Bleomycin Regulation of Transforming Growth Factor-β mRNA in Rat Lung Fibroblasts

Abstract

Pulmonary fibrosis is a well-known toxic response to bleomycin treatment. Here we demonstrate the direct effects of bleomycin on lung fibroblasts that resulted in a marked increase of collagen synthesis as compared with total noncollagen protein synthesis. Bleomycin treatment of rat lung fibroblast cultures resulted in an increase of total cellular transforming growth factor-beta (TGF-beta) mRNA and increased secretion of TGF-beta protein into the conditioned media. beta 2-Microglobulin was measured as an mRNA that did not increase with bleomycin treatment. The bleomycin-induced increase of TGF-beta mRNA was decreased by cells cultured in the presence of either cycloheximide, an inhibitor of protein synthesis, or 2-mercapto-1-(beta-4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis. To assess the mechanism underlying increased steady-state mRNA levels, the nuclear fraction was isolated from bleomycin-treated cells and the TGF-beta transcripts were determined. Transcription of TGF-beta mRNA was increased 12 h after bleomycin treatment, whereas the transcription of type I procollagen, type III procollagen, and beta-actin mRNAs were increased after 48 h of bleomycin treatment. beta 2-Microglobulin mRNA synthesis was not increased within this time frame. These results suggest bleomycin regulation of TGF-beta at both the mRNA and protein levels. Rats lung fibroblasts were separated by cell sorting into two subpopulations. One population of fibroblasts demonstrated increased procollagen type I mRNAs, whereas fibroblasts in the other population had increased procollagen type III mRNA. Following bleomycin treatment, TGF-beta mRNA was shown to be located more prominently in those fibroblasts that contain primarily collagen type I mRNAs.

Disruption of tumor necrosis factor receptor p55 impairs collagen turnover in experimentally induced sclerodermic skin fibroblasts

“…A recent study showed that TGF␤ is a mediator of the fibrotic effect of bleomycin at the transcriptional level and that the TGF␤ response element is required for bleomycin stimulation of the pro␣1(I) collagen promoter (30). These previously published results (27)(28)(29)(30) indicate that bleomycin increases extracellular matrix production even without the involvement of immune cells.…”

“…It was shown that bleomycin up-regulates type I collagen and fibronectin mRNA in cultured normal skin fibroblasts (28). Exposure of rat lung fibroblast cultures to bleomycin results in elevated TGF␤ mRNA synthesis and TGF␤ protein, which is followed by increased procollagen gene transcription (29). A recent study showed that TGF␤ is a mediator of the fibrotic effect of bleomycin at the transcriptional level and that the TGF␤ response element is required for bleomycin stimulation of the pro␣1(I) collagen promoter (30).…”

T cell independence of bleomycin-induced pulmonary fibrosis

“…A variety of cytokines have been implicated, such as IL-1 [13,14], TNF-␣ [15], and TFG-␤ [24]. Lung macrophages have been reported as the primary source of these cytokines [13][14][15][16][17][18][19][20][21][22], but other cell types such as fibroblasts [23], eosinophils [18], and endothelial cells [24] also have been shown to secrete TGF-␤ after BLM stimulation. In addition to TGF-␤, the pleiotropic cytokine TNF-␣ also promotes the growth of fibroblasts [36].…”

Interleukin-10 Secretion by Alveolar Macrophages and Monocytes in Sarcoidosis

“…Through its fibroblast-stimulating properties [22], TGF-β may play a role in granuloma formation [23]as well as in the resolution of inflammation [24]. Because TGF-β is known to be secreted in an inactive form, it was no surprise that there was no bioactivity in any SN.…”