ACTIVIN B PRODUCED BY SINUSOIDAL ENDOTHELIAL/KUPFFER CELLS IN RESPONSE TO INFLAMMATORY STIMULI INDUCES HEPCIDIN EXPRESSION THROUGH UNCANONICAL SIGNALING IN HEPATOCYTES
(Abstract release date: 05/19/16)
EHA Library. Kanamori Y. 06/09/16; 135377; LB2266
Mr. Yohei Kanamori
Contributions
Contributions
Abstract
Abstract: LB2266
Type: Eposter Presentation
Background
Hepcidin, a liver-derived peptide hormone, is involved in the pathogenesis of anemia under inflammation. Activins and BMPs bind to corresponding type I and type II receptors and phosphorylate Smad2/3 and Smad1/5/8, respectively, as canonical signaling pathway. BMPs are well known to up-regulate hepcidin expression through the canonical signaling pathway when hepatocytes accumulate excess iron. On the other hand, activin B is suggested as a key molecule responsible for the up-regulation of hepcidin in inflammation based on the following two evidences: (1) the elevation of activin B mRNA level in the livers of LPS-injected mice, and (2) the stimulation of hepcidin expression by activin B in hepatocytes. Furthermore, activin B has been shown to activate uncanonical Smad1/5/8 signaling as well as canonical Smad2/3 signaling in hepatocytes.
Aims
We examine the following three points in the present study: (1) which cell-types in the liver are responsible for the up-regulation of activin B by inflammatory stimuli, (2) which receptor combination is responsible for the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B, and (3) whether activin B induces uncanonical Smad1/5/8 signaling in extrahepatic cells.
Methods
A male Sprague-Dawley rat was intraperitoneally injected with LPS (5 mg/kg). After 2.5 h of injection, hepatocytes and non-parenchymal cells were collected from the liver by collagenase-perfusion. Sinusoidal endothelial and Kupffer cell-rich fraction was isolated from non-parenchymal cells by a two-step density gradient centrifugation. The expression of activin B was measured by RT-qPCR.Rat primary hepatocytes, HepG2 human hepatoma cells, C2C12 myoblasts, 3T3-L1 pre-adipocytes, and RAW264.7 murine macrophage-like cells were stimulated with 2 nM of activin B. Phosphorylation of Smad1/5/8 and mRNA level of hepcidin or Id1 were examined.HepG2 cells were transfected with siRNAs of type I or type II receptors and luciferase reporter of murine hepcidin promoter. After transfection, cells were treated with activin B. Transcriptional activity of hepcidin was assayed.
Results
Significant expression of activin B was detected in sinusoidal endothelial and Kupffer cell-enriched fraction isolated from the liver of a rat injected with LPS, while expression of activin B in hepatocyte-enriched fraction was almost negligible, suggesting that sinusoidal endothelial cells or Kupffer cells are the major sources of hepatic activin B under inflammation. Knockdown of the BMP type I receptor, ALK2 or ALK3, indicated that ALK2, but not ALK3, is involved in the responsiveness of the hepcidin promoter to activin B through the activation of Smad1/5/8 signaling in HepG2 cells. Knockdown of the type II receptor ActRIIA inhibited activin B-mediated Smad1/5/8 phosphorylation and hepcidin transcription. These results suggest that upon the binding of activin B, the receptor complex, ALK2 and ActRIIA, stimulates Smad1/5/8 signaling, followed by induction of hepcidin. Unexpectedly, the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B was resistant to LDN-193189, an inhibitor of ALK2 and ALK3, whereas those actions of BMP2 was sensitive to the inhibitor. These results implicate that unknown mechanisms distinct from BMP signaling are involved in the modulation of Smad1/5/8 signaling by activin B. We also showed that activin B increased phosphorylation of Smad1/5/8 and mRNA level of Id1, a representative BMP response gene, in C2C12 cells, 3T3-L1 cells and RAW264.7 cells, implicating that activin B-induced uncanonical Smad1/5/8 signaling is independent of cell-type.
Conclusion
Upon inflammatory stimuli, sinusoidal endothelial cells and/or Kupffer cells up-regulate activin B expression in the liver, followed by induction of Smad1/5/8 phosphorylation and hepcidin expression in hepatocytes via the receptor complex of ALK2 and ActRIIA. In addition, activin B-induced Smad1/5/8 signaling is not limited to hepatocytes. The present study provide novel insights into the molecular mechanisms underlying induction of hepcidin by inflammation and the possible roles of activin B-mediated Smad1/5/8 signaling in several tissues.
