestinal mucosal barrier function in inflammatory bowel disease. Cell Commun Signal 2024; 22(1): 474.

[37] Müller S, Lory J, Corazza N, et al. Activated CD4+ and CD8+ cytotoxic cells are present in increased numbers in the intestinal mucosa from patients with active inflammatory bowel disease. Am J Pathol. 1998; 152(1): 261-268.

[38] Santiago L, Castro M, Sanz-Pamplona R, et al. Extracellular granzyme A promotes colorectal cancer development by enhancing gut inflammation. Cell Rep. 2020; 32(1):107847.

[39] Scott JI, Cheng Z, Thompson EJ, et al. A chemiluminescence assay targeting granzyme A activity for monitoring inflammatory bowel disease. Nat Biomed Eng. 2026.

[40] Zhong X, Su Y, Zhou Z, et al. Exosite-mediated targeting of GSDMB by dimeric granzyme A in lymphocyte pyroptotic killing. Immunity. 2026; 59(2): 257-269.e6.

[41] Uranga-Murillo I, Tapia E, Garzón-Tituaña M, et al. Biological relevance of granzymes A and K during E. coli sepsis. Theranostics. 2021; 11(20): 9873-9883.

[42] Chiang HS, Zhao Y, Song JH, et al. GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses. Nat Immunol. 2014; 15(1): 63-71.

[43] Zhao Y, Zagani R, Park SM, Yoshida N, Shah P, Reinecker HC. Microbial recognition by GEF-H1 controls IKKε mediated activation of IRF5. Nat Commun. 2019; 10(1): 1349.

[44] Kashyap AS, Fernandez-Rodriguez L, Zhao Y, et al. GEF-H1 signaling upon microtubule destabilization is required for dendritic cell activation and specific anti-tumor responses. Cell Rep. 2019; 28(13): 3367-3380.e8.

[45] Balda MS, Whitney JA, Flores C, González S, Cereijido M, Matter K. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol. 1996; 134(4): 1031-1049.

[46] Michikawa H, Fujita-Yoshigaki J, Sugiya H. Enhancement of barrier function by overexpression of claudin-4 in tight junctions of submandibular gland cells. Cell Tissue Res. 2008; 334(2): 255-264.

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