Prolyl-3-hydroxylase 4 (P3H4), a novel player in collagen synthesis and secretion in lung fibroblasts

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterized by fibroblast activation and accumulation of collagen and other extracellular matrix (ECM) components. We have previously proposed the collagen chaperone FK506-binding protein 10 (FKBP10) as a potential therapeutic target for IPF. The genes encoding FKBP10 and prolyl-3-hydroxylase 4 (P3H4), a novel component of the ER-resident collagen folding complex in skin and bone, are controlled by a common bidirectional promoter. Regulation and function of P3H4 in lung fibrosis, however, is unknown. Objective: To assess regulation and function of P3H4 in the lung and in an in vitro model of lung fibrosis. Methods: Functional analysis was carried out in an established in vitro model of lung fibrosis where primary human lung fibroblasts (phLF) are treated with transforming growth factor-β1 (TGF-β1) in presence of 2-phosphoascorbate. Following knockdown of FKBP10 or P3H4 in phLF, gene expression of profibrotic markers and total collagen secretion was assessed using qRT-PCR, immunoblotting, and Sircol assay. We performed transcriptomic and proteomic analysis of embryonic lungs from FKBP10 knockout mice and littermate controls. Results: TGF-β1 induced P3H4 expression in phLF. Similar to knockdown of FKBP10, knockdown of P3H4 in phLF significantly decreased synthesis and secretion of collagen. Interestingly, knockdown of P3H4 resulted in downregulation of FKBP10 and vice versa. Also transcriptomic and proteomic analysis of FKBP10 knockout mouse lungs revealed downregulation of P3H4. Measurements and Main Results: P3H4 is coregulated with FKBP10 and represents a novel modulator of collagen biosynthesis in lung fibroblasts.