Session topic: E-poster
Keyword(s): Anemia, Hepcidin, Inflammation
Type: Eposter Presentation
Background
Hepcidin, a liver-derived peptide hormone, is involved in the pathogenesis of anemia under inflammation. Activins and BMPs bind to corresponding type I and type II receptors and phosphorylate Smad2/3 and Smad1/5/8, respectively, as canonical signaling pathway. BMPs are well known to up-regulate hepcidin expression through the canonical signaling pathway when hepatocytes accumulate excess iron. On the other hand, activin B is suggested as a key molecule responsible for the up-regulation of hepcidin in inflammation based on the following two evidences: (1) the elevation of activin B mRNA level in the livers of LPS-injected mice, and (2) the stimulation of hepcidin expression by activin B in hepatocytes. Furthermore, activin B has been shown to activate uncanonical Smad1/5/8 signaling as well as canonical Smad2/3 signaling in hepatocytes.
Aims
We examine the following three points in the present study: (1) which cell-types in the liver are responsible for the up-regulation of activin B by inflammatory stimuli, (2) which receptor combination is responsible for the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B, and (3) whether activin B induces uncanonical Smad1/5/8 signaling in extrahepatic cells.
Methods
A male Sprague-Dawley rat was intraperitoneally injected with LPS (5 mg/kg). After 2.5 h of injection, hepatocytes and non-parenchymal cells were collected from the liver by collagenase-perfusion. Sinusoidal endothelial and Kupffer cell-rich fraction was isolated from non-parenchymal cells by a two-step density gradient centrifugation. The expression of activin B was measured by RT-qPCR.Rat primary hepatocytes, HepG2 human hepatoma cells, C2C12 myoblasts, 3T3-L1 pre-adipocytes, and RAW264.7 murine macrophage-like cells were stimulated with 2 nM of activin B. Phosphorylation of Smad1/5/8 and mRNA level of hepcidin or Id1 were examined.HepG2 cells were transfected with siRNAs of type I or type II receptors and luciferase reporter of murine hepcidin promoter. After transfection, cells were treated with activin B. Transcriptional activity of hepcidin was assayed.
Results
Significant expression of activin B was detected in sinusoidal endothelial and Kupffer cell-enriched fraction isolated from the liver of a rat injected with LPS, while expression of activin B in hepatocyte-enriched fraction was almost negligible, suggesting that sinusoidal endothelial cells or Kupffer cells are the major sources of hepatic activin B under inflammation. Knockdown of the BMP type I receptor, ALK2 or ALK3, indicated that ALK2, but not ALK3, is involved in the responsiveness of the hepcidin promoter to activin B through the activation of Smad1/5/8 signaling in HepG2 cells. Knockdown of the type II receptor ActRIIA inhibited activin B-mediated Smad1/5/8 phosphorylation and hepcidin transcription. These results suggest that upon the binding of activin B, the receptor complex, ALK2 and ActRIIA, stimulates Smad1/5/8 signaling, followed by induction of hepcidin. Unexpectedly, the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B was resistant to LDN-193189, an inhibitor of ALK2 and ALK3, whereas those actions of BMP2 was sensitive to the inhibitor. These results implicate that unknown mechanisms distinct from BMP signaling are involved in the modulation of Smad1/5/8 signaling by activin B. We also showed that activin B increased phosphorylation of Smad1/5/8 and mRNA level of Id1, a representative BMP response gene, in C2C12 cells, 3T3-L1 cells and RAW264.7 cells, implicating that activin B-induced uncanonical Smad1/5/8 signaling is independent of cell-type.
Conclusion
Upon inflammatory stimuli, sinusoidal endothelial cells and/or Kupffer cells up-regulate activin B expression in the liver, followed by induction of Smad1/5/8 phosphorylation and hepcidin expression in hepatocytes via the receptor complex of ALK2 and ActRIIA. In addition, activin B-induced Smad1/5/8 signaling is not limited to hepatocytes. The present study provide novel insights into the molecular mechanisms underlying induction of hepcidin by inflammation and the possible roles of activin B-mediated Smad1/5/8 signaling in several tissues.
