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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 degradation under hypoxia is facilitated by calpain. a-f Macrophages were stimulated with TGFß under normoxia vs. hypoxia for 8 h in the presence of bafilomycin a, b, MG132 c, d, or lactacystin e, f. 2 h after TGFß, inhibitors were added and incubations went on for 6 h. Western blot analysis and statistical evaluation is presented. g mRNA expression of the calpain regulatory subunit (CAPNS1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). h Western blot analysis of the calpain regulatory subunit after exposure to TGFß and/or hypoxia for 8 h. i mRNA expression of the calpain 1 catalytic subunit (CAPN1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). j Proteolytic cleavage of CAPN1 under TGFß/hypoxia was followed in macrophages by Western blot analysis after 8 h. k Macrophages were exposed to the calpain inhibitor 1 (CPI) to follow SMAD2 degradation after stimulating cells for 8 h with TGFß under normoxia vs. hypoxia
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Fig4: SMAD2 degradation under hypoxia is facilitated by calpain. a-f Macrophages were stimulated with TGFß under normoxia vs. hypoxia for 8 h in the presence of bafilomycin a, b, MG132 c, d, or lactacystin e, f. 2 h after TGFß, inhibitors were added and incubations went on for 6 h. Western blot analysis and statistical evaluation is presented. g mRNA expression of the calpain regulatory subunit (CAPNS1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). h Western blot analysis of the calpain regulatory subunit after exposure to TGFß and/or hypoxia for 8 h. i mRNA expression of the calpain 1 catalytic subunit (CAPN1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). j Proteolytic cleavage of CAPN1 under TGFß/hypoxia was followed in macrophages by Western blot analysis after 8 h. k Macrophages were exposed to the calpain inhibitor 1 (CPI) to follow SMAD2 degradation after stimulating cells for 8 h with TGFß under normoxia vs. hypoxia

Mentions: Considering increased degradation of SMAD2 under TGFß/hypoxia, we analyzed pathways being involved. Using bafilomycin A1 to inhibit lysosomal functions, SMAD2 degradation remained unaffected thus, ruling the involvement of the lysosomal compartment out (Fig. 4a, b). We then used MG132 to block proteasomal- and calpain-dependent degradation systems. MG132 rescued SMAD2 degradation in response to TGFß/hypoxia, while lactacystin, a more specific proteasomal inhibitor, not affecting calpain, failed to restore SMAD2 expression under these conditions (Fig. 4c-f). Calpain is a calcium-dependent non-lysosomal cysteine proteolytic system that comprises a small regulatory subunit (CAPNS1, also known as calpain reg) and a large catalytic subunit (μ-calpain/m-calpain, also known as CAPN). We tested mRNA expression of CAPNS1 in macrophages stimulated with TGFß under normoxia and hypoxia. CAPNS1 mRNA was significantly upregulated by hypoxia or hypoxia/TGFß compared to normoxia or TGFß-stimulation (Fig. 4g). The mRNA increase became also apparent at the protein level (Fig. 4h). Expression of the catalytic subunit (CAPN1) was not affected by TGFβ or hypoxia nor their combination (Fig. 4i). Following macrophage activation with hypoxia/TGFß, holo-calpain (80KD) is proteolytically processed and the removal of 14 or 28 amino acids forms active calpain. Western blot analysis detected active calpain in macrophages stimulated with TGFß/hypoxia but not with TGFβ or hypoxia alone (Fig. 4j). Experiments with the specific calpain inhibitor 1 CPI (10 μM), showed that decreased SMAD2 expression seen under hypoxia/TGFß was fully recovered when calpain activity was blocked (Fig. 4k). Conclusively, in TGFß-stimulated macrophages hypoxia promotes degradation of SMAD2 by increasing calpain activity.Fig. 4


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 degradation under hypoxia is facilitated by calpain. a-f Macrophages were stimulated with TGFß under normoxia vs. hypoxia for 8 h in the presence of bafilomycin a, b, MG132 c, d, or lactacystin e, f. 2 h after TGFß, inhibitors were added and incubations went on for 6 h. Western blot analysis and statistical evaluation is presented. g mRNA expression of the calpain regulatory subunit (CAPNS1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). h Western blot analysis of the calpain regulatory subunit after exposure to TGFß and/or hypoxia for 8 h. i mRNA expression of the calpain 1 catalytic subunit (CAPN1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). j Proteolytic cleavage of CAPN1 under TGFß/hypoxia was followed in macrophages by Western blot analysis after 8 h. k Macrophages were exposed to the calpain inhibitor 1 (CPI) to follow SMAD2 degradation after stimulating cells for 8 h with TGFß under normoxia vs. hypoxia
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: SMAD2 degradation under hypoxia is facilitated by calpain. a-f Macrophages were stimulated with TGFß under normoxia vs. hypoxia for 8 h in the presence of bafilomycin a, b, MG132 c, d, or lactacystin e, f. 2 h after TGFß, inhibitors were added and incubations went on for 6 h. Western blot analysis and statistical evaluation is presented. g mRNA expression of the calpain regulatory subunit (CAPNS1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). h Western blot analysis of the calpain regulatory subunit after exposure to TGFß and/or hypoxia for 8 h. i mRNA expression of the calpain 1 catalytic subunit (CAPN1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy). j Proteolytic cleavage of CAPN1 under TGFß/hypoxia was followed in macrophages by Western blot analysis after 8 h. k Macrophages were exposed to the calpain inhibitor 1 (CPI) to follow SMAD2 degradation after stimulating cells for 8 h with TGFß under normoxia vs. hypoxia
Mentions: Considering increased degradation of SMAD2 under TGFß/hypoxia, we analyzed pathways being involved. Using bafilomycin A1 to inhibit lysosomal functions, SMAD2 degradation remained unaffected thus, ruling the involvement of the lysosomal compartment out (Fig. 4a, b). We then used MG132 to block proteasomal- and calpain-dependent degradation systems. MG132 rescued SMAD2 degradation in response to TGFß/hypoxia, while lactacystin, a more specific proteasomal inhibitor, not affecting calpain, failed to restore SMAD2 expression under these conditions (Fig. 4c-f). Calpain is a calcium-dependent non-lysosomal cysteine proteolytic system that comprises a small regulatory subunit (CAPNS1, also known as calpain reg) and a large catalytic subunit (μ-calpain/m-calpain, also known as CAPN). We tested mRNA expression of CAPNS1 in macrophages stimulated with TGFß under normoxia and hypoxia. CAPNS1 mRNA was significantly upregulated by hypoxia or hypoxia/TGFß compared to normoxia or TGFß-stimulation (Fig. 4g). The mRNA increase became also apparent at the protein level (Fig. 4h). Expression of the catalytic subunit (CAPN1) was not affected by TGFβ or hypoxia nor their combination (Fig. 4i). Following macrophage activation with hypoxia/TGFß, holo-calpain (80KD) is proteolytically processed and the removal of 14 or 28 amino acids forms active calpain. Western blot analysis detected active calpain in macrophages stimulated with TGFß/hypoxia but not with TGFβ or hypoxia alone (Fig. 4j). Experiments with the specific calpain inhibitor 1 CPI (10 μM), showed that decreased SMAD2 expression seen under hypoxia/TGFß was fully recovered when calpain activity was blocked (Fig. 4k). Conclusively, in TGFß-stimulated macrophages hypoxia promotes degradation of SMAD2 by increasing calpain activity.Fig. 4

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