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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 reduces SMAD2-dependent transcription. a TGFß-induced transcriptional activity in J774A.1 cells transfected with a SBE4-Luc (4xSMAD binding element coupled to luciferase) as well as a Renilla-Luc plasmid. Stimulation was with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h. mRNA expression of b thrombospondin1 (TSP1), c dystonin DST, d matrix metalloproteinase (MMP2), e cystatin (CST6), and (f) plasminogen activator inhibitor 1 (PAI1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy)
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Fig5: Hypoxia reduces SMAD2-dependent transcription. a TGFß-induced transcriptional activity in J774A.1 cells transfected with a SBE4-Luc (4xSMAD binding element coupled to luciferase) as well as a Renilla-Luc plasmid. Stimulation was with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h. mRNA expression of b thrombospondin1 (TSP1), c dystonin DST, d matrix metalloproteinase (MMP2), e cystatin (CST6), and (f) plasminogen activator inhibitor 1 (PAI1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy)

Mentions: To explore how hypoxia affects TGFß-induced transcriptional activity, we used a SMAD-specific reporter assay. Due to difficulties in transfecting primary human macrophages we used the J774A.1 mouse macrophage cell line, which responded with SMAD2 phosphorylation in response to TGFß, also showing a decrease in SMAD2 expression with TGFß/hypoxia under longer exposure times (Additional file 1: Figure S1C, D). Stimulation of J774A.1 cells, transfected with the SMAD reporter plasmid SEB4-Luc, with TGFß under hypoxia significantly reduced transcriptional activity compared to TGFß supplied under normoxia (Fig. 5a). We then analyzed expression of SAMD2 and SMAD3 target genes in primary human macrophages. Thrombospondin 1 (TSP1), dystonin (DST), and matrix metalloproteinase 2 (MMP2) were reported to be selectively regulated by SMAD2 [25–27] and we confirmed their responsiveness to TGFβ (Fig. 5b, c, d). Induction was significantly reduced in macrophages stimulated with TGFβ at 1 % O2, reflecting the reduced expression of SMAD2. In contrast induction of SMAD3 target genes such as cystatin (CST6) and plasminogen activator inhibitor 1 (PAI1) [25] was not reduced under hypoxia (Fig. 5e, f). Apparently, only transcriptional responses of SMAD2 are selectively reduced by hypoxia in TGFβ-stimulated human macrophages.Fig. 5


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 reduces SMAD2-dependent transcription. a TGFß-induced transcriptional activity in J774A.1 cells transfected with a SBE4-Luc (4xSMAD binding element coupled to luciferase) as well as a Renilla-Luc plasmid. Stimulation was with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h. mRNA expression of b thrombospondin1 (TSP1), c dystonin DST, d matrix metalloproteinase (MMP2), e cystatin (CST6), and (f) plasminogen activator inhibitor 1 (PAI1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Hypoxia reduces SMAD2-dependent transcription. a TGFß-induced transcriptional activity in J774A.1 cells transfected with a SBE4-Luc (4xSMAD binding element coupled to luciferase) as well as a Renilla-Luc plasmid. Stimulation was with TGFß under normoxia (nor) vs. hypoxia (hy) for 8 h. mRNA expression of b thrombospondin1 (TSP1), c dystonin DST, d matrix metalloproteinase (MMP2), e cystatin (CST6), and (f) plasminogen activator inhibitor 1 (PAI1) was analyzed by quantitative PCR in macrophages exposed for 8 h to TGFß under normoxia (nor) vs. hypoxia (hy)
Mentions: To explore how hypoxia affects TGFß-induced transcriptional activity, we used a SMAD-specific reporter assay. Due to difficulties in transfecting primary human macrophages we used the J774A.1 mouse macrophage cell line, which responded with SMAD2 phosphorylation in response to TGFß, also showing a decrease in SMAD2 expression with TGFß/hypoxia under longer exposure times (Additional file 1: Figure S1C, D). Stimulation of J774A.1 cells, transfected with the SMAD reporter plasmid SEB4-Luc, with TGFß under hypoxia significantly reduced transcriptional activity compared to TGFß supplied under normoxia (Fig. 5a). We then analyzed expression of SAMD2 and SMAD3 target genes in primary human macrophages. Thrombospondin 1 (TSP1), dystonin (DST), and matrix metalloproteinase 2 (MMP2) were reported to be selectively regulated by SMAD2 [25–27] and we confirmed their responsiveness to TGFβ (Fig. 5b, c, d). Induction was significantly reduced in macrophages stimulated with TGFβ at 1 % O2, reflecting the reduced expression of SMAD2. In contrast induction of SMAD3 target genes such as cystatin (CST6) and plasminogen activator inhibitor 1 (PAI1) [25] was not reduced under hypoxia (Fig. 5e, f). Apparently, only transcriptional responses of SMAD2 are selectively reduced by hypoxia in TGFβ-stimulated human macrophages.Fig. 5

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