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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

SMAD2 protein stability is reduced in response to TGFß/hypoxia. a mRNA expression of SMAD2 followed by quantitative PCR in macrophages stimulated for 8 h with TGFß under normoxia (nor) vs. hypoxia (hy). b Macrophages were stimulated with TGFß for 4 h, followed by the addition of cycloheximide for 0 h to 4 h and subsequent Western blot analysis of SMAD2 expression. c Half-life determination of SMAD2 in TGFß-stimulated macrophages under normoxia (nor) vs. hypoxia (hy) as a result of experiments performed in Fig. 3b
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Fig3: SMAD2 protein stability is reduced in response to TGFß/hypoxia. a mRNA expression of SMAD2 followed by quantitative PCR in macrophages stimulated for 8 h with TGFß under normoxia (nor) vs. hypoxia (hy). b Macrophages were stimulated with TGFß for 4 h, followed by the addition of cycloheximide for 0 h to 4 h and subsequent Western blot analysis of SMAD2 expression. c Half-life determination of SMAD2 in TGFß-stimulated macrophages under normoxia (nor) vs. hypoxia (hy) as a result of experiments performed in Fig. 3b

Mentions: Next, we considered phosphatases to decrease SMAD2 phosphorylation. It was reported that PP2A dephosphorylates p-SMAD3 under hypoxia [24]. Supplying the PP2A inhibitor okadaic acid (OA) 1 h prior to addition of TGFß for 8 h failed to rescue the decrease of phoshorylated or total SAMD2 under TGFß/hypoxia (Fig. 2h). Excluding SMAD2 activation or dephoshorylation as an explanation for its decreased protein amount, we went on to analyze SMAD2 mRNA. In general, protein expression is either regulated by an altered mRNA expression, reduced translation, and/or enhanced degradation. SMAD2 mRNA expression was slightly induced but not reduced by hypoxia (Fig. 3a). Therefore, we examined protein stability by suppressing translation using 10 μg/ml cycloheximide (CHX). SMAD2 was degraded faster, comparing hypoxia to normoxia (Fig. 3b). The half-life of SAMD2 under normoxia/TGFß was calculated to be 8.9 ± 1.2 h, while it was reduced to 5.1 ± 0.6 h under hypoxia/TGFß (Fig. 3c). In conclusion, SMAD2 is destabilized under hypoxia in TGFß-stimulated macrophages.Fig. 3


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

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

SMAD2 protein stability is reduced in response to TGFß/hypoxia. a mRNA expression of SMAD2 followed by quantitative PCR in macrophages stimulated for 8 h with TGFß under normoxia (nor) vs. hypoxia (hy). b Macrophages were stimulated with TGFß for 4 h, followed by the addition of cycloheximide for 0 h to 4 h and subsequent Western blot analysis of SMAD2 expression. c Half-life determination of SMAD2 in TGFß-stimulated macrophages under normoxia (nor) vs. hypoxia (hy) as a result of experiments performed in Fig. 3b
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491253&req=5

Fig3: SMAD2 protein stability is reduced in response to TGFß/hypoxia. a mRNA expression of SMAD2 followed by quantitative PCR in macrophages stimulated for 8 h with TGFß under normoxia (nor) vs. hypoxia (hy). b Macrophages were stimulated with TGFß for 4 h, followed by the addition of cycloheximide for 0 h to 4 h and subsequent Western blot analysis of SMAD2 expression. c Half-life determination of SMAD2 in TGFß-stimulated macrophages under normoxia (nor) vs. hypoxia (hy) as a result of experiments performed in Fig. 3b
Mentions: Next, we considered phosphatases to decrease SMAD2 phosphorylation. It was reported that PP2A dephosphorylates p-SMAD3 under hypoxia [24]. Supplying the PP2A inhibitor okadaic acid (OA) 1 h prior to addition of TGFß for 8 h failed to rescue the decrease of phoshorylated or total SAMD2 under TGFß/hypoxia (Fig. 2h). Excluding SMAD2 activation or dephoshorylation as an explanation for its decreased protein amount, we went on to analyze SMAD2 mRNA. In general, protein expression is either regulated by an altered mRNA expression, reduced translation, and/or enhanced degradation. SMAD2 mRNA expression was slightly induced but not reduced by hypoxia (Fig. 3a). Therefore, we examined protein stability by suppressing translation using 10 μg/ml cycloheximide (CHX). SMAD2 was degraded faster, comparing hypoxia to normoxia (Fig. 3b). The half-life of SAMD2 under normoxia/TGFß was calculated to be 8.9 ± 1.2 h, while it was reduced to 5.1 ± 0.6 h under hypoxia/TGFß (Fig. 3c). In conclusion, SMAD2 is destabilized under hypoxia in TGFß-stimulated macrophages.Fig. 3

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