Controlled immune responses provide an effective defense system to invading organisms in the lung without permanently damaging it, but, if left uncontrolled, this process often leads to dysfunction. However, the lung has evolved mechanisms to maintain lung homeostasis. The Clara cells are the non-ciliated secretory cells that line the bronchiolar epithelium of the lung and produce a small 10kDa protein, CC10. A feedback loop in which high levels of the pro-inflammatory cytokine interferon gamma, IFN-γ, can induce expression of the CC10 gene has been discovered, suggesting that CC10 is critical for regulating inflammation in the lung. Previous analysis demonstrated two pathways potentially responsible for signaling to the CC10 gene. A direct pathway, utilizing a gamma activated sequence (GAS) site at position -284 to -292, and an indirect pathway, that involved increases in the HNF-3β transcription factor which binds the CC10 promoter at two sites in the proximal promoter region. We have utilized a cell line established from mouse transformed Clara cells, the mtCC1-2 cell line, to investigate the molecular mechanisms that control this signaling. The mtCC1-2 cell line maintains expression of the CC10 protein, as well as, all the known relavant transcription factors. Using transient transfection analysis, the pathways that are activated in the Clara cell by IFN-γ were investigated.

Bacterial chloramphenicol acetyl transferase gene (CAT) reporter constructions were generated that contain 800bp, 166bp or 144bp of mouse CC10 promoter region all were induced by IFN-γ (1000 U/ml) after 20 hours in transient transfections in mtCC1-2 cells. Linker-scanner mutations were constructed within the full length 800bp construction at each of two HNF-3 sites or at the GAS site to determine the contribution of these cis-elements to CC10 promoter activity in response to IFN-γ. Transient transfections performed in the mtCC1-2 cells show that destruction of either HNF-3 site abolished promoter activity, as well as, induction by IFN-γ. Destruction of the GAS site reduced the basal levels of the promoter, however, this construction maintained the ability to be induced by IFN-γ. These results suggest that the indirect pathway, through activation by HNF-3β acting specifically on the CC10 promoter may be the major pathway that is responsible for controlling the levels of CC10 in the lung to regulate the inflammatory effects of IFN-γ. Future analyses will involve determining the factors that restrict the response to IFN-γ specifically to the CC10 promoter.