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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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Reversal of the phenotype in −/− lung fibroblasts by stable expression of human LTBP-4. Human LTBP-4 or LTBP-1 expression vector was stably transfected into LTBP-4 hypomorphic (−/−) lung fibroblasts. (A) Serum-free conditioned medium was collected from lung fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold, and LTBP-4 (LTBP-4–transfected −/− fibroblasts, top) or LTBP-1 (LTBP-1–transfected −/− fibroblasts, bottom) secretion was analyzed by immunoblotting. The migration of the molecular mass markers (kD) is indicated on the left. (B) wt (+/+), −/−, and LTBP-4– or LTBP-1–transfected −/− 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. (C) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of CTGF, PAI-1, TGF-β2, and TGF-β3 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading.
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fig4: Reversal of the phenotype in −/− lung fibroblasts by stable expression of human LTBP-4. Human LTBP-4 or LTBP-1 expression vector was stably transfected into LTBP-4 hypomorphic (−/−) lung fibroblasts. (A) Serum-free conditioned medium was collected from lung fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold, and LTBP-4 (LTBP-4–transfected −/− fibroblasts, top) or LTBP-1 (LTBP-1–transfected −/− fibroblasts, bottom) secretion was analyzed by immunoblotting. The migration of the molecular mass markers (kD) is indicated on the left. (B) wt (+/+), −/−, and LTBP-4– or LTBP-1–transfected −/− 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. (C) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of CTGF, PAI-1, TGF-β2, and TGF-β3 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading.

Mentions: Because the −/− fibroblasts secreted increased amounts of latent TGF-β, we analyzed the expression and secretion of all three isoforms of TGF-β. Northern analyses of mRNA expression indicated that TGF-β1 levels were not significantly altered, whereas TGF-β2 and -β3 levels were highly up-regulated in −/− fibroblasts (Fig. 1 C and see Fig. 4 C). In accordance with mRNA data, their protein levels were clearly up-regulated (Fig. 1 D), whereas the total levels of TGF-β1 did not change appreciably. Although the mechanism of this induction is unclear, one possible explanation is that low levels of TGF-β1 secretion induced up-regulation of the other isoforms. Importantly, TGF-β1 is a potent repressor of TGF-β2 and -β3 expression (Bascom et al., 1989).


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)

Reversal of the phenotype in −/− lung fibroblasts by stable expression of human LTBP-4. Human LTBP-4 or LTBP-1 expression vector was stably transfected into LTBP-4 hypomorphic (−/−) lung fibroblasts. (A) Serum-free conditioned medium was collected from lung fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold, and LTBP-4 (LTBP-4–transfected −/− fibroblasts, top) or LTBP-1 (LTBP-1–transfected −/− fibroblasts, bottom) secretion was analyzed by immunoblotting. The migration of the molecular mass markers (kD) is indicated on the left. (B) wt (+/+), −/−, and LTBP-4– or LTBP-1–transfected −/− 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. (C) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of CTGF, PAI-1, TGF-β2, and TGF-β3 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Reversal of the phenotype in −/− lung fibroblasts by stable expression of human LTBP-4. Human LTBP-4 or LTBP-1 expression vector was stably transfected into LTBP-4 hypomorphic (−/−) lung fibroblasts. (A) Serum-free conditioned medium was collected from lung fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold, and LTBP-4 (LTBP-4–transfected −/− fibroblasts, top) or LTBP-1 (LTBP-1–transfected −/− fibroblasts, bottom) secretion was analyzed by immunoblotting. The migration of the molecular mass markers (kD) is indicated on the left. (B) wt (+/+), −/−, and LTBP-4– or LTBP-1–transfected −/− 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. (C) Total RNA was isolated from cultured lung fibroblasts, and mRNA expression levels of CTGF, PAI-1, TGF-β2, and TGF-β3 were analyzed by Northern blotting. mRNA expression of a constant gene, GADPH, was used to control loading.
Mentions: Because the −/− fibroblasts secreted increased amounts of latent TGF-β, we analyzed the expression and secretion of all three isoforms of TGF-β. Northern analyses of mRNA expression indicated that TGF-β1 levels were not significantly altered, whereas TGF-β2 and -β3 levels were highly up-regulated in −/− fibroblasts (Fig. 1 C and see Fig. 4 C). In accordance with mRNA data, their protein levels were clearly up-regulated (Fig. 1 D), whereas the total levels of TGF-β1 did not change appreciably. Although the mechanism of this induction is unclear, one possible explanation is that low levels of TGF-β1 secretion induced up-regulation of the other isoforms. Importantly, TGF-β1 is a potent repressor of TGF-β2 and -β3 expression (Bascom et al., 1989).

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