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Disruption of LTBP-4 function reduces TGF-beta activation and enhances BMP-4 signaling in the lung.

Koli K, Wempe F, Sterner-Kock A, Kantola A, Komor M, Hofmann WK, von Melchner H, Keski-Oja J - J. Cell Biol. (2004)

Bottom Line: These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts.Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin.Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Haartman Institute and Helsinki University Hospital, University of Helsinki, 00014 Helsinki, Finland. katri.koli@helsinki.fi

ABSTRACT
Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.

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Loss of LTBP-4 results in increased production of ECM in cultured fibroblasts and in vivo mouse lung. (A) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of sodium deoxycholate insoluble matrices (ECM). Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) and the molecular mass markers (kD) are indicated. (B) Lung fibroblasts were cultured for 5 d on class coverslips, after which they were fixed with methanol (−20°C) and stained for fibronectin. (C) Lung sections from the mice were stained for fibronectin and fibrillin-1. Positive staining is reddish-brown. (D) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase–2 (MMP-2), FN, and PAI-1 were analyzed by Northern blotting. mRNA expression of a constant gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH), was used to control loading.
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fig2: Loss of LTBP-4 results in increased production of ECM in cultured fibroblasts and in vivo mouse lung. (A) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of sodium deoxycholate insoluble matrices (ECM). Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) and the molecular mass markers (kD) are indicated. (B) Lung fibroblasts were cultured for 5 d on class coverslips, after which they were fixed with methanol (−20°C) and stained for fibronectin. (C) Lung sections from the mice were stained for fibronectin and fibrillin-1. Positive staining is reddish-brown. (D) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase–2 (MMP-2), FN, and PAI-1 were analyzed by Northern blotting. mRNA expression of a constant gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH), was used to control loading.

Mentions: TGF-β1 is a potent regulator of ECM production and degradation that generally causes an accumulation of ECM. Therefore, we analyzed whether or not the lack of LTBP-4–mediated TGF-β1 targeting would reduce ECM production. Cells were metabolically labeled for 2 d, after which extracellular matrices were prepared and analyzed by SDS-PAGE. Unexpectedly, the amount of sodium deoxycholate–resistant matrix was considerably higher in −/− fibroblasts (Fig. 2 A), and the amount of the 250-kD fibronectin polypeptide was particularly increased. Immunofluorescence analyses of 5-d-old fibroblast cultures indicated that in wt cells, fibronectin was localized into fibrillar structures in the matrices, whereas in −/− cells fibronectin was more abundant and formed a uniform sheetlike structure around the cells (Fig. 2 B). Because the altered ECM structure may change the sodium deoxycholate solubility of the ECM-associated fibronectin, the massive increase seen in Fig. 2 A is plausibly due to both increased protein deposition and decreased solubility. Neither fibronectin mRNA levels in vitro (Fig. 2 D) or in vivo (not depicted) nor secreted protein levels in −/− fibroblasts (not depicted) were up-regulated, suggesting increased deposition and/or decreased turnover. The cell surface expression levels of the fibronectin receptor α5β1 integrin were not changed in −/− fibroblasts (unpublished data). Accordingly, immunohistochemical analysis of lung tissues indicated that fibronectin levels were highly up-regulated in the −/− mice (Fig. 2 C). LTBP-4 binds to fibrillin-1 (Isogai et al., 2003) and loss of fibrillin fibers in mice leads to changes in the levels of active TGF-β (Neptune et al., 2003). Therefore, fibrillin-1 distribution was evaluated, but no significant changes were observed (Fig. 2 C).


Disruption of LTBP-4 function reduces TGF-beta activation and enhances BMP-4 signaling in the lung.

Koli K, Wempe F, Sterner-Kock A, Kantola A, Komor M, Hofmann WK, von Melchner H, Keski-Oja J - J. Cell Biol. (2004)

Loss of LTBP-4 results in increased production of ECM in cultured fibroblasts and in vivo mouse lung. (A) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of sodium deoxycholate insoluble matrices (ECM). Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) and the molecular mass markers (kD) are indicated. (B) Lung fibroblasts were cultured for 5 d on class coverslips, after which they were fixed with methanol (−20°C) and stained for fibronectin. (C) Lung sections from the mice were stained for fibronectin and fibrillin-1. Positive staining is reddish-brown. (D) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase–2 (MMP-2), FN, and PAI-1 were analyzed by Northern blotting. mRNA expression of a constant gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH), was used to control loading.
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Related In: Results  -  Collection

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fig2: Loss of LTBP-4 results in increased production of ECM in cultured fibroblasts and in vivo mouse lung. (A) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of sodium deoxycholate insoluble matrices (ECM). Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) and the molecular mass markers (kD) are indicated. (B) Lung fibroblasts were cultured for 5 d on class coverslips, after which they were fixed with methanol (−20°C) and stained for fibronectin. (C) Lung sections from the mice were stained for fibronectin and fibrillin-1. Positive staining is reddish-brown. (D) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase–2 (MMP-2), FN, and PAI-1 were analyzed by Northern blotting. mRNA expression of a constant gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH), was used to control loading.
Mentions: TGF-β1 is a potent regulator of ECM production and degradation that generally causes an accumulation of ECM. Therefore, we analyzed whether or not the lack of LTBP-4–mediated TGF-β1 targeting would reduce ECM production. Cells were metabolically labeled for 2 d, after which extracellular matrices were prepared and analyzed by SDS-PAGE. Unexpectedly, the amount of sodium deoxycholate–resistant matrix was considerably higher in −/− fibroblasts (Fig. 2 A), and the amount of the 250-kD fibronectin polypeptide was particularly increased. Immunofluorescence analyses of 5-d-old fibroblast cultures indicated that in wt cells, fibronectin was localized into fibrillar structures in the matrices, whereas in −/− cells fibronectin was more abundant and formed a uniform sheetlike structure around the cells (Fig. 2 B). Because the altered ECM structure may change the sodium deoxycholate solubility of the ECM-associated fibronectin, the massive increase seen in Fig. 2 A is plausibly due to both increased protein deposition and decreased solubility. Neither fibronectin mRNA levels in vitro (Fig. 2 D) or in vivo (not depicted) nor secreted protein levels in −/− fibroblasts (not depicted) were up-regulated, suggesting increased deposition and/or decreased turnover. The cell surface expression levels of the fibronectin receptor α5β1 integrin were not changed in −/− fibroblasts (unpublished data). Accordingly, immunohistochemical analysis of lung tissues indicated that fibronectin levels were highly up-regulated in the −/− mice (Fig. 2 C). LTBP-4 binds to fibrillin-1 (Isogai et al., 2003) and loss of fibrillin fibers in mice leads to changes in the levels of active TGF-β (Neptune et al., 2003). Therefore, fibrillin-1 distribution was evaluated, but no significant changes were observed (Fig. 2 C).

Bottom Line: These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts.Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin.Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Haartman Institute and Helsinki University Hospital, University of Helsinki, 00014 Helsinki, Finland. katri.koli@helsinki.fi

ABSTRACT
Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.

Show MeSH
Related in: MedlinePlus