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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 and secretion of TGF-β by mouse lung fibroblasts. (A) RT-PCR analyses of LTBP-4 mRNA levels in wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. The migration of the molecular mass markers (bp) is shown on the left. (B) Serum-free conditioned medium was collected from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts for 24 h, and the amount of TGF-β in medium (active TGF-β) or heat-treated medium (total TGF-β) was analyzed. Pan-specific neutralizing antibody against TGF-β was added as a specificity control where indicated (anti–TGF-β). The results are expressed as relative TGF-β activity (the activity in medium from +/+ cells equals one). The error bars represent the SEM of the samples. (C) Total RNA was isolated from lung fibroblasts, and mRNA expression levels of TGF-β1–3 were analyzed by Northern blotting. The results were quantified by phosphoimager analyses and are expressed as relative value of mRNA (see Materials and methods). The mRNA levels in control (+/+) cells equal one. (D) Serum-free conditioned medium was collected from lung fibroblasts for 24 h. Aliquots of the media were concentrated and analyzed by immunoblotting using specific antibodies against TGF-β1–3.
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fig1: Expression and secretion of TGF-β by mouse lung fibroblasts. (A) RT-PCR analyses of LTBP-4 mRNA levels in wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. The migration of the molecular mass markers (bp) is shown on the left. (B) Serum-free conditioned medium was collected from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts for 24 h, and the amount of TGF-β in medium (active TGF-β) or heat-treated medium (total TGF-β) was analyzed. Pan-specific neutralizing antibody against TGF-β was added as a specificity control where indicated (anti–TGF-β). The results are expressed as relative TGF-β activity (the activity in medium from +/+ cells equals one). The error bars represent the SEM of the samples. (C) Total RNA was isolated from lung fibroblasts, and mRNA expression levels of TGF-β1–3 were analyzed by Northern blotting. The results were quantified by phosphoimager analyses and are expressed as relative value of mRNA (see Materials and methods). The mRNA levels in control (+/+) cells equal one. (D) Serum-free conditioned medium was collected from lung fibroblasts for 24 h. Aliquots of the media were concentrated and analyzed by immunoblotting using specific antibodies against TGF-β1–3.

Mentions: To determine if the functional inactivation of LTBP-4 interferes with TGF-β secretion and/or activation, we established lung fibroblast cultures from wt and LTBP-4 −/− mice (Sterner-Kock et al., 2002). As expected, LTBP-4 mRNA was not expressed in the −/− fibroblasts (Fig. 1 A). TGF-β activity was quantified from cell-conditioned medium using TGF-β responsive reporter cells that produce luciferase activity in response to TGF-β (Abe et al., 1994). Fibroblast-conditioned medium (harvested at 24 h) was incubated with the reporter cells overnight, after which the luciferase activity was measured. The amount of active TGF-β in the conditioned medium of wt cells was ∼50 pg/ml/106 cells, whereas the amount of active TGF-β was down-regulated ∼50% in the −/− fibroblasts (Fig. 1 B). Neutralizing anti–TGF-β antibodies drastically reduced the activity of the medium. These results correlated well with previous in vivo findings, which indicated significantly less TGF-β signaling in the lungs of the −/− mice (Sterner-Kock et al., 2002). Latent forms of TGF-β from the cell-conditioned media were then activated by heat treatment (Brown et al., 1990) and analyzed with the reporter cell system. In contrast to the reduced levels of active TGF-β, the secretion of latent TGF-βs was prominently increased in the −/− fibroblasts (Fig. 1 B). This indicated that the reduced TGF-β activity cannot be attributed to decreased secretion. The results imply that the TGF-β activation process is significantly impaired in LTBP-4 −/− fibroblasts, probably due to perturbed targeting.


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 and secretion of TGF-β by mouse lung fibroblasts. (A) RT-PCR analyses of LTBP-4 mRNA levels in wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. The migration of the molecular mass markers (bp) is shown on the left. (B) Serum-free conditioned medium was collected from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts for 24 h, and the amount of TGF-β in medium (active TGF-β) or heat-treated medium (total TGF-β) was analyzed. Pan-specific neutralizing antibody against TGF-β was added as a specificity control where indicated (anti–TGF-β). The results are expressed as relative TGF-β activity (the activity in medium from +/+ cells equals one). The error bars represent the SEM of the samples. (C) Total RNA was isolated from lung fibroblasts, and mRNA expression levels of TGF-β1–3 were analyzed by Northern blotting. The results were quantified by phosphoimager analyses and are expressed as relative value of mRNA (see Materials and methods). The mRNA levels in control (+/+) cells equal one. (D) Serum-free conditioned medium was collected from lung fibroblasts for 24 h. Aliquots of the media were concentrated and analyzed by immunoblotting using specific antibodies against TGF-β1–3.
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fig1: Expression and secretion of TGF-β by mouse lung fibroblasts. (A) RT-PCR analyses of LTBP-4 mRNA levels in wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts. The migration of the molecular mass markers (bp) is shown on the left. (B) Serum-free conditioned medium was collected from wt (+/+) and LTBP-4 hypomorphic (−/−) lung fibroblasts for 24 h, and the amount of TGF-β in medium (active TGF-β) or heat-treated medium (total TGF-β) was analyzed. Pan-specific neutralizing antibody against TGF-β was added as a specificity control where indicated (anti–TGF-β). The results are expressed as relative TGF-β activity (the activity in medium from +/+ cells equals one). The error bars represent the SEM of the samples. (C) Total RNA was isolated from lung fibroblasts, and mRNA expression levels of TGF-β1–3 were analyzed by Northern blotting. The results were quantified by phosphoimager analyses and are expressed as relative value of mRNA (see Materials and methods). The mRNA levels in control (+/+) cells equal one. (D) Serum-free conditioned medium was collected from lung fibroblasts for 24 h. Aliquots of the media were concentrated and analyzed by immunoblotting using specific antibodies against TGF-β1–3.
Mentions: To determine if the functional inactivation of LTBP-4 interferes with TGF-β secretion and/or activation, we established lung fibroblast cultures from wt and LTBP-4 −/− mice (Sterner-Kock et al., 2002). As expected, LTBP-4 mRNA was not expressed in the −/− fibroblasts (Fig. 1 A). TGF-β activity was quantified from cell-conditioned medium using TGF-β responsive reporter cells that produce luciferase activity in response to TGF-β (Abe et al., 1994). Fibroblast-conditioned medium (harvested at 24 h) was incubated with the reporter cells overnight, after which the luciferase activity was measured. The amount of active TGF-β in the conditioned medium of wt cells was ∼50 pg/ml/106 cells, whereas the amount of active TGF-β was down-regulated ∼50% in the −/− fibroblasts (Fig. 1 B). Neutralizing anti–TGF-β antibodies drastically reduced the activity of the medium. These results correlated well with previous in vivo findings, which indicated significantly less TGF-β signaling in the lungs of the −/− mice (Sterner-Kock et al., 2002). Latent forms of TGF-β from the cell-conditioned media were then activated by heat treatment (Brown et al., 1990) and analyzed with the reporter cell system. In contrast to the reduced levels of active TGF-β, the secretion of latent TGF-βs was prominently increased in the −/− fibroblasts (Fig. 1 B). This indicated that the reduced TGF-β activity cannot be attributed to decreased secretion. The results imply that the TGF-β activation process is significantly impaired in LTBP-4 −/− fibroblasts, probably due to perturbed targeting.

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