Smad7 interrupts TGF-β signaling in intestinal macrophages and promotes inflammatory activation of these cells during necrotizing enterocolitis

Mohan Krishnan, Kopperuncholan Namachivayam, Kalyan C. Chapalamadugu, Steven A. Garzon, Muralidhar H. Premkumar, Srinivas M. Tipparaju, Akhil Maheshwari

Research output: Contribution to journalArticle

Abstract

Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-β signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusion: Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

Original languageEnglish (US)
Pages (from-to)951-961
Number of pages11
JournalPediatric Research
Volume79
Issue number6
DOIs
StatePublished - Jun 1 2016
Externally publishedYes

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Necrotizing Enterocolitis
Transforming Growth Factors
Macrophages
NF-kappa B
I-kappa B Kinase
Bacterial Load
Chromatin Immunoprecipitation
Luciferases
Premature Infants
Transcriptional Activation
Lipopolysaccharides
Necrosis
Phosphotransferases
Western Blotting
Immunohistochemistry
Cytokines
Polymerase Chain Reaction
Wounds and Injuries

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health

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Smad7 interrupts TGF-β signaling in intestinal macrophages and promotes inflammatory activation of these cells during necrotizing enterocolitis. / Krishnan, Mohan; Namachivayam, Kopperuncholan; Chapalamadugu, Kalyan C.; Garzon, Steven A.; Premkumar, Muralidhar H.; Tipparaju, Srinivas M.; Maheshwari, Akhil.

In: Pediatric Research, Vol. 79, No. 6, 01.06.2016, p. 951-961.

Research output: Contribution to journalArticle

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abstract = "Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-β signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusion: Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.",
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AU - Namachivayam, Kopperuncholan

AU - Chapalamadugu, Kalyan C.

AU - Garzon, Steven A.

AU - Premkumar, Muralidhar H.

AU - Tipparaju, Srinivas M.

AU - Maheshwari, Akhil

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N2 - Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-β signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusion: Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

AB - Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-β signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusion: Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

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