Fibroblasts or myofibroblasts in fibroblast foci stained largely negative for PKD3. Taken together, these findings indicate that the expression of PKD family kinases is increased in bronchiolar and alveolar epithelia as well as macrophages in IPF lungs. To identify agonists that activate PKD in lung epithelial cells, we next treated primary human small airway epithelial cells and A549 alveolar cell line with various receptor ligands or stimuli and performed Western blotting analysis to assess PKD activation by using the phospho-specific PKD-pSer744/748 antibody. Interestingly, we found that PKD was predominantly activated by poly-Larginine, LPA, and thrombin through a strong phosphorylation of PKD on Ser-744/748 in both primary airway epithelial and A549 alveolar cells. LPA and thrombin are profibrotic factors and have been shown to play important roles in the pathogenesis of pulmonary fibrosis. Poly-L-arginine is a highly charged cationic NCX 466 polypeptide that is similar in structure and function to the active moiety of major basic protein secreted from eosinophils. In contrast, the phosphorylation of PKD on Ser-744/748 was only slightly increased by TNFa, EGF, FGF, interlukin-6, as well as TLR ligands and LPS) in A549 cells but not in primary airway epithelial cells. Since PKD family kinases are increased and activated in IPF bronchiolar and alveolar epithelia, we next sought to assess the effect of PKD overexpression on lung epithelial cell biology. We have recently shown that overexpression of PKD family kinases disrupts the formation of apical intercellular junctions and their reassembly, impairs the development of TEER, and increases paracellular permeability to sodium fluorescein in 16HBE14ohuman airway epithelial monolayers. As lung epithelial cells interact with proximate fibroblasts and endothelial cells, we next assessed whether PKD could also promote lung epithelial barrier dysfunction in the presence of co-cultured primary lung fibroblasts or endothelial cells. TEER reflects the paracellular and transcellular resistance and is a sensitive measure of barrier integrity. We found that TEER of control 16HBE14o- cell monolayers on the Transwell inserts was significantly increased by co-culturing with primary lung fibroblasts PF 06465469 derived from IPF lungs and normal subjects or with HPAECs in the bottom chamber. Moreover, 16HBE14o- cells overexpressing GFP-PKD3 developed a low TEER, and the TEER was increased but not reversed to the level of control GFP cells in the presence of co-cultured lung fibroblasts or HPAECs.