Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and coimmunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated fulllength mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser 80 , Ser 108 , and Ser 114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser 80 and Ser 108 were major phosphorylation sites. The alanine mutations of Ser 80 and Ser 108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser 80 of ARD1 was phosphorylated by PKCβI and PKCζ isotypes with the highest selectivity, while Ser 108 and/or Ser 114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.

Alveolar organoids (AOs), derived from human pluripotent stem cells (hPSCs) exhibit lung-specific functions. Therefore, the application of AOs in pulmonary disease modeling is a promising tool for understanding disease pathogenesis. However, the lack of immune cells in organoids limits the use of human AOs as models of inflammatory diseases. In this study, we generated AOs containing a functional macrophage derived from hPSCs based on human fetal lung development using biomimetic strategies. We optimized culture conditions to maintain the iMACs (induced hPSC-derived macrophages) AOs for up to 14 days. In lipopolysaccharide (LPS)-induced inflammatory conditions, IL-1β, MCP-1 and TNF-α levels were significantly increased in iMAC-AOs, which were not detected in AOs. In addition, chemotactic factor IL-8, which is produced by mononuclear phagocytic cells, was induced by LPS treatment in iMACs-AOs. iMACs-AOs can be used to understand pulmonary infectious diseases and is a useful tool in identifying the mechanism of action of therapeutic drugs in humans. Our study highlights the importance of immune cell presentation in AOs for modeling inflammatory pulmonary diseases.