Abstract Rationale Pulmonary fibrosis is characterized by excessive collagen deposition by activated fibroblasts, which disrupts tissue architecture and lung function. While FKBP10 has been identified as a key regulator of collagen biosynthesis in lung fibroblasts, less is known about other related proteins such as Endoplasmic reticulum (ER) protein SC65 (P3H4, LEPREL4). This study investigated the potential role of SC65 in lung fibrosis, with a focus on its interaction with FKBP10 and its involvement in collagen biosynthesis and fibroblast activation. Methods RNA and protein were isolated from lungs of FKBP10 knockout (KO) mice and wildtype (WT) littermates. Label-free mass spectrometry-based proteomics and gene expression microarray analysis were performed to identify potential downstream targets of FKBP10. SC65 expression was further examined in primary human lung fibroblasts (phLF) following siRNA-mediated FKBP10 knockdown. In vivo expression of SC65 was assessed in available single-cell RNA sequencing (scRNA-Seq) data and by immunofluorescence (IF) staining of idiopathic pulmonary fibrosis (IPF) lung samples and of mouse lung samples model of bleomycin-induced lung fibrosis. Functional assays were conducted on phLF with siRNA-mediated SC65 knockdown, assessing profibrotic markers and collagen secretion using qRT-PCR, immunoblotting, and Sircol assay. Proximity ligation assays (PLA) and IF were used to explore protein interactions. Results SC65 was identified as one of the top downregulated genes in FKBP10 KO mice, and knockdown of FKBP10 in phLF similarly downregulated SC65 expression. In IPF and in the mouse model of bleomycin-induced fibrosis, SC65 was upregulated and localized to activated interstitial fibroblasts and, interestingly, to secretory cells in the bronchial epithelium. SiRNA-mediated knockdown of SC65 in phLF reduced expression of fibroblast activation markers such as FKBP10, CTHRC1, α-SMA, fibronectin, and type I collagen, as well as decreased total collagen secretion. PLA and IF revealed colocalization of FKBP10 and SC65 in the ER of phLF. Conclusions SC65 colocalizes with FKBP10 in activated lung fibroblasts, where it regulates profibrotic gene expression and collagen secretion. This highlights SC65 as a potential drug target for fibrosis therapy. Additionally, its FKBP10-independent role in secretory cells of the bronchial epithelium, warrants further investigation This abstract is funded by: None
Onursal et al. (Fri,) studied this question.