A74-30 The Hippo Pathway Component Amotl2 Impacts Lung Epithelial Cell Reprogramming in Pulmonary Fibrosis

Rationale Idiopathic pulmonary fibrosis (IPF) is a fatal, age-related lung disease with few treatments, marked by excess ECM, tissue stiffening, and alveolar loss from failed stem cell differentiation. In IPF, alveolar type 2 (AT2) cells struggle to regenerate properly, contributing to disease progression. Single-cell RNA sequencing identified abnormal AT2 cells in IPF, called basaloid cells, and similar alveolar differentiation intermediate (ADI) cells in mouse models. We and others have recently demonstrated that the Hippo pathway effector protein and mechanotransducer Yes-associated protein 1 (YAP) is upregulated in AT2 cells in IPF and contributes to ADI cells and ECM crosslinking. The mechanisms that lead to YAP upregulation in fibrosis, however, are largely unknown. Here, we focus on the YAP regulator, AMOTL2 (a scaffolding protein called angiomotin-like protein 2) and hypothesize that AMOTL2 downregulation in AT2 cells leads to AT2 cell reprogramming. Methods We examined localization of AMOTL2 in human lung tissue from healthy and IPF donors (n = 5/group -IPF vs control) via immunofluorescent (IF) staining of AMOTL2 alongside specific AT2 cell-markers. We additionally assessed AMOTL2 expression and localization in MLE12 cells and primary mouse AT2 cells as well as whole lung tissue from bleomycin-treated mice compared to control (n = 4-8 each group). Using primary mouse AT2 cell cultures, we temporally defined AMOTL2 expression and their ADI phenotypes using RT-qPCR and Western blotting. We performed AMOTL2 loss-of-function studies in MLE12 and AT2 cells using AMOTL2 siRNA and analyzed fibrotic and ADI phenotypes using RT-qPCR and Western Blotting (n = 4). Results We found decreased expression of AMOTL2 in AT2 cells from IPF patients and experimental mouse models of lung fibrosis compared to their respective controls. We see an increased expression of ADI genes in AT2 cells from bleomycin treated mice, such as Claudin 4 and Keratin 8, and fibrosis-related genes such as Col1a1, confirming the presence of a fibrotic-ADI phenotype. Our in vitro knockdown results show successful inhibition of AMOTL2 expression accompanied by an increased expression of ADI marker Keratin 8 and Claudin 4. Conclusion AMOTL2 reduction leads to AT2 cell reprogramming and thus might contribute to the development and progression of pulmonary fibrosis. This abstract is funded by: Three Lakes Foundation, R