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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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Expression of BMP-4, gremlin, and BMP target genes Id1 and -2 in lung fibroblasts. (A) Total RNA was isolated from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. On the basis of microarray analyses (Table I), mRNA expression levels of BMP-4, gremlin, and inhibitors of differentiation (Id)1 and -2 were analyzed by Northern blotting. mRNA expression of the constant gene, GADPH, was used to control loading. (B) Serum-free conditioned medium was harvested from fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold and analyzed by immunoblotting using a specific antibody against BMP-4. Recombinant human BMP-4 (rhBMP-4, 1 ng) was used as a control. The migration of the molecular mass markers (kD) is indicated on the left.
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fig5: Expression of BMP-4, gremlin, and BMP target genes Id1 and -2 in lung fibroblasts. (A) Total RNA was isolated from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. On the basis of microarray analyses (Table I), mRNA expression levels of BMP-4, gremlin, and inhibitors of differentiation (Id)1 and -2 were analyzed by Northern blotting. mRNA expression of the constant gene, GADPH, was used to control loading. (B) Serum-free conditioned medium was harvested from fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold and analyzed by immunoblotting using a specific antibody against BMP-4. Recombinant human BMP-4 (rhBMP-4, 1 ng) was used as a control. The migration of the molecular mass markers (kD) is indicated on the left.

Mentions: A prominent observation in the −/− fibroblasts was a fivefold increase in the expression of BMP-4, in parallel with almost complete loss of expression of the BMP inhibitor gremlin. Northern analyses confirmed these observations (Fig. 5 A). Immunoblotting of cell-conditioned medium revealed increased levels of BMP-4 protein in the medium of −/− fibroblasts (Fig. 5 B). In addition, the BMP-4 target gene inhibitor of differentiation (Id)2 was induced in the gene array. Northern analyses of Id2 as well as Id1 indicated 5–10-fold increases in expression levels (Fig. 5 A). The expression levels of the BMP-4 receptors BMPR-IA (ALK-3), BMPR-IB (ALK-6), and BMPR-II were comparable in wt and −/− fibroblasts (gene array analyses; unpublished data). These results suggest that BMP-4 signaling is considerably increased in LTBP-4 −/− fibroblasts.


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)

Expression of BMP-4, gremlin, and BMP target genes Id1 and -2 in lung fibroblasts. (A) Total RNA was isolated from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. On the basis of microarray analyses (Table I), mRNA expression levels of BMP-4, gremlin, and inhibitors of differentiation (Id)1 and -2 were analyzed by Northern blotting. mRNA expression of the constant gene, GADPH, was used to control loading. (B) Serum-free conditioned medium was harvested from fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold and analyzed by immunoblotting using a specific antibody against BMP-4. Recombinant human BMP-4 (rhBMP-4, 1 ng) was used as a control. 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

fig5: Expression of BMP-4, gremlin, and BMP target genes Id1 and -2 in lung fibroblasts. (A) Total RNA was isolated from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. On the basis of microarray analyses (Table I), mRNA expression levels of BMP-4, gremlin, and inhibitors of differentiation (Id)1 and -2 were analyzed by Northern blotting. mRNA expression of the constant gene, GADPH, was used to control loading. (B) Serum-free conditioned medium was harvested from fibroblasts at 24 h. Aliquots of the media were concentrated 10-fold and analyzed by immunoblotting using a specific antibody against BMP-4. Recombinant human BMP-4 (rhBMP-4, 1 ng) was used as a control. The migration of the molecular mass markers (kD) is indicated on the left.
Mentions: A prominent observation in the −/− fibroblasts was a fivefold increase in the expression of BMP-4, in parallel with almost complete loss of expression of the BMP inhibitor gremlin. Northern analyses confirmed these observations (Fig. 5 A). Immunoblotting of cell-conditioned medium revealed increased levels of BMP-4 protein in the medium of −/− fibroblasts (Fig. 5 B). In addition, the BMP-4 target gene inhibitor of differentiation (Id)2 was induced in the gene array. Northern analyses of Id2 as well as Id1 indicated 5–10-fold increases in expression levels (Fig. 5 A). The expression levels of the BMP-4 receptors BMPR-IA (ALK-3), BMPR-IB (ALK-6), and BMPR-II were comparable in wt and −/− fibroblasts (gene array analyses; unpublished data). These results suggest that BMP-4 signaling is considerably increased in LTBP-4 −/− fibroblasts.

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