Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription

Abstract

Objective: Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA.

Materials and methods: Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-microm pore size), were analyzed using species-specific primers (for rat or mouse).

Results: These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena.

Conclusion: These studies suggest that cellular systems are more phenotypically labile than previously considered.

Figure 1. CM induces mRNA expression changes, contains microvesicles

(A) mRNA expression scale vs. control cells (WBM cultured alone). (B) mRNA in WBM co-cultured with CM vs. control. (C) mRNA in CM ultracentrifuged pellet vs. control. Electron microscopy of (D) lung, (E) brain, (F) heart, (G) liver CM ultracentrifuged pellets. Bar=100 nm, * P<0.05.

Figure 2. LDMV isolation, characterization

(A–C) LCM is made from lung cultured on cell-impermeable membranes, which is then ultracentrifuged. The pellet is labeled with PKH26, CFSE and separated by FACS to isolate LDMV. (D) Electron microscopy of LDMV. Populations of LDMV express (E) CXCR4, (F) LAMP-1, (H) α6 and (I) α5 integrin. (G,J) isotype controls.

Figure 3. LDMV affect marrow cell mRNA content

(A) LDMV are incubated with WBM, myeloid or lymphoid cells. Cells containing LDMV (R2, PKH26+/CFSE+) or no LDMV (R1, PKH−/CFSE−) are isolated by FACS. (B) Sp-B, C and CCSP mRNA levels of cells containing LDMV or no LDMV vs. control cells (cells not exposed to LDMV in culture).

Figure 4. Transcriptional blocking studies

WBM co-cultured with (A) lung or (C) LDMV is then cultured alone in media containing Actinomycin-D (Act-D,10ug/ml) or diluent (DMSO), (E) α-Amantin (10ug/ml) or diluent. (B,D,F) mRNA levels of co-cultured WBM vs. WBM cultured without lung, LDMV and transcriptional blocking agents. * P<0.01. No error bars displayed in graphs with only experiment.

Figure 5. Hybrid rat/mouse co-cultures

Mouse, rat-specific Sp-B, C mRNA in mouse WBM co-cultured with (A) mouse, (B) rat lung or in rat WBM co-cultured with (C) rat, (D) mouse lung. Mouse, rat-specific albumin mRNA in mouse WBM co-cultured with (E) mouse, (F) rat liver or in rat WBM co-cultured with (G) rat, (H) mouse liver. *P<0.01.

Figure 6. RNase studies, microRNA content of LDMV

WBM co-cultured with RNase-treated, untreated (A) LCM, (C) LDMV. (B,D) mRNA expression in co-cultured WBM vs. WBM co-cultured with untreated LCM, LDMV. *P < 0.05. microRNA expression in (E) LDMV, (F) WBM co-cultured with LDMV vs. WBM, microRNA species with homologies to pulmonary epithelial cell genes (yellow box).

Figure 7. LDMV-mediated transfer of genetic phenotype from lung to marrow: A proposed mechanism

LDMV enter marrow cells in co-culture. Pulmonary epithelial cell-specific mRNAs found in co-cultured marrow cells are a reflection of LDMV-derived mRNA species and mRNA induced from target cells by LDMV-based transcription factors. LDMV-derived microRNA degrades mRNA to further modulate the system.