Tiotropium bromide inhibits TGF-ß-induced MMP production from lung fibroblasts by interfering with Smad and MAPK pathways in vitro

Kazuhito Asano¹, Yusuke Shikama², Naruo Shoji³, Kojiro Hirano³, Harumi Suzaki³, Hiroaki Nakajima<sup>2

1</sup>Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University, Yokohama, Japan; ²Department of Respiratory Diseases, Showa University Northern Yokohamashi Hospital, Yokohama, Japan; ³Department of Otorhinolaryngology, School of Medicine, Showa University, Tokyo, Japan

Background: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and structural alterations (ie, tissue remodeling) throughout the conducting airways, parenchyma, and pulmonary vasculature. Matrix metalloproteinases (MMPs) are extracellular degrading enzymes that play a critical role in inflammatory cell infiltration and tissue ­remodeling, but the influence of the agents that are used for the treatment of COPD on the production of MMPs is not well understood.

Purpose: The present study aimed to examine the influence of tiotropium bromide hydrate (TBH) on the production of MMPs from lung fibroblasts (LFs) induced by transforming growth factor (TGF)-ß in vitro.

Methods: LFs, at a concentration of 5 × 10⁵ cells·mL^-1, were stimulated with TGF-ß in the presence of various concentrations of TBH. MMP-1 and MMP-2 levels in culture ­supernatants were examined by enzyme-linked immunosorbent assay (ELISA), and MMP messenger ribonucleic acid (mRNA) expression was examined by real-time polymerase chain reaction (RT-PCR). The influence of TBH on TGF-ß signaling pathways was also analyzed by ­examining Smad activation and signaling protein phosphorylation by ELISA.

Results: TBH at more than 15 pg·mL^-1 inhibited the production of MMP-1 and MMP-2, but not tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2, from LFs, after TGF-ß stimulation. TBH also suppressed MMP mRNA expression through the inhibition of Smad activation and signaling protein, extracellular-signal-regulated kinase (ERK) 1 and 2, and c-Jun N-terminal kinase (JNK), phosphorylation.

Conclusion: These results may suggest that TBH suppresses MMP production from LFs, through interference of TGF-ß-mediated signaling pathways and results in favorable modification of the clinical status of COPD.

Keywords: tiotropium bromide, matrix metalloproteinases, lung fibroblast, TGF-ß, inhibition, in vitro