Hedgehog interacting protein (HHIP) represses airway remodeling and metabolic reprogramming in COPD-derived airway smooth muscle cells

Abstract

Although HHIP locus has been consistently associated with the susceptibility to COPD including airway remodeling and emphysema in genome-wide association studies, the molecular mechanism underlying this genetic association remains incompletely understood. By utilizing Hhip^+/- mice and primary human airway smooth muscle cells (ASMCs), here we aim to determine whether HHIP haploinsufficiency increases airway smooth muscle mass by reprogramming glucose metabolism, thus contributing to airway remodeling in COPD pathogenesis. The mRNA levels of HHIP were compared in normal and COPD-derived ASMCs. Mitochondrial oxygen consumption rate and lactate levels in the medium were measured in COPD-derived ASMCs with or without HHIP overexpression as readouts of glucose oxidative phosphorylation and aerobic glycolysis rates. The proliferation rate was measured in healthy and COPD-derived ASMCs treated with or without 2-DG. Smooth muscle mass around airways was measured by immunofluorescence staining for α-smooth muscle actin (α-SMA) in lung sections from Hhip^+/- mice and their wild type littermates, Hhip^+/+ mice. Airway remodeling was assessed in Hhip^+/- and Hhip^+/- mice exposed to 6 months of cigarette smoke. Our results show HHIP inhibited aerobic glycolysis and represses cell proliferation in COPD-derived ASMCs. Notably, knockdown of HHIP in normal ASMCs increased PKM2 activity. Importantly, Hhip^+/- mice demonstrated increased airway remodeling and increased intensity of α-SMA staining around airways compared to Hhip^+/+ mice. In conclusion, our findings suggest that HHIP represses aerobic glycolysis and ASMCs hyperplasia, which may contribute to the increased airway remodeling in Hhip^+/- mice.

Figure 1

COPD-derived ASMCs demonstrated decreased oxygen consumption rate (OCR), increased lactate production, and increased sensitivity to glycolysis inhibition as well as its associated growth inhibition. (A,B) Oxygen consumption rate (OCR) in ASMCs from a healthy individual and COPD patient measured by Seahorse Mitostress assay. (C) Relative levels of lactate in the culture medium from healthy ASMCs (N = 4) and COPD-derived ASMCs (N = 3) normalized to DNA content in corresponding wells. (D) Relative levels of lactate were measured in the culture medium of normal ASMCs (N = 4) and COPD-derived ASMCs (N = 3) treated with 2-Deoxy-D-glucose (2-DG) at various concentrations (0-2 mM) for 24 h. Relative cell growth curve of normal ASMCs (N = 4) (E) or COPD-derived ASMCs (N = 3) (F) with or without 2-DG treatment (0.5 mM). *P < 0.05, **P < 0.01 by unpaired t-test. Means ± SEM from biological or technical repeats shown for each group.

Figure 2

HHIP repressed proliferation and glycolytic metabolic reprogramming of COPD-derived ASMCs. (A) The HHIP mRNA levels were measured in ASMCs from healthy individuals (N = 4) and COPD patients (N = 4) by RT-qPCR. Red dots indicate data from female subjects, and blue dots indicate data from male subjects. (B–F) COPD-derived ASMCs were transfected with either HHIP overexpressing plasmid or empty vector, followed by measurements of (B) Lactate levels, (C) proliferation rates, (D) basal OCR, (E) HHIP mRNA levels, and (F) HHIP protein levels. *P < 0.05, **P < 0.01 by unpaired t-test. Means ± SEM from 3–4 biological and technical repeats are shown for each group.

Figure 3

PKM2 activity is essential for metabolic reprogramming towards aerobic glycolysis in COPD-derived ASMCs. (A) Pyruvate kinase activity was measured in normal ASMCs with si-RNA mediated HHIP and/or PKM2 knockdown with normalization to protein concentration. (B) The PKM2 expression levels were examined by Western blot after HHIP knockdown in normal ASMCs (Left panel) or HHIP overexpression in COPD-derived ASMCs (Right panel). (C) Lactate levels in the culture medium were measured in COPD-derived ASMCs transfected with si-Ctrl or si-PKM2. (D) Basal OCR was measured in COPD-derived ASMCs transfected with si-Ctrl or siHHIP and/or si-PKM2. (E)The knockdown efficiency of si-PKM2 was shown by Western blotting. si-Ctrl: control siRNA; si-PKM2, PKM2 specific siRNA pool. (F) The knockdown efficiency of si-HHIP was shown by RT-PCR results. *P < 0.05, **P < 0.01 by unpaired t-test. Means ± SEM from 3 independent experiments shown for each group.

Figure 4

Hhip is expressed in ASMCs (airway lung) as indicated by LacZ staining in Hhip^+/- mice. A representative image of histology of lung sections from unchallenged Hhip^+/- mice at 2 months of age. LacZ staining (left panel), α-smooth muscle actin staining (middle panel), and DAPI staining (right panel) are demonstrated. Scale bars, 100 μm. Arrows indicate the localization of Hhip in the lungs. Arrowheads indicate vascular smooth muscle cells.

Figure 5

Immunostaining and lung function measurement in Hhip^+/- (HET) and Hhip^+/+ (WT) mice. (A) Representative immunofluorescence staining of α-smooth muscle actin (SMA) in lung sections from Hhip^+/- (HET) vs. Hhip^+/+ (WT) at the age of 8 months (Scale bars, 100 μm). Green color, α-SMA staining; Blue color, DAPI staining. Magnified figures from areas indicated by arrowheads are shown at the top right corner (Scale bars, 10 μm). (B) Quantification on α-SMA staining intensity around airways (N = 10–12 airways) in mice from each group (4–5 mice per group). Airways around 300–699 μm in diameter were chosen for quantification. Mean ± SEM are from airways (N = 10–12 airways) in each group. Lung mechanics including (C) Respiratory system resistance (Rrs) and (D) Newtonian resistance were measured in WT (Hhip^+/+) and HET (Hhip^+/-) mice at the age of 8 months (N = 14 vs. 19 mice). *P < 0.05 by Mann–Whitney test. (E) Trichrome staining in lung sections from 8-month-old female Hhip^+/+ and Hhip^+/− mice with or without 6 months of CS exposure. (F) Quantification of Trichrome staining in murine airways (N = 10–12 airways) from each group (5–7 mice per group). Mean ± SEM are from airways (N = 10–12 airways) per group. Scale bars, 100 μm. WT, Hhip^+/+ mice; HET: Hhip^+/- mice; CS, cigarette smoke. *P < 0.05 by one-way ANOVA. (Diameter of the airways assessed is 300–600 μm).