TGF-ß2 decreases baseline and IL-13-stimulated mucin production by primary human bronchial epithelial cells.

INTRODUCTION: Mucus hypersecretion is a major contributor to asthma pathology and occurs as part of a spectrum of structural changes termed airway wall remodeling. Transforming growth factor (TGF)-ß is proposed to play a key role in regulating airway matrix remodeling although less is known about the specific action of TGF-ß isoforms in regulating mucus production. METHODS: Primary human bronchial epithelial (HBE) cells cultured at air-liquid interface were treated with exogenous TGF-ß(1), TGF-ß(2), and/or a Th2 cytokine, interleukin (IL)-13. Expression and production of respiratory mucins, MUC5AC and MUC5B, were analyzed by real-time PCR, agarose gel electrophoresis, and western blotting. A murine-transformed Clara cell line (mtCC1-2) transfected with a luciferase reporter driven by the Muc5ac promoter containing Smad4 site-mutated cis sequences was used to determine whether exogenous TGF-ß(2) affects Muc5ac promoter function. RESULTS: Surprisingly, TGF-ß(1) showed no measurable effect on MUC5AC or MUC5B production by HBE cells whereas TGF-ß(2) caused a decrease in both MUC5AC and MUC5B mRNA and protein. Dual treatment with TGF-ß(2) and IL-13 partially attenuated the increase in mucin production found with IL-13 alone. This effect was confirmed by using mtCC1-2 cells where addition of TGF-ß(2) reduced the ability of IL-13/EGF to induce Muc5ac promoter expression in wild-type cells; however, this decrease was absent in mutant promoter-transfected cells. DISCUSSION AND CONCLUSION: Findings suggest that normal regulation of MUC5AC and MUC5B production by HBE cells is TGF-ß isoform-specific and that TGF-ß(2) downregulates both MUC5AC and MUC5B. Furthermore, TGF-ß(2) controls baseline and IL-13-driven Muc5ac promoter function in murine Clara cells via an endogenous Smad4 recognition motif.

Detecting, visualising, and quantifying mucins.

The extreme size, extensive glycosylation, and gel-forming nature of mucins make them a challenge to work with, and methodologies for the detection of mucins must take into consideration these features to ensure that one obtains both accurate and meaningful results. In understanding and appreciating the nature of mucins, this affords the researcher a valuable toolkit which can be used to full advantage in detecting, quantifying, and visualising mucins. The employment of a combinatorial approach to mucin detection, using antibody, chemical, and lectin detection methods, allows important information to be gleaned regarding the size, extent of glycosylation, specific mucin species, and distribution of mucins within a given sample. In this chapter, the researcher is guided through considerations into the structure of mucins and how this both affects the detection of mucins and can be used to full advantage. Techniques including ELISA, dot/slot blotting, and Western blotting, use of lectins and antibodies in mucin detection on membranes as well as immunohistochemistry and immunofluorescence on both tissues and cells grown on Transwell™ inserts are described. Notes along with each section advice the researcher on best practice and describe any associated limitations of a particular technique from which the researcher can further develop a particular protocol.