Hypoxia-inducible factor pathway activation promotes bone-type collagen cross-linking in Idiopathic Pulmonary Fibrosis

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.