Silica is a fibrogenic agent capable of inducing fibroblast proliferation and excess collagen production, causing a spectrum of pulmonary diseases known collectively as lung fibrosis or silicosis. The key event in the genesis of silicosis is the interaction of the silica particle with alveolar macrophages (AM). AMs from silicotic patients release fibrogenic factors and cytokines, including TNF-alpha, IL-1 alpha and IL-1 beta. In vitro, and probably in vivo, treatment of AMs with highly fibrogenic silica induces apoptosis whereas treatment with poorly fibrogenic silica does not. The principal investigator hypothesizes that this apoptosis depends on activation of the JNK/SAPK signaling pathway, mediated in part by proinflammatory cytokines including IL-1 alpha and IL-1 beta, and that activated JNK phosphorylates transcription factors leading to the induction of apoptosis. The investigator further hypothesizes that defining and inhibiting the apoptotic signaling pathway may provide new therapeutic strategies against silica-induced lung injury. Her three specific aims test this hypothesis in cultured monocytic cell lines, human peripheral blood monocytes, AM from human BAL, paraffin-embedded human lung tissue and normal and knockout mice.
Specific aim 1 will explore the role of the JNK pathway in silica-induced apoptosis through in vitro studies. The investigator will first determine the kinetics of silica-induced JNK activation and apoptosis in vitro; and explore the roles of the proinflammatory cytokines IL-1 beta and TNF-alpha in these responses. The relationship of JNK activation to apoptosis will be studied in transfectants where JNK activation is inhibited by the introduction of dominant-negative upstream signaling molecules (MEKK1, SEK, JNK) or potentially activated by overexpression of the PAK-like kinase, HPK1.
Specific aim 2 will investigate the association of apoptosis with silicosis in vivo through studies of paraffin sections of lung from silicotic patients and through further studies in the normal and IL-1 beta KO mice.
Specific aim 3 will look for inhibitors of cysteine proteases and scavenger receptors that can block JNK activation and apoptosis in vitro. Successful compounds will be tested in vivo in mice for their ability to attenuate or reverse silica-induced lung injury.
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