Abstract
We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems. We find that irradiation to murine lung and lung-derived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines. The functional classification of transferred proteins was broad and included transcription factors, mediators of basic cellular processes and components of the nucleosome remodeling and deacetylase complex, including metastasis associated protein 3 and retinoblastoma-binding protein 7. In further analysis we find that retinoblastoma-binding protein 7 is a transcriptional activator of E-cadherin and that its intercellular transfer leads to decreased gene expression of downstream targets such as N-cadherin and vimentin. SILAC-generated data sets offer a valuable tool to identify and validate potential paracrine networks that may impact relevant biologic processes associated with phenotypic and genotypic signatures of health and disease.
Highlights
We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems
We find that irradiation to murine lung and lungderived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines
The technique of stable isotope labeling with amino acids in cell culture (SILAC)3 has been introduced as a mass spectrometry (MS)-based strategy to quantitatively interrogate the proteome [3]
Summary
Candidate proteins thought to undergo transfer from MLG to FDCP1 cells based upon MS data. Shown are the seven proteins, their molecular masses, number of heavy peptides, as well as number of peptides associated with each protein. We required that each protein be associated with at least two heavy peptides and protein ratios Ͼ0.5 to be considered for downstream evaluation. In-depth analysis of MS data for candidate proteins, including MTA3 and RBBP7 are shown in supplemental Table S3
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