Abstract Background Enhanced glycolysis and lactate accumulation in endothelium are hallmarks of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Emerging evidence highlights the critical role of lactylation in ALI/ARDS. Our preliminary data revealed elevated lactate as well as lactylation levels in ALI, particularly in pulmonary endothelial cells, which are highly glycolytic and central to vascular barrier dysfunction. Yet, the molecular mechanisms remain unclear. Methods Using multi-omics, we investigated Kla sites and protein levels in control or lipopolysaccharide (LPS)-induced mouse lungs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The functional relevance of specific Kla sites was assessed in vitro, ex vivo, and in vivo. We developed lung endothelium-targeted (CGSPGWVRC) inhibitory peptides against SRSF4 K95 lactylation by screening a random peptide library. Results Integrative lactylome and proteome analyses identified 944 upregulated and 196 downregulated proteins, and 1034 upregulated and 24 downregulated Kla sites on proteins in injured murine lungs, with most proteins localized to the nucleus. Lung injury triggers preferential Kla of proteins involved in (regulation of) RNA splicing and endothelial permeability, which occur largely independently of protein-level changes in the same proteins. Notably, we screened that serine/arginine-rich splicing factor 4 (SRSF4), a crucial splicing factor, is lactylated at K95 in response to ALI. Modulating site-specific Kla of SRSF4 at K95 in pulmonary endothelium (AAV-QuadYF/Tie1-SRSF4-K95R) attenuated the functional, histological and inflammatory hallmarks of LPS-induced ALI, and the platelet-activating factor (PAF)-induced vascular permeability in isolated lungs. Mechanistically, integrated multi-omics analysis (scRNA-seq, RNA-seq, and eCLIP-seq) implicated SMPD1, which encodes acid sphingomyelinase (ASM), as a downstream target of SRSF4 lactylation. SRSF4 K95 lactylation enhances its binding to SMPD1 exon splicing enhancers (ESEs), suppressing the exon skipping of SMPD1 and hence increasing the enzymatically active ASM-1 expression. SRSF4 K95 lactylation shifts SMPD1 splicing toward the active isoform, elevating ASM activity and ceramide production, ultimately exacerbating vascular barrier dysfunction. Compared to healthy controls, the alternative splicing of SMPD1 was significantly lower, yet the ASM activity was higher, in ARDS patients. We further identified the “writers” and “erasers” responsible for SRSF4 lactylation. Finally, lung endothelial-targeted peptides that specifically block K95 lactylation phenocopy the effects of SRSF4 K95 mutant (SRSF4 K95R) on double-hit induced lung injury. Conclusions Our results unveil a novel regulatory axis that lactate-induced SRSF4 K95 lactylation drives lung injury by regulating SMPD1 alternative splicing, providing new insights into the pathogenesis of ALI/ARDS. This abstract is funded by: National Natural Science Foundation of China (Grant No. 82300108, 82200090, 82241013, 82350127); China Postdoctoral Science Foundation (Grant No. 2023M730657, 2023TQ0071).
Jiang et al. (Fri,) studied this question.