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Fibulin-4 deficiency increases TGF-β signalling in aortic smooth muscle cells due to elevated TGF-β2 levels.

Ramnath NW, Hawinkels LJ, van Heijningen PM, te Riet L, Paauwe M, Vermeij M, Danser AH, Kanaar R, ten Dijke P, Essers J - Sci Rep (2015)

Bottom Line: Fibulins are extracellular matrix proteins associated with elastic fibres.These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels.TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-β signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-β pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-β neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-β signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-β isoforms. These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels. Significantly increased TGF-β2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-β signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-β1, but especially TGF-β2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-β pathway regulation in the pathogenesis of aortic aneurysms.

No MeSH data available.


Related in: MedlinePlus

Increased TGF-β signalling in Fibulin-4 deficient SMCs.(a) Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs show similar proliferation rates in the time course of the experiment (MTS proliferation assay) (b) Transfection with green fluorescent protein (GFP) encoding plasmids display no difference in percentage of transfected SMCs between Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (c) The TGF-β response assay reveals a gradual increase in TGF-β activity in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, after stimulation with exogenous TGF-β. Data represent fold change relative to unstimulated Fibulin-4+/+ SMCs. Addition of the ALK-5 kinase inhibitor (SB431542) abolishes the TGF-β response in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (d) Western blot analyses for TGF-β signalling downstream mediators pSMAD2 and pSMAD3 on TGF-β stimulated SMCs show a gradual increase in TGF-β signalling in Fibulin-4 deficient SMCs compared to Fibulin-4+/+ SMCs. (e) Measurement of basal TGF-β activity (no stimulation with exogenous TGF-β) by the CAGA-luciferase reporter show increased TGF-β activity in Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, which can be inhibited by the TβRI kinase inhibitor SB431542. (f) These data were confirmed by western blot analyses for pSMAD2 and pSMAD3. All data shown are representative for in total n = 3 independent experiments and all performed under serum starved conditions (*p < 0.05, **p < 0.01).
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f3: Increased TGF-β signalling in Fibulin-4 deficient SMCs.(a) Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs show similar proliferation rates in the time course of the experiment (MTS proliferation assay) (b) Transfection with green fluorescent protein (GFP) encoding plasmids display no difference in percentage of transfected SMCs between Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (c) The TGF-β response assay reveals a gradual increase in TGF-β activity in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, after stimulation with exogenous TGF-β. Data represent fold change relative to unstimulated Fibulin-4+/+ SMCs. Addition of the ALK-5 kinase inhibitor (SB431542) abolishes the TGF-β response in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (d) Western blot analyses for TGF-β signalling downstream mediators pSMAD2 and pSMAD3 on TGF-β stimulated SMCs show a gradual increase in TGF-β signalling in Fibulin-4 deficient SMCs compared to Fibulin-4+/+ SMCs. (e) Measurement of basal TGF-β activity (no stimulation with exogenous TGF-β) by the CAGA-luciferase reporter show increased TGF-β activity in Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, which can be inhibited by the TβRI kinase inhibitor SB431542. (f) These data were confirmed by western blot analyses for pSMAD2 and pSMAD3. All data shown are representative for in total n = 3 independent experiments and all performed under serum starved conditions (*p < 0.05, **p < 0.01).

Mentions: Since we observed that TGF-β neutralizing antibodies revert the decreased proliferation rates of Fibulin-4R/R SMCs, we further analysed transcriptional consequences of increased TGF-β signalling in these cells using a SMAD3/SMAD4 dependent promoter transcriptional reporter construct (CAGA-luciferase)28. Although there was a difference in proliferation between different genotypes at later time points, this was not observed during the shorter duration of this assay (Fig. 3a). To determine whether transfection efficiency was similar between the different genotypes a green fluorescent protein (GFP) expressing construct was transfected and GFP expression determined. Flow cytometric analysis showed no differences between the percentages of GFP expressing Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs (Fig. 3b) and thus no differences in transfection efficiencies among these different genotype. Next, we used the CAGA-luciferase reporter construct to assess TGF-β signalling activity in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. Stimulation with TGF-β of Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs showed a strong induction of luciferase activity, which was increased in a Fibulin-4 dose-dependent manner (Fig. 3c). Addition of the TβRI kinase inhibitor SB431542, a compound selectively blocking TGF-β type-I receptor kinase activity29, abolished TGF-β-induced transcriptional responses. Analysis of downstream pSMAD2 and pSMAD3 by western blotting revealed a gradual increase in SMAD2 and SMAD3 phosphorylation after stimulation with TGF-β in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs (Fig. 3d), confirming the CAGA-luciferase reporter data. These data indicate that Fibullin-4 deficient cells show increased signalling upon exogenous TGF-β stimulation.


Fibulin-4 deficiency increases TGF-β signalling in aortic smooth muscle cells due to elevated TGF-β2 levels.

