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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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Inhibition of BMP-4 signaling pathway partially rescues the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing mouse gremlin (+gremlin) were transiently transfected with (BRE)2-luc promoter, treated with indicated concentrations of BMP-4, and analyzed for luciferase activity as in Fig. 3 (see Materials and methods). The results are expressed as relative luciferase activities (untreated −/− fibroblast samples equal one). (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. (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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fig7: Inhibition of BMP-4 signaling pathway partially rescues the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing mouse gremlin (+gremlin) were transiently transfected with (BRE)2-luc promoter, treated with indicated concentrations of BMP-4, and analyzed for luciferase activity as in Fig. 3 (see Materials and methods). The results are expressed as relative luciferase activities (untreated −/− fibroblast samples equal one). (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. (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: Although the amount of active TGF-β in the conditioned medium was decreased, the −/− fibroblasts exhibited a phenotype similar to TGF-β–treated wt cells. TGF-β induces the production of ECM and enhances the expression of PAI-1 and CTGF in wt cells (unpublished data). The enhanced secretion of latent forms of TGF-β led us to speculate whether or not TGF-β might be activated by a distinct mechanism. For example, direct activation of TGF-β and subsequent binding to cell surface receptors may occur without release into the medium. To address this possibility, TGF-β signaling was inhibited by stable expression of a dominant-negative TGF-β type II receptor (ΔRII) in −/− fibroblasts (see Materials and methods). This form of the receptor lacks the kinase domain–containing cytoplasmic tail, and is therefore unable to transduce TGF-β signals (Wieser et al., 1993). To verify that the transgene prevented TGF-β signaling, the cells were transiently transfected with a TGF-β responsive promoter ((CAGA)12-luc; Dennler et al., 1998), followed by overnight treatment with TGF-β1. In −/− fibroblasts, TGF-β1 (100 pg/ml) induced the activity of (CAGA)12-luc promoter by ∼30-fold, whereas in cells expressing ΔRII the response was almost completely lost (Fig. 3 A). Analyses of ECM production revealed that the inhibition of TGF-β signaling in −/− fibroblasts did not lead to reversal of the phenotype (Fig. 3 B and see Fig. 7), which suggested that enhanced TGF-β activity is not involved in the induction of this phenotype.


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)

Inhibition of BMP-4 signaling pathway partially rescues the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing mouse gremlin (+gremlin) were transiently transfected with (BRE)2-luc promoter, treated with indicated concentrations of BMP-4, and analyzed for luciferase activity as in Fig. 3 (see Materials and methods). The results are expressed as relative luciferase activities (untreated −/− fibroblast samples equal one). (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. (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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172518&req=5

fig7: Inhibition of BMP-4 signaling pathway partially rescues the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing mouse gremlin (+gremlin) were transiently transfected with (BRE)2-luc promoter, treated with indicated concentrations of BMP-4, and analyzed for luciferase activity as in Fig. 3 (see Materials and methods). The results are expressed as relative luciferase activities (untreated −/− fibroblast samples equal one). (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. (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: Although the amount of active TGF-β in the conditioned medium was decreased, the −/− fibroblasts exhibited a phenotype similar to TGF-β–treated wt cells. TGF-β induces the production of ECM and enhances the expression of PAI-1 and CTGF in wt cells (unpublished data). The enhanced secretion of latent forms of TGF-β led us to speculate whether or not TGF-β might be activated by a distinct mechanism. For example, direct activation of TGF-β and subsequent binding to cell surface receptors may occur without release into the medium. To address this possibility, TGF-β signaling was inhibited by stable expression of a dominant-negative TGF-β type II receptor (ΔRII) in −/− fibroblasts (see Materials and methods). This form of the receptor lacks the kinase domain–containing cytoplasmic tail, and is therefore unable to transduce TGF-β signals (Wieser et al., 1993). To verify that the transgene prevented TGF-β signaling, the cells were transiently transfected with a TGF-β responsive promoter ((CAGA)12-luc; Dennler et al., 1998), followed by overnight treatment with TGF-β1. In −/− fibroblasts, TGF-β1 (100 pg/ml) induced the activity of (CAGA)12-luc promoter by ∼30-fold, whereas in cells expressing ΔRII the response was almost completely lost (Fig. 3 A). Analyses of ECM production revealed that the inhibition of TGF-β signaling in −/− fibroblasts did not lead to reversal of the phenotype (Fig. 3 B and see Fig. 7), which suggested that enhanced TGF-β activity is not involved in the induction of this phenotype.

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