(30 lines): Dysregulated pulmonary inflammation is a hallmark of several diseases, such as ARDS, sepsis, and pneumonia. Inflammation causes ineffective alveolar gas-exchange, hypoxemia, and thrombocytosis, which can lead to cardiac dysfunction and pulmonary failure. Interventions for patients with pulmonary inflammation are limited, and ARDS patients suffer 40% mortality. Further, the underlying mechanisms regulating the propagation of inflammation remain unclear. Thus, there is an unmet need for research into inflammatory pathways to help promote drug development. Inflammation in the lung is driven by cytokine signaling pathways such as the NF-?B pathway, which amplifies proinflammatory cytokine release. These cytokines facilitate the recruitment of immune cells, contributing to capillary damage, the disruption of pulmonary barrier function, and alveolar damage. Several protein families repress this signaling, such as the suppressor of cytokine signaling (SOCS) proteins. SOCS2 has been shown to suppress the NF-?B pathway, and deletion of SOCS2 in mouse models causes uncontrolled cytokine storm. Despite these studies, there is little research into the cellular regulation of this potent anti-inflammatory protein?s stability. A main regulation of protein stability is the process of ubiquitination. The selective tagging of proteins with ubiquitin functions to fate substrates for degradation, often via the proteasome. Ubiquitin E3 ligase proteins are essential to this process, and several groups have shown the pathological association between lung disease and E3 ligase activity. Preliminary data suggested that the E3 ligase KIAA0317 potently regulates SOCS2 stability, leading to enhanced NF-?B signaling. The central hypothesis is that KIAA0317 degrades SOCS2 protein via ubiquitination, enhancing pro- inflammatory NF-?B signaling. The role of ubiquitin and KIAA0317 in SOCS2 degradation will be investigated, along with the biological consequences to cytokine signaling and pulmonary inflammation and injury.
Aim 1 will determine if SOCS2 is ubiquitinated and degraded through the proteasome, and the mechanisms of KIAA0317 targeting.
Aim 2 will probe the biological effect of KIAA0317 regulation of SOCS2 through in vitro promoter assays, cell models of cytokine release, and RNAi. Additionally, the novel Kiaa0317 KO mouse line will be used to investigate potential resistance to experimental pulmonary inflammation induced by LPS. Further, KIAA0317 will be expressed in KO mice, and SOCS2 will be silenced, and responses will be measured. Endpoints for these studies will include markers of pulmonary inflammation such as protein, cytokine, and cell concentrations in bronchoalveolar lavage fluid, and histological analysis of immune infiltrates. Finally, aim 3 will characterize and test the effect of the KIAA0317 small-molecule inhibitor BC-1365 in vitro and in vivo.
The aims of this study will lay the groundwork for the development of novel small-molecules against KIAA0317 to lessen deleterious pulmonary inflammation.
Dysregulated inflammation is the cause of several lung diseases such as ARDS, and a major cause of morbidity and mortality in critically-ill patients. In this proposal, we will study how the deleterious E3 ligase KIAA0317 degrades the protective anti-inflammatory protein SOCS2 in injured lung tissue, and attempt to pharmacologically inhibit this pathway. Understanding of the mechanism by which KIAA0317 degrades SOCS2 will help to lead future drug discovery efforts toward an effective therapeutic for lung inflammation and injury.
