USP13 Deficiency Aggravates Cigarette-smoke-induced Alveolar Space Enlargement

Abstract

Alveolar enlargement is a pathological feature of emphysema. Long-term exposure to cigarette smoke (CS) is a high-risk factor for the development of emphysema. Abnormal protein ubiquitination has been implicated to regulate the development of human disorders, however, the role of protein ubiquitination in emphysema has not been well-studied. In this study, we attempted to investigate if a deubiquitinase, USP13, regulates the development of emphysema. Under a mild CS exposure condition, USP13-deficient mice show significant increases in alveolar chord length, indicating that USP13-deficient mice are susceptible to CS-induced alveolar enlargement. It has been shown that USP13 knockout reduced fibronectin expression in lungs. Here, we found that collagen levels were reduced in USP13 siRNA-transfected lung fibroblast cells. This suggests that a loss of extracellular matrix in connective tissues contributes to alveolar enlargement in USP13-deficient mice in response to CS exposure. Further, we investigated the role of USP13 in the expression of oxidative stress markers TXNIP and HMOX1. An increase in HMOX1 abundance was observed in USP13 knockdown lung fibroblast and epithelial cells. Overexpression of USP13 reduced HMOX1 protein levels in lung fibroblast cells, suggesting that modulation of USP13 levels may affect oxidative stress. Knockdown of USP13 significantly reduced TXNIP levels in lungs or lung fibroblast cells. A protein stability pulse-chase assay showed that TXNIP is instable within USP13 knockdown lung fibroblast cells. Further, the reduction of TXNIP was observed in USP13 inhibitor-treated lung epithelial cells. USP13-deficient mice also show higher levels of IgG in bronchoalveolar lavage fluid. This study provides evidence showing that USP13 deficiency plays a role in alveolar space enlargement.

Fig. 1

USP13 knockout mice exhibit increased alveolar spaces in response to cigarette smoke (CS) exposure. A Male C57BL/6 wild-type mice were exposed to four non-filtered cigarettes, 5 days per week, for a total of 6 months. Lung tissue lysates were analyzed with γ-H2AX, Elastin, and β-actin immunoblotting. B Intensities of γ-H2AX and Elastin immunoblots were analyzed by Image J software (n = 3–4). C Male C57BL/6 wild type and USP13KO mice were exposed to four non-filtered cigarettes, 5 days per week, for a total of 6 months. The lungs were fixed in formalin and then embedded in paraffin. Serial midsagittal sections were obtained and stained with modified Gill’s stain. D Mean alveolar chord length was measured using Scion Image software (n = 5–6)

Fig. 2

USP13 regulates HMOX1 levels in lung fibroblast and epithelial cells. A Male C57BL/6 wild type were exposed to four non-filtered cigarettes, 5 days per week, for a total of 6 months. Lung tissue lysates were analyzed with HMOX1 and β-actin immunoblotting. HMOX1 intensities were analyzed by Image J software (n = 3–4). B Lung fibroblast Mrc5 cells were transfected with control siRNA or USP13 siRNA for 1 day, followed by TGF-β1 treatment for an additional 1–3 days. USP13, HMOX1, and β-actin were analyzed by immunoblotting. C Mrc5 cells were transfected with USP13-V5 plasmid for 48 h, cell lysates were analyzed by V5, HMOX1, and β-actin immunoblotting. A549 cells (D) and BEAS-2B cells (E) were transfected with control siRNA or USP13 siRNA for 3 days, and then cell lysates were analyzed by HMOX1, USP13, and β-actin immunoblotting

Fig. 3

TXNIP levels are regulated by USP13 in lung cells. A TXNIP and β-actin levels in lung tissues from C57BL/6 and USP13KO mice were analyzed by immunoblotting. B Mrc5 were transfected with control siRNA and USP13 siRNA for 3 days, followed by cycloheximide (CHX) treatment for an additional 0–24 h. Collagen, TXNIP, USP13, and β-actin levels were analyzed by immunoblotting

Fig. 4

Spautin-1 decreases TNXIP levels. Lung epithelial cells (MLE12) were treated with USP13 inhibitor Spautin-1 for 24 h. TXNIP and β-actin levels were analyzed by immunoblotting. TXNIP intensities were analyzed by Image J software (n = 3)

Fig. 5

IgG levels are increased in USP13KO mice. Mouse IgG levels in lung tissues from C57BL/6 and USP13KO mice were analyzed by immunoblotting. IgG heavy chain intensities were analyzed by Image J software (n = 4)