Abstract Rationale Chronic obstructive pulmonary disease (COPD) is a refractory inflammatory disease caused by long-term inhalation of harmful substances such as cigarette smoke. The R-spondin (RSPO) family, consisting of RSPO1-4, acts as a potent enhancer of canonical WNT signaling by binding to LGR receptors and preventing WNT receptor degradation. WNT signaling plays a crucial role in lung tissue repair and alveolar regeneration after injury. RSPOs have been implicated in inflammatory diseases of several organs. In human lungs, RSPOs are primarily expressed in mesenchymal cells, including fibroblasts, and their expression patterns and regulation in COPD remain largely unexplored. The aim of this study was to elucidate the expression and regulatory mechanisms of RSPOs in the lungs of patients with COPD. Methods Between April 2024 and January 2025, lung fibroblasts were isolated and cultured from non-tumorous peripheral lung tissues obtained from patients undergoing surgical resection for early-stage lung cancer at the National Defense Medical College Hospital. Patients were grouped into three categories based on their smoking history, pulmonary function tests, and chest CT findings: non-smoking healthy, smoking healthy, and smoking COPD. The expression levels of RSPO1-4 in the isolated fibroblasts were analyzed using quantitative real-time PCR, western blotting and immunostaining. Additionally, cultured fibroblasts were exposed to cigarette smoke extract (CSE) or COPD-related cytokines to assess changes in RSPO expression. Results A total of 39 participants were enrolled: non-smoking healthy (n = 19), smoking healthy (n = 9), and smoking COPD (n = 9). RSPO2 mRNA expression was significantly reduced in lung fibroblasts derived from patients with COPD compared to that in non-smoking controls (p 0.05). Exposure of fibroblasts to CSE similarly resulted in decreased RSPO2 expression. Further analysis revealed that CSE-induced RSPO2 downregulation occurred through activation of the aryl hydrocarbon receptor (AhR) pathway, as demonstrated by the reversal of RSPO2 suppression upon treatment with the AhR inhibitor CH-223191 and by siRNA-mediated AhR knockdown. In addition, stimulation with TGF-β or TNF-α led to further suppression of RSPO2 expression, indicating that inflammatory cytokines contribute to this regulatory mechanism. Intracellular flow cytometry revealed that RSPO2 was expressed in PDGFRA+THY1− alveolar fibroblasts among human lung cells. Conclusions RSPO2 expression is decreased in lung fibroblasts from patients with COPD, likely due to cigarette smoke-induced AhR activation and the pro-inflammatory cytokines TGF-β and TNF-α. Given the role of RSPO in amplifying WNT signaling, its downregulation may lead to impaired WNT pathway activity, potentially contributing to defective alveolar repair and regeneration in COPD. This abstract is funded by: None
Ogawa et al. (Fri,) studied this question.