TET1-Mediated DNA hydroxymethylation of KLF6 promotes LPS-induced pulmonary fibrosis by activating the S1PR3/RhoA/ROCK signaling pathway.

BACKGROUND: Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) plays crucial roles in organ injury and fibrosis, but its role and regulatory mechanism in pulmonary fibrosis remain to be elucidated. METHODS: Gene and protein expression levels were assessed using RT-qPCR, Western blot and immunohistochemistry. CCK-8 and TUNEL were used to detect cell viability and apoptosis, respectively. The DNA methylation of Kruppel-like factor 6 (KLF6) was detected by MSP. 5-hydroxymethylcytosine (5hmC) level of KLF6 was detected by the corresponding kit. ChIP assay or dual luciferase reporter gene assay was applied to detect the binding relationship between TET1 and KLF6 promoter/KLF6 and Sphingosine-1-phosphate receptor 3 (S1PR3) promoter. Histological examination, including H&E and Masson trichrome staining, was conducted to analyze lung injury and fibrosis. RESULTS: TET1 knockdown attenuated lipopolysaccharide (LPS)-induced endothelial-mesenchymal transition (EndMT) and apoptosis in HPMECs by restoring viability, reducing apoptosis, and reversing changes in endothelial and mesenchymal markers. TET1 knockdown reduced KLF6 expression via promoting DNA methylation. KLF6 overexpression reversed the effect of TET1 knockdown on EndMT and apoptosis in LPS-induced HPMECs. KLF6 knockdown inactivated RhoA/ROCK signaling and reduced EndMT of LPS-induced HPMECs cells by inhibiting S1PR3 transcription. TET1 knockdown alleviated LPS-induced pulmonary fibrosis via suppressing KLF6 and inactivating S1PR3/RhoA/ROCK. CONCLUSION: TET1 promoted LPS-induced pulmonary fibrosis by regulating KLF6 and activating the S1PR3/RhoA/ROCK signaling pathway.