Metabolomic profiling in a Hedgehog Interacting Protein (Hhip) murine model of chronic obstructive pulmonary disease

Abstract

Genetic variants annotated to the hedgehog interacting protein (HHIP) are robustly associated with chronic obstructive pulmonary disease (COPD). Hhip haploinsufficiency in mice leads to increased susceptibility towards the development of emphysema following exposure to chronic cigarette smoke (CS). To explore the molecular pathways which contribute to increased susceptibility, we performed metabolomic profiling using high performance liquid chromatography tandem mass spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip +/− heterozygotes and wild type (Hhip +/+) C57/BL6 mice exposed to either room-air or CS for six months. Univariate comparisons between groups were made with a combined fold change ≥2 and Student’s t-test p-value < 0.05 to denote significance; associations with mean alveolar chord length (MACL), a quantitative measure of emphysema, and gene-by-environment interactions were examined using empiric Bayes-mediated linear models. Decreased urinary excretion of cotinine despite comparable plasma levels was observed in Hhip +/− heterozygotes; a strong gene-by-smoking association was also observed. Correlations between MACL and markers of oxidative stress such as urinary methionine sulfoxide were observed in Hhip +/− but not in Hhip +/+ mice. Metabolite set enrichment analyses suggest reduced antioxidant capacity and alterations in macronutrient metabolism contribute to increased susceptibility to chronic CS-induced oxidative stress in Hhip haploinsufficiency states.

Airway epithelial cell differentiation relies on deficient Hedgehog signalling in COPD

“…Firstly, not all the known elements of HH pathway were investigated. We focused on the three main transcription factors Glis and the two receptors Ptch1 and Smo but of particular interest would be also Suppressor of Fused Homolog (SuFu) which is part of a regulating complex allowing the activation of HH pathway [74]; HH Interacting Protein (HHIP) acting as a ligand receptor and associated with COPD [75], [76], [77]; Ptch2, Gas1, Cdo and Boc, all identified as HH receptorsome but not fully characterized in any physiological context [75,[78], [79], [80]]. The second limitation is the experimental approach with AB5E1: maintaining efficient silencing through si/shRNA or selecting clones with CRISPR-Cas9 would improve the robustness of the results but it would be particularly challenging in the context of lung research with primary cells originating from patients where cell differentiation is monitored during several weeks or months [81], [82], [83].…”

Participation of HHIP Gene Variants in COPD Susceptibility, Lung Function, and Serum and Sputum Protein Levels in Women Exposed to Biomass-Burning Smoke

“…In a haploinsufficient murine model exposed to cigarette smoke, the importance of the HHIP protein in lung development was demonstrated; homozygous (Hhip-/-) mice died in the short term after birth due to defects in lung morphogenesis. In contrast, heterozygous mice (Hhip+/−) were viable with normal lung development but with an approximately 33% decrease in protein expression and an increase in functional and histological emphysema [ 35 ].…”

The SNP rs13147758 in the HHIP Gene Is Associated With COPD Susceptibility, Serum, and Sputum Protein Levels in Smokers

“…Two proteins are upregulated in response to hedgehog pathway activation: the hedgehog receptor protein patched (Ptch), which is vertebrate-specific, and Hedgehog interacting protein (HHIP). Both proteins form a negative feedback loop of hedgehog signaling necessary for the development of the lungs and other organs, since decreased protein levels are related to specific lung defects that result in neonatal lethality in a murine model (Wan et al, 2017) They are also involved in the response of pulmonary epithelial cells to smoking (Van Tuyl and Post, 2000;Chuang et al, 2003;Wang et al, 2013). This pathway plays a central role in the repair and regeneration of multiple tissues (Watkins et al, 2003;Rubin and de Sauvage, 2006;Wang et al, 2013).…”

“…Experimental studies have been performed in a murine Hhip haploinsufficient model to explore smoking as a risk factor for COPD. According to Wan et al, homozygous mice (Hhip −/−) die quickly after birth due to defects in their pulmonary morphogenesis, and while heterozygous mice (Hhip ±, haploinsufficient) are viable and exhibit normal pulmonary development, they do exhibit a ∼33% decrease in Hhip protein expression (Wan et al, 2017). However, no previous studies have described protein levels in the lung microenvironment in humans.…”