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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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TGF-β1 stimulation restores BMP-4 and gremlin expression to wt levels. Lung −/− fibroblasts were cultured in the presence of 0.5 ng/ml TGF-β1 for 2 wk (+TGF-β1). (A) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of gremlin and BMP-4 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading. (B) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of the ECM. Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of the molecular mass markers (kD) is indicated on the left.
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fig9: TGF-β1 stimulation restores BMP-4 and gremlin expression to wt levels. Lung −/− fibroblasts were cultured in the presence of 0.5 ng/ml TGF-β1 for 2 wk (+TGF-β1). (A) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of gremlin and BMP-4 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading. (B) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of the ECM. Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of the molecular mass markers (kD) is indicated on the left.

Mentions: Lack of LTBP-4 in lung fibroblasts leads to decreased activation of TGF-β and increased BMP-4 signaling. To evaluate the relationship between these changes, we analyzed whether or not restoring TGF-β1 activity to −/− fibroblasts rescues BMP-4 and gremlin expression to wt levels. Induced levels of TGF-β activity are reported to up-regulate the expression of gremlin in other cell systems (McMahon et al., 2000). To restore TGF-β1 activity into −/− fibroblasts, they were cultured for 2 wk in the presence of a low concentration (0.5 ng/ml) of TGF-β1 in normal serum-containing medium. Cells cultured in the presence of TGF-β1 expressed wt levels of gremlin and BMP-4 mRNAs (Fig. 9 A). The expression of gremlin was especially increased, from undetectable to high levels, which probably controls BMP-4 signaling levels. As expected, ECM production was significantly decreased in −/− fibroblasts after restoration of TGF-β1 activity (Fig. 9 B).


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)

TGF-β1 stimulation restores BMP-4 and gremlin expression to wt levels. Lung −/− fibroblasts were cultured in the presence of 0.5 ng/ml TGF-β1 for 2 wk (+TGF-β1). (A) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of gremlin and BMP-4 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading. (B) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of the ECM. Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of the molecular mass markers (kD) is indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172518&req=5

fig9: TGF-β1 stimulation restores BMP-4 and gremlin expression to wt levels. Lung −/− fibroblasts were cultured in the presence of 0.5 ng/ml TGF-β1 for 2 wk (+TGF-β1). (A) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of gremlin and BMP-4 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading. (B) Lung fibroblasts were cultured in the presence of labeled amino acids for 2 d followed by isolation of the ECM. Polypeptides of the ECM preparations were separated by SDS-PAGE under reducing conditions and visualized by fluorography. The migration of the molecular mass markers (kD) is indicated on the left.
Mentions: Lack of LTBP-4 in lung fibroblasts leads to decreased activation of TGF-β and increased BMP-4 signaling. To evaluate the relationship between these changes, we analyzed whether or not restoring TGF-β1 activity to −/− fibroblasts rescues BMP-4 and gremlin expression to wt levels. Induced levels of TGF-β activity are reported to up-regulate the expression of gremlin in other cell systems (McMahon et al., 2000). To restore TGF-β1 activity into −/− fibroblasts, they were cultured for 2 wk in the presence of a low concentration (0.5 ng/ml) of TGF-β1 in normal serum-containing medium. Cells cultured in the presence of TGF-β1 expressed wt levels of gremlin and BMP-4 mRNAs (Fig. 9 A). The expression of gremlin was especially increased, from undetectable to high levels, which probably controls BMP-4 signaling levels. As expected, ECM production was significantly decreased in −/− fibroblasts after restoration of TGF-β1 activity (Fig. 9 B).

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