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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 TGF-β signaling does not rescue the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing a dominant-negative TGF-β type II receptor (+ΔRII) were transiently transfected with (CAGA)12-luc promoter construct (see Materials and methods). The next day (24 h after transfection), the indicated concentrations of TGF-β1 were added to the medium and the cells were incubated overnight. Luciferase activities were measured 48 h after transfection. The activities were normalized by comparing them with the activities of cotransfected Renilla luciferase. The results are expressed as relative luciferase activities (nontreated −/− 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.
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fig3: Inhibition of TGF-β signaling does not rescue the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing a dominant-negative TGF-β type II receptor (+ΔRII) were transiently transfected with (CAGA)12-luc promoter construct (see Materials and methods). The next day (24 h after transfection), the indicated concentrations of TGF-β1 were added to the medium and the cells were incubated overnight. Luciferase activities were measured 48 h after transfection. The activities were normalized by comparing them with the activities of cotransfected Renilla luciferase. The results are expressed as relative luciferase activities (nontreated −/− 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.

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 TGF-β signaling does not rescue the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing a dominant-negative TGF-β type II receptor (+ΔRII) were transiently transfected with (CAGA)12-luc promoter construct (see Materials and methods). The next day (24 h after transfection), the indicated concentrations of TGF-β1 were added to the medium and the cells were incubated overnight. Luciferase activities were measured 48 h after transfection. The activities were normalized by comparing them with the activities of cotransfected Renilla luciferase. The results are expressed as relative luciferase activities (nontreated −/− 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.
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Related In: Results  -  Collection

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

fig3: Inhibition of TGF-β signaling does not rescue the LTBP-4 hypomorphic (−/−) fibroblast phenotype. (A) −/− lung fibroblasts expressing a dominant-negative TGF-β type II receptor (+ΔRII) were transiently transfected with (CAGA)12-luc promoter construct (see Materials and methods). The next day (24 h after transfection), the indicated concentrations of TGF-β1 were added to the medium and the cells were incubated overnight. Luciferase activities were measured 48 h after transfection. The activities were normalized by comparing them with the activities of cotransfected Renilla luciferase. The results are expressed as relative luciferase activities (nontreated −/− 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.
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