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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

Hypoxia attenuates TGFß-induced SMAD2 phosphorylation in macrophages. Time-dependent Western blot analysis b, d and the corresponding statistical evaluation a, c of SMAD2 a, b and SMAD3 c, d phosphorylation in human primary macrophages exposed to TGFß under normoxia (−; nor) or hypoxia (1 % O2; hy). Macrophages were exposed to TGFß under normoxia vs. hypoxia for 8, 16, and 24 h, followed by (e) Western blot analysis of total SMAD2 and SMAD2 phosphorylation and corresponding statistical analysis f, g. Tubulin served as a loading control
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Fig1: Hypoxia attenuates TGFß-induced SMAD2 phosphorylation in macrophages. Time-dependent Western blot analysis b, d and the corresponding statistical evaluation a, c of SMAD2 a, b and SMAD3 c, d phosphorylation in human primary macrophages exposed to TGFß under normoxia (−; nor) or hypoxia (1 % O2; hy). Macrophages were exposed to TGFß under normoxia vs. hypoxia for 8, 16, and 24 h, followed by (e) Western blot analysis of total SMAD2 and SMAD2 phosphorylation and corresponding statistical analysis f, g. Tubulin served as a loading control

Mentions: Macrophages in the tumor microenvironment are exposed to hypoxia and TGFß but their signaling crosstalk has not been explored in detail. Therefore, we time-dependently stimulated primary human macrophages with 10 ng/mL TGFß under normoxia vs. hypoxia and followed phosphorylation of SMAD2 (p-SMAD2) and SMAD3 (p-SMAD3) by Western blot analysis. Resting macrophage did not show SMAD2 phosphorylation (Fig. 1a, b), whereas TGFß provoked a rapid SMAD2 phosphorylation within 15 min. Phospho-SMAD2 reached its maximum after 1 h both under normoxia and hypoxia. After 4 h, the level of p-SMAD2 gradually declined under hypoxia compared to normoxia. These differences became significant after 8 h (Fig. 1a, b). In contrast, the amount of p-SMAD3 rapidly increased upon TGFß addition, with phosphorylation not being affected by hypoxia. Although we noticed declining levels of p-SMAD3 at 4 to 8 h, this occurred irrespective to the presence or absence of oxygen (Fig. 1c, d). Apparently, in TGFβ-stimulated macrophages, only SMAD2, but not SMAD3, was sensitive to hypoxia. To explore this response for longer incubation times, we stimulated cells with TGFβ for 8 to 24 h at 20 % or 1 % O2. We observed decreased SMAD2 phosphorylation comparing 8 to 16 or 24 h, an effect drastically enhanced by hypoxia (Fig. 1e, f). Using an antibody to detect total SMAD2 protein showed a progressively reduced expression under hypoxia from 8 to 24 h (Fig. 1e, g). Apparently, hypoxia reduced phosphorylation of SMAD2 at 8 to 24 h following TGFβ-stimulation because of reduced SMAD2 protein expression.Fig. 1


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

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

Hypoxia attenuates TGFß-induced SMAD2 phosphorylation in macrophages. Time-dependent Western blot analysis b, d and the corresponding statistical evaluation a, c of SMAD2 a, b and SMAD3 c, d phosphorylation in human primary macrophages exposed to TGFß under normoxia (−; nor) or hypoxia (1 % O2; hy). Macrophages were exposed to TGFß under normoxia vs. hypoxia for 8, 16, and 24 h, followed by (e) Western blot analysis of total SMAD2 and SMAD2 phosphorylation and corresponding statistical analysis f, g. Tubulin served as a loading control
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Hypoxia attenuates TGFß-induced SMAD2 phosphorylation in macrophages. Time-dependent Western blot analysis b, d and the corresponding statistical evaluation a, c of SMAD2 a, b and SMAD3 c, d phosphorylation in human primary macrophages exposed to TGFß under normoxia (−; nor) or hypoxia (1 % O2; hy). Macrophages were exposed to TGFß under normoxia vs. hypoxia for 8, 16, and 24 h, followed by (e) Western blot analysis of total SMAD2 and SMAD2 phosphorylation and corresponding statistical analysis f, g. Tubulin served as a loading control
Mentions: Macrophages in the tumor microenvironment are exposed to hypoxia and TGFß but their signaling crosstalk has not been explored in detail. Therefore, we time-dependently stimulated primary human macrophages with 10 ng/mL TGFß under normoxia vs. hypoxia and followed phosphorylation of SMAD2 (p-SMAD2) and SMAD3 (p-SMAD3) by Western blot analysis. Resting macrophage did not show SMAD2 phosphorylation (Fig. 1a, b), whereas TGFß provoked a rapid SMAD2 phosphorylation within 15 min. Phospho-SMAD2 reached its maximum after 1 h both under normoxia and hypoxia. After 4 h, the level of p-SMAD2 gradually declined under hypoxia compared to normoxia. These differences became significant after 8 h (Fig. 1a, b). In contrast, the amount of p-SMAD3 rapidly increased upon TGFß addition, with phosphorylation not being affected by hypoxia. Although we noticed declining levels of p-SMAD3 at 4 to 8 h, this occurred irrespective to the presence or absence of oxygen (Fig. 1c, d). Apparently, in TGFβ-stimulated macrophages, only SMAD2, but not SMAD3, was sensitive to hypoxia. To explore this response for longer incubation times, we stimulated cells with TGFβ for 8 to 24 h at 20 % or 1 % O2. We observed decreased SMAD2 phosphorylation comparing 8 to 16 or 24 h, an effect drastically enhanced by hypoxia (Fig. 1e, f). Using an antibody to detect total SMAD2 protein showed a progressively reduced expression under hypoxia from 8 to 24 h (Fig. 1e, g). Apparently, hypoxia reduced phosphorylation of SMAD2 at 8 to 24 h following TGFβ-stimulation because of reduced SMAD2 protein expression.Fig. 1

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