The pulmonary alveolar epithelium forms the largest surface of interaction of the human body with the external environment. Injury or disruption of normal function in alveolar epithelial cells (AEC) has important consequences for pulmonary gas exchange (resulting in acute lung injury), for determining normal or aberrant lung repair following lung injury, and for pulmonary innate immunity. Granulocyte-macrophage colony stimulating factor (GM-CSF) is an endogenous pulmonary cytokine produced by normal AEC that has emerged as a key defender of the lung. GM-CSF is protective against acute lung injury induced by oxidative stress and is an anti-apoptotic factor for AEC. Insults, such as exposure to bleomycin, that result in pulmonary fibrosis are associated with decreased AEC expression of GM-CSF;fibrosis is reduced following treatment with GM-CSF. GM-CSF is essential for normal function of alveolar macrophages, critical components of the pulmonary innate immunity that are also responsible for maintenance of normal surfactant homeostasis. Oxidative stress experienced by AEC in the setting of lung injury leads to impaired AEC GM-CSF expression and increased susceptibility to lethal pneumonia. This process is reversed by treatment with GM-CSF. Despite these protective effects, GM-CSF occupies a complex niche, with suggestions that inappropriate expression may contribute to pathologic inflammatory states such as rheumatoid arthritis or COPD. Our data demonstrate that regulation of GM-CSF expression in the alveolar epithelium differs in fundamental ways from regulation of this growth factor in other cells. Thus it is essential to understand the details of regulation of this pluripotent cytokine in the alveolar epithelium. Because lung epithelial cell lines poorly replicate the pattern of expression of GM-CSF by primary AEC, it is important to study the regulation of GM-CSF expression in primary AEC and to extend these studies to the intact lung. We hypothesize that AEC GM-CSF expression is regulated both post-transcriptionally and at the level of transcription. This proposal will examine both of these components in detail. Our preliminary studies indicate that mRNA stability is an important determinant of GM-CSF expression in the setting of oxidative stress. We have identified a group of microRNAs (miRNAs) whose behavior suggests that they are candidates to participate in GM-CSF mRNA destabilization in the setting of oxidative stress and have confirmed our ability to manipulate miRNA in primary murine AEC using lentviral transduction.
Specific Aim 1 of this proposal will determine the regulatory roles of these miRNA, using a reporter construct for the GM-CSF 3'untranslated region and miRNA mimics and knockdown. We also have evidence that a zinc finger protein, ZFP36 or tristetraprolin (TTP), may contribute to GM-CSF mRNA degradation.
Specific Aim 2 will define the role of TTP in the regulation of GM-CSF mRNA stability in AEC, with particular reference to miRNA effects on TTP expression.
Specific Aim 3 will define the contribution of chromosomal accessibility and histone acetylation in determining the pattern of GM-CSF transcription in normal AEC and in the setting of injury and repair. These studies will determine critical features of the regulation of GM-CSF expression in a key cell type, the alveolar epithelial cell, and are essential for understanding th biology of GM-CSF in the contexts of defense of, and diseases of the human lung. They will offer the opportunity for targeted manipulation of endogenous GM-CSF expression within the alveolar space to promote normal repair following lung injury and restore or augment pulmonary host defense against pneumonia. Thus, they have the potential to provide new therapeutic approaches to restore alveolar epithelial integrity while limiting unintended adverse effects.
Acute lung injury causes major morbidity and mortality among Veterans. Veterans have a high incidence of conditions that are major risk factors for developing acute lung injury, including sepsis, pneumonia, alcoholism and illnesses requiring major surgical procedures. Survivors of acute lung injury commonly require long periods of acute care followed by extensive rehabilitation. Similarly, relatively large numbers of Veterans are afflicted with pulmonary fibrosis, a chronic and progressive condition that is thought to be a consequence of abnormal healing after an initial injury and for which there are very limited effective therapies. The processes that promote normal recovery after lung injury or that lead to progressive respiratory failure and disability remain poorly understood. The studies proposed in this Merit Review application will provide improved understanding of the regulation of pulmonary defense and repair mechanisms that are impaired by these processes and will identify new interventions to initiate or speed recovery.