Abstract
Transforming growth factor-β1 (TGF-β1) plays a premier role in fibrosis. To understand the molecular events underpinning TGF-β1-induced fibrogenesis, we examined the proteomic profiling of a TGF-β1-induced in vitro model of fibrosis in NRK-49F normal rat kidney fibroblasts. Mass spectrometric analysis indicated that 628 cell-lysate proteins enriched in 44 cellular component clusters, 24 biological processes and 27 molecular functions were regulated by TGF-β1. Cell-lysate proteins regulated by TGF-β1 were characterised by increased ribosomal proteins and dysregulated proteins involved in multiple metabolic pathways, including reduced Aldh3a1 and induced Enpp1 and Impdh2, which were validated by enzyme-linked immunosorbent assays (ELISA). In conditioned media, 62 proteins enriched in 20 cellular component clusters, 40 biological processes and 7 molecular functions were regulated by TGF-β1. Secretomic analysis and ELISA uncovered dysregulated collagen degradation regulators (induced PAI-1 and reduced Mmp3), collagen crosslinker (induced Plod2), signalling molecules (induced Ccn1, Ccn2 and Tsku, and reduced Ccn3) and chemokines (induced Ccl2 and Ccl7) in the TGF-β1 group. We conclude that TGF-β1-induced fibrogenesis in renal fibroblasts is an intracellular metabolic disorder and is inherently coupled with inflammation mediated by chemokines. Proteomic profiling established in this project may guide development of novel anti-fibrotic therapies in a network pharmacology approach.
Highlights
Transforming growth factor-β1 (TGF-β1) plays a premier role in fibrosis
This study has a few major limitations: (i) it did not examine conditioned media, which could provide crucial insights into fibrogenesis, a well-established disorder characterised by the accumulation of collagen in the extracellular compartment; (ii) TGF-β1 treatment for 6 h was not sufficient to induce an established model of fibrosis and the authors did not show accumulation of total collagens in their samples for proteomic analysis; and (iii) 2D-gel electrophoresis detected more than 1500 protein spots, among which 150 were differentially displayed, but they only selected 78 for proteomic analysis
Gene Ontology (GO) function enrichment analysis of TGF-β1-regulated proteins was performed on Database for Annotation, Visualization and Integrated Discovery (DAVID; https://david.ncifcrf.gov/)
Summary
Transforming growth factor-β1 (TGF-β1) plays a premier role in fibrosis. To understand the molecular events underpinning TGF-β1-induced fibrogenesis, we examined the proteomic profiling of a TGFβ1-induced in vitro model of fibrosis in NRK-49F normal rat kidney fibroblasts. This study has a few major limitations: (i) it did not examine conditioned media, which could provide crucial insights into fibrogenesis, a well-established disorder characterised by the accumulation of collagen in the extracellular compartment; (ii) TGF-β1 treatment for 6 h was not sufficient to induce an established model of fibrosis and the authors did not show accumulation of total collagens in their samples for proteomic analysis; and (iii) 2D-gel electrophoresis detected more than 1500 protein spots, among which 150 were differentially displayed, but they only selected 78 for proteomic analysis. They failed to establish an un-biased complete proteomic view on TGF-β1-regulated p roteins[24]
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