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Hypoxia induces calpain activity and degrades SMAD2 to attenuate TGFβ signaling in macrophages.

Cui W, Zhou J, Dehne N, Brüne B - Cell Biosci (2015)

Bottom Line: Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation.The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2.Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, 100875 Beijing, China ; Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany.

ABSTRACT

Background: Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming growth factor β (TGFβ) shaping their functional plasticity. TGFβ is among the factors causing alternative macrophage activation, which contributes to tissue regeneration and thus, resolution of inflammation but may also provoke tumor progression. However, the signal crosstalk between TGFβ and hypoxia is ill defined.

Results: Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation. This early TGFβ-signaling remained unaffected by hypoxia. However, with prolonged exposure periods to TGFβ/hypoxia the expression of SMAD2 declined because of decreased protein stability. In parallel, hypoxia increased mRNA and protein amount of the calpain regulatory subunit, with the further notion that TGFβ/hypoxia elicited calpain activation. The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2. Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.

Conclusions: Hypoxia interferes with TGFβ signaling in macrophages by calpain-mediated proteolysis of the central signaling component SMAD2.

No MeSH data available.


Related in: MedlinePlus

Expression of TGFBR2 and SMAD7 in response to TGFß/hypoxia. Macrophages were transfected with non-targeting siRNA constructs (ctr), siRNA-HIF1α (si1α), or siRNA-HIF2α (si2α) and mRNA expression of TGFBR2 a or SMAD7 b were analyzed by quantitative PCR after treatments with TGFß under normoxia (nor) or hypoxia (hy) for 8 h. c Cell surface expression of TGFBR2 was followed by flow cytometry after stimulation of macrophages with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h and d mean fluorescence intensity was calculated from 4 individual experiments. e, f Time-dependent Western blot analysis and statistical evaluation of SMAD7 expression in macrophages exposed to TGFß under normoxia or hypoxia. g, h Macrophages were exposed to the phosphatase inhibitor okadaic acid (OA) 1 h prior to adding TGFß for 8 h. Phosphorylation and total protein expression of SMAD2 was followed by Western blot analysis, using tubulin as a loading control
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Fig2: Expression of TGFBR2 and SMAD7 in response to TGFß/hypoxia. Macrophages were transfected with non-targeting siRNA constructs (ctr), siRNA-HIF1α (si1α), or siRNA-HIF2α (si2α) and mRNA expression of TGFBR2 a or SMAD7 b were analyzed by quantitative PCR after treatments with TGFß under normoxia (nor) or hypoxia (hy) for 8 h. c Cell surface expression of TGFBR2 was followed by flow cytometry after stimulation of macrophages with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h and d mean fluorescence intensity was calculated from 4 individual experiments. e, f Time-dependent Western blot analysis and statistical evaluation of SMAD7 expression in macrophages exposed to TGFß under normoxia or hypoxia. g, h Macrophages were exposed to the phosphatase inhibitor okadaic acid (OA) 1 h prior to adding TGFß for 8 h. Phosphorylation and total protein expression of SMAD2 was followed by Western blot analysis, using tubulin as a loading control

Mentions: Several publications link SMAD activation to its subsequent degradation [21, 22]. Therefore, we started to validate TGFΒR2 and SMAD7 as potential HIF target genes that were previously identified by ChIP-Seq and mRNA array experiments [23]. TGFΒR2 is the receptor subunit that binds TGFß and phosphorylates the co-receptor TGFΒR1 to initiate intracellular signaling. An altered expression level of TGFBR2 will alter the ratio of TGFβ to its receptor, thereby modulating downstream signaling. SMAD7 is an inhibitory SMAD, which competes with SMAD2/3 at the TGFß receptor to control TGFß signal transmission. Increased expression under hypoxia may reduce SMAD2 activation. First, we validated microarray data by quantitative PCR 8 h after TGFß and/or hypoxia stimulation and verified hypoxic induction of TGFΒR2 mRNA (Fig. 2a). The knockdown of HIF-1α or HIF-2α, which was verified by Western blot analysis (Additional file 1: Figure S1A, B), reduced hypoxic TGFBR2 mRNA induction. mRNA expression of TGFBR2 under hypoxia/TGFß was lower compared to the hypoxic response. In contrast, SMAD7 was predominantly induced by TGFß, with the signal not being altered by a HIF-knockdown (Fig. 2b). Hypoxia only showed a small mRNA induction of SMAD7, while induction by hypoxia/TGFß was comparable to TGFß alone. A knockdown of either HIF-1α or HIF-2α slightly reduced hypoxic induction of SMAD7 and significantly reduced induction by hypoxia/TGFß, suggesting the involvement of HIF in hypoxic SMAD7 mRNA regulation.Fig. 2


Hypoxia induces calpain activity and degrades SMAD2 to attenuate TGFβ signaling in macrophages.

