Actinomycin D Blocks Transcription of Surfactant B mRNA in Marrow Cells Co-Cultured with Lung

Abstract

We have previously described that RNA-filled microvesicles are shed from lung cells and are capable of entering whole bone marrow (WBM) cells in culture (Aliotta et al, Stem Cells , 2007; 25(9):2245–56). WBM cells cultured with these lung-derived microvesicles appear to express lung cell-specific genes after 7 days in culture. However, it is not known if the lung cell-specific mRNA detected in these cells represents de novo transcription or previously transcribed mRNA carried into the cell via microvesicles. To address this issue, we co-cultured WBM cells extracted from C57BL/6 mice and co-cultured them opposite lung from C57BL/6 mice exposed to 500 centigrey (cGy) of total body irradiation (TBI) or no radiation. The two cell types were separated by a cell-impermeable membrane (pore size, 0.4μm). After 7 days of co-culture, WBM cells were removed, extensively washed and cultured alone for an additional 36 hours, with or without the transcriptional blocking agent Actinomycin-D (Act-D, 10 ug/ml). Control WBM cells were co-cultured without lung and not exposed to Act-D (control). WBM cells were then analyzed for the presence of a variety of hematopoietic and lung-specific markers by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). WBM cells co-cultured with or without lung expressed high levels of hematopoietic markers including Sca-1, c-kit and CD45. This expression was nearly or completely eliminated after exposure to Act-D in culture. Expression of the surfactant protein B (Sp-B) gene was also significantly decreased in WBM co-cultured with lung (irradiated or non-irradiated) after exposure to Act-D (mean ± SE, from 16±4.9 to 1.1±0.4-fold increase vs. control, p=0.012). Interestingly, exposure to Act-D resulted in an increase in the expression of other lung-specific genes, including surfactant protein C (Sp-C) and clara cell specific protein (CCSP), in WBM co-cultured with lung (from 165±103 to 3,700±1,625 and 2±0.9 to 93±38-fold increases vs. control, p<0.015). In separate experiments, WBM cells co-cultured with cell-free lung conditioned media (CM) that was pre-treated with RNase had nearly 70% lower Sp-B expression compared with WBM cells co-cultured with untreated cell-free CM. These data suggest that lung cells release a transcriptional factor that induces marrow cell expression of the Sp-B gene in culture. This transcriptional factor is RNase-sensitive and could potentially represent microRNA transferred to marrow cells via lung-derived microvesicles. The increase in expression of other lung-specific genes after Act-D exposure suggests alternative transcriptional control mechanisms for lung-specific genes other than Sp-B.