Involvement of Creatine Kinase B in Cigarette Smoke–Induced Bronchial Epithelial Cell Senescence

Abstract

Cigarette smoke induces damage to proteins and organelles by oxidative stress, resulting in accelerated epithelial cell senescence in the lung, which is implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Although the detailed molecular mechanisms are not fully understood, cellular energy status is one of the most crucial determinants for cell senescence. Creatine kinase (CK) is a constitutive enzyme, playing regulatory roles in energy homeostasis of cells. Among two isozymes, brain-type CK (CKB) is the predominant CK in lung tissue. In this study, we investigated the role of CKB in cigarette smoke extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBECs). Primary HBECs and Beas2B cells were used. Protein carbonylation was evaluated as a marker of oxidative protein damage. Cellular senescence was evaluated by senescence-associated β-galactosidase staining. CKB inhibition was examined by small interfering RNA and cyclocreatine. Secretion of IL-8, a hallmark of senescence-associated secretary phenotype, was measured by ELISA. CKB expression levels were reduced in HBECs from patients with COPD compared with that of HBECs from nonsmokers. CSE induced carbonylation of CKB and subsequently decreased CKB protein levels, which was reversed by a proteasome inhibitor. CKB inhibition alone induced cell senescence, and further enhanced CSE-induced cell senescence and IL-8 secretion. CSE-induced oxidation of CKB is a trigger for proteasomal degradation. Concomitant loss of enzymatic activity regulating energy homeostasis may lead to the acceleration of bronchial epithelial cell senescence, which is implicated in the pathogenesis of COPD.

Autophagy Induction by SIRT6 through Attenuation of Insulin-like Growth Factor Signaling Is Involved in the Regulation of Human Bronchial Epithelial Cell Senescence

“…Hence, mapping the molecular mechanisms for CS-induced acceleration of cellular senescence may offer clues into COPD pathogenesis. Although the regulatory mechanisms for cellular senescence are complex and incompletely understood, recent advances, including our findings, support the notion that a homeostatic balance of energy status and removal of damaged intracellular components through autophagic degradation are critical for prevention of cellular senescence induced by CS exposure (4)(5)(6).…”

“…Increased cellular senescence is a major feature of aging and has been widely implicated in the pathogenesis of COPD by impairing cell repopulation and by aberrant cytokine secretion, the so-called senescence-associated secretory phenotype (3)(4)(5). Hence, mapping the molecular mechanisms for CS-induced acceleration of cellular senescence may offer clues into COPD pathogenesis.…”

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

“…7,45 PARK2 immunostaining were assessed by counting total and positively staining cells in small airways at a magnification of £400 and percentage of positive staining was scored in a semi-quantitative fashion; 0 (less than 10%), 1 (11 to 49%), and 2 (more than 50%). Immunofluorescence staining was also performed as previously described.…”

“…Immunofluorescence staining was also performed as previously described. 7,45 BEAS-2B cells expressing EGFP-LC3B grown on 8-well culture slides were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.03% Triton X-100 (Wako, 160-24751) for 60 min. After blocking with 0.1% BSA (SigmaAldrich, A2153) for 60 min, the primary and secondary antibodies were applied according to the manufacturer's instructions.…”

“…[1][2][3] Recent advances COPD have implicated acceleration of cell senescence in both alveolar and airway epithelial cells, which is functionally characterized by impaired cell regeneration and aberrant cytokine secretion of the senescence-associated secretory phenotype (SASP), in COPD pathogenesis. 2,[4][5][6][7][8][9] Although the molecular mechanism underlying cell senescence regulation is still unknown, reactive oxygen species (ROS) released during mitochondrial respiration has been generally implicated in the progression of cellular senescence. 3,10 Mitochondrial respiratory chain dysfunction accompanying enhanced ROS production can be attributed to cigarette smoke (CS) exposure, a main cause for COPD development.…”