Protein profiling of cultured primary alveolar epithelial type II cells reveals a rapid loss of the alveolar phenotype

The alveolar epithelium is organized mainly by two cell types, alveolar epithelial type I (AECI) and type II cells (AECII). AECII are the main cell type involved in regeneration of the alveolar epithelium, largely via proliferation and trans(differentiation) into AECI. In vitro, defining the AECI and AECII phenotype remains a challenging task, although primary AECII have been proposed to (trans)differentiate into AECI-like cells over time. Using 2D-gel electrophoresis and mass spectrometry, we characterized protein expression profiles of primary mouse AEC cultured in the presence/absence of fibronectin (FN) over time. Proteins that exhibited a differential expression pattern included lung carbonyl reductase 2 and peroxiredoxin 6, which were downregulated with time. In contrast, keratin 8, 19, and heat shock proteins 47 and 70 were upregulated with time. The expression of calreticulin and annexin 1 and 2 was independent of culture time. These data were confirmed by quantitative (q)RT-PCR and immunofluorescence analysis. In addition, qRT-PCR detected a rapid loss of the AECI and II markers surfactant protein A, B, C, TTF-1, and aquaporine V, whereas mRNA levels of E-cadherin, ZO-1, and T1alpha remained stable during culture. Interestingly, the mRNA and protein expression of these markers was independent of the presence of FN coating. Our proteomic study thus uncovered novel regulatory proteins during primary culture of AEC. While the mRNA expression of well-known AEC markers (independent of AECI or II) is lost upon culture, irrespective of fibronectin, structural epithelial proteins as well as newly identified proteins were maintained with time. This suggests that primary culture conditions lead to a loss of the alveolar, but not a general, epithelial phenotype.