Ramnath NW, Hawinkels LJ, van Heijningen PM, te Riet L, Paauwe M, Vermeij M, Danser AH, Kanaar R, ten Dijke P, Essers J - Sci Rep (2015)

Increased TGF-β signalling in Fibulin-4 deficient SMCs.(a) Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs show similar proliferation rates in the time course of the experiment (MTS proliferation assay) (b) Transfection with green fluorescent protein (GFP) encoding plasmids display no difference in percentage of transfected SMCs between Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (c) The TGF-β response assay reveals a gradual increase in TGF-β activity in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, after stimulation with exogenous TGF-β. Data represent fold change relative to unstimulated Fibulin-4+/+ SMCs. Addition of the ALK-5 kinase inhibitor (SB431542) abolishes the TGF-β response in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (d) Western blot analyses for TGF-β signalling downstream mediators pSMAD2 and pSMAD3 on TGF-β stimulated SMCs show a gradual increase in TGF-β signalling in Fibulin-4 deficient SMCs compared to Fibulin-4+/+ SMCs. (e) Measurement of basal TGF-β activity (no stimulation with exogenous TGF-β) by the CAGA-luciferase reporter show increased TGF-β activity in Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, which can be inhibited by the TβRI kinase inhibitor SB431542. (f) These data were confirmed by western blot analyses for pSMAD2 and pSMAD3. All data shown are representative for in total n = 3 independent experiments and all performed under serum starved conditions (*p < 0.05, **p < 0.01).
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f3: Increased TGF-β signalling in Fibulin-4 deficient SMCs.(a) Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs show similar proliferation rates in the time course of the experiment (MTS proliferation assay) (b) Transfection with green fluorescent protein (GFP) encoding plasmids display no difference in percentage of transfected SMCs between Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (c) The TGF-β response assay reveals a gradual increase in TGF-β activity in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, after stimulation with exogenous TGF-β. Data represent fold change relative to unstimulated Fibulin-4+/+ SMCs. Addition of the ALK-5 kinase inhibitor (SB431542) abolishes the TGF-β response in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. (d) Western blot analyses for TGF-β signalling downstream mediators pSMAD2 and pSMAD3 on TGF-β stimulated SMCs show a gradual increase in TGF-β signalling in Fibulin-4 deficient SMCs compared to Fibulin-4+/+ SMCs. (e) Measurement of basal TGF-β activity (no stimulation with exogenous TGF-β) by the CAGA-luciferase reporter show increased TGF-β activity in Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs, which can be inhibited by the TβRI kinase inhibitor SB431542. (f) These data were confirmed by western blot analyses for pSMAD2 and pSMAD3. All data shown are representative for in total n = 3 independent experiments and all performed under serum starved conditions (*p < 0.05, **p < 0.01).
Mentions: Since we observed that TGF-β neutralizing antibodies revert the decreased proliferation rates of Fibulin-4R/R SMCs, we further analysed transcriptional consequences of increased TGF-β signalling in these cells using a SMAD3/SMAD4 dependent promoter transcriptional reporter construct (CAGA-luciferase)28. Although there was a difference in proliferation between different genotypes at later time points, this was not observed during the shorter duration of this assay (Fig. 3a). To determine whether transfection efficiency was similar between the different genotypes a green fluorescent protein (GFP) expressing construct was transfected and GFP expression determined. Flow cytometric analysis showed no differences between the percentages of GFP expressing Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs (Fig. 3b) and thus no differences in transfection efficiencies among these different genotype. Next, we used the CAGA-luciferase reporter construct to assess TGF-β signalling activity in Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs. Stimulation with TGF-β of Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R SMCs showed a strong induction of luciferase activity, which was increased in a Fibulin-4 dose-dependent manner (Fig. 3c). Addition of the TβRI kinase inhibitor SB431542, a compound selectively blocking TGF-β type-I receptor kinase activity29, abolished TGF-β-induced transcriptional responses. Analysis of downstream pSMAD2 and pSMAD3 by western blotting revealed a gradual increase in SMAD2 and SMAD3 phosphorylation after stimulation with TGF-β in Fibulin-4+/R and Fibulin-4R/R SMCs compared to Fibulin-4+/+ SMCs (Fig. 3d), confirming the CAGA-luciferase reporter data. These data indicate that Fibullin-4 deficient cells show increased signalling upon exogenous TGF-β stimulation.

Bottom Line: Fibulins are extracellular matrix proteins associated with elastic fibres.These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels.TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cancer Genomics Centre Netherlands, Erasmus MC, Rotterdam, The Netherlands.

ABSTRACT
Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-β signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-β pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-β neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-β signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-β isoforms. These data revealed slightly increased TGF-β1 and markedly increased TGF-β2 levels. Significantly increased TGF-β2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-β2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-β signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-β1, but especially TGF-β2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-β pathway regulation in the pathogenesis of aortic aneurysms.

No MeSH data available.


Related in: MedlinePlus