Cui W, Zhou J, Dehne N, Brüne B - Cell Biosci (2015)

Expression of TGFBR2 and SMAD7 in response to TGFß/hypoxia. Macrophages were transfected with non-targeting siRNA constructs (ctr), siRNA-HIF1α (si1α), or siRNA-HIF2α (si2α) and mRNA expression of TGFBR2 a or SMAD7 b were analyzed by quantitative PCR after treatments with TGFß under normoxia (nor) or hypoxia (hy) for 8 h. c Cell surface expression of TGFBR2 was followed by flow cytometry after stimulation of macrophages with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h and d mean fluorescence intensity was calculated from 4 individual experiments. e, f Time-dependent Western blot analysis and statistical evaluation of SMAD7 expression in macrophages exposed to TGFß under normoxia or hypoxia. g, h Macrophages were exposed to the phosphatase inhibitor okadaic acid (OA) 1 h prior to adding TGFß for 8 h. Phosphorylation and total protein expression of SMAD2 was followed by Western blot analysis, using tubulin as a loading control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4491253&req=5

Fig2: Expression of TGFBR2 and SMAD7 in response to TGFß/hypoxia. Macrophages were transfected with non-targeting siRNA constructs (ctr), siRNA-HIF1α (si1α), or siRNA-HIF2α (si2α) and mRNA expression of TGFBR2 a or SMAD7 b were analyzed by quantitative PCR after treatments with TGFß under normoxia (nor) or hypoxia (hy) for 8 h. c Cell surface expression of TGFBR2 was followed by flow cytometry after stimulation of macrophages with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h and d mean fluorescence intensity was calculated from 4 individual experiments. e, f Time-dependent Western blot analysis and statistical evaluation of SMAD7 expression in macrophages exposed to TGFß under normoxia or hypoxia. g, h Macrophages were exposed to the phosphatase inhibitor okadaic acid (OA) 1 h prior to adding TGFß for 8 h. Phosphorylation and total protein expression of SMAD2 was followed by Western blot analysis, using tubulin as a loading control
Mentions: Several publications link SMAD activation to its subsequent degradation [21, 22]. Therefore, we started to validate TGFΒR2 and SMAD7 as potential HIF target genes that were previously identified by ChIP-Seq and mRNA array experiments [23]. TGFΒR2 is the receptor subunit that binds TGFß and phosphorylates the co-receptor TGFΒR1 to initiate intracellular signaling. An altered expression level of TGFBR2 will alter the ratio of TGFβ to its receptor, thereby modulating downstream signaling. SMAD7 is an inhibitory SMAD, which competes with SMAD2/3 at the TGFß receptor to control TGFß signal transmission. Increased expression under hypoxia may reduce SMAD2 activation. First, we validated microarray data by quantitative PCR 8 h after TGFß and/or hypoxia stimulation and verified hypoxic induction of TGFΒR2 mRNA (Fig. 2a). The knockdown of HIF-1α or HIF-2α, which was verified by Western blot analysis (Additional file 1: Figure S1A, B), reduced hypoxic TGFBR2 mRNA induction. mRNA expression of TGFBR2 under hypoxia/TGFß was lower compared to the hypoxic response. In contrast, SMAD7 was predominantly induced by TGFß, with the signal not being altered by a HIF-knockdown (Fig. 2b). Hypoxia only showed a small mRNA induction of SMAD7, while induction by hypoxia/TGFß was comparable to TGFß alone. A knockdown of either HIF-1α or HIF-2α slightly reduced hypoxic induction of SMAD7 and significantly reduced induction by hypoxia/TGFß, suggesting the involvement of HIF in hypoxic SMAD7 mRNA regulation.Fig. 2

Bottom Line: Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation.The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2.Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, 100875 Beijing, China ; Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany.

ABSTRACT

Background: Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming growth factor β (TGFβ) shaping their functional plasticity. TGFβ is among the factors causing alternative macrophage activation, which contributes to tissue regeneration and thus, resolution of inflammation but may also provoke tumor progression. However, the signal crosstalk between TGFβ and hypoxia is ill defined.

Results: Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation. This early TGFβ-signaling remained unaffected by hypoxia. However, with prolonged exposure periods to TGFβ/hypoxia the expression of SMAD2 declined because of decreased protein stability. In parallel, hypoxia increased mRNA and protein amount of the calpain regulatory subunit, with the further notion that TGFβ/hypoxia elicited calpain activation. The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2. Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.

Conclusions: Hypoxia interferes with TGFβ signaling in macrophages by calpain-mediated proteolysis of the central signaling component SMAD2.

No MeSH data available.


Related in: MedlinePlus