Session topic: E-poster
Keyword(s): Anemia, Hepcidin, Inflammation
Abstract: LB2266
Type: Eposter Presentation
Background
Hepcidin, a liver-derived peptide hormone, is involved in the pathogenesis of anemia under inflammation. Activins and BMPs bind to corresponding type I and type II receptors and phosphorylate Smad2/3 and Smad1/5/8, respectively, as canonical signaling pathway. BMPs are well known to up-regulate hepcidin expression through the canonical signaling pathway when hepatocytes accumulate excess iron. On the other hand, activin B is suggested as a key molecule responsible for the up-regulation of hepcidin in inflammation based on the following two evidences: (1) the elevation of activin B mRNA level in the livers of LPS-injected mice, and (2) the stimulation of hepcidin expression by activin B in hepatocytes. Furthermore, activin B has been shown to activate uncanonical Smad1/5/8 signaling as well as canonical Smad2/3 signaling in hepatocytes.
Aims
We examine the following three points in the present study: (1) which cell-types in the liver are responsible for the up-regulation of activin B by inflammatory stimuli, (2) which receptor combination is responsible for the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B, and (3) whether activin B induces uncanonical Smad1/5/8 signaling in extrahepatic cells.
Methods
A male Sprague-Dawley rat was intraperitoneally injected with LPS (5 mg/kg). After 2.5 h of injection, hepatocytes and non-parenchymal cells were collected from the liver by collagenase-perfusion. Sinusoidal endothelial and Kupffer cell-rich fraction was isolated from non-parenchymal cells by a two-step density gradient centrifugation. The expression of activin B was measured by RT-qPCR.Rat primary hepatocytes, HepG2 human hepatoma cells, C2C12 myoblasts, 3T3-L1 pre-adipocytes, and RAW264.7 murine macrophage-like cells were stimulated with 2 nM of activin B. Phosphorylation of Smad1/5/8 and mRNA level of hepcidin or Id1 were examined.HepG2 cells were transfected with siRNAs of type I or type II receptors and luciferase reporter of murine hepcidin promoter. After transfection, cells were treated with activin B. Transcriptional activity of hepcidin was assayed.
Results
Significant expression of activin B was detected in sinusoidal endothelial and Kupffer cell-enriched fraction isolated from the liver of a rat injected with LPS, while expression of activin B in hepatocyte-enriched fraction was almost negligible, suggesting that sinusoidal endothelial cells or Kupffer cells are the major sources of hepatic activin B under inflammation. Knockdown of the BMP type I receptor, ALK2 or ALK3, indicated that ALK2, but not ALK3, is involved in the responsiveness of the hepcidin promoter to activin B through the activation of Smad1/5/8 signaling in HepG2 cells. Knockdown of the type II receptor ActRIIA inhibited activin B-mediated Smad1/5/8 phosphorylation and hepcidin transcription. These results suggest that upon the binding of activin B, the receptor complex, ALK2 and ActRIIA, stimulates Smad1/5/8 signaling, followed by induction of hepcidin. Unexpectedly, the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B was resistant to LDN-193189, an inhibitor of ALK2 and ALK3, whereas those actions of BMP2 was sensitive to the inhibitor. These results implicate that unknown mechanisms distinct from BMP signaling are involved in the modulation of Smad1/5/8 signaling by activin B. We also showed that activin B increased phosphorylation of Smad1/5/8 and mRNA level of Id1, a representative BMP response gene, in C2C12 cells, 3T3-L1 cells and RAW264.7 cells, implicating that activin B-induced uncanonical Smad1/5/8 signaling is independent of cell-type.
Conclusion
Upon inflammatory stimuli, sinusoidal endothelial cells and/or Kupffer cells up-regulate activin B expression in the liver, followed by induction of Smad1/5/8 phosphorylation and hepcidin expression in hepatocytes via the receptor complex of ALK2 and ActRIIA. In addition, activin B-induced Smad1/5/8 signaling is not limited to hepatocytes. The present study provide novel insights into the molecular mechanisms underlying induction of hepcidin by inflammation and the possible roles of activin B-mediated Smad1/5/8 signaling in several tissues.
Session topic: E-poster
Keyword(s): Anemia, Hepcidin, Inflammation
Type: Eposter Presentation
Background
Hepcidin, a liver-derived peptide hormone, is involved in the pathogenesis of anemia under inflammation. Activins and BMPs bind to corresponding type I and type II receptors and phosphorylate Smad2/3 and Smad1/5/8, respectively, as canonical signaling pathway. BMPs are well known to up-regulate hepcidin expression through the canonical signaling pathway when hepatocytes accumulate excess iron. On the other hand, activin B is suggested as a key molecule responsible for the up-regulation of hepcidin in inflammation based on the following two evidences: (1) the elevation of activin B mRNA level in the livers of LPS-injected mice, and (2) the stimulation of hepcidin expression by activin B in hepatocytes. Furthermore, activin B has been shown to activate uncanonical Smad1/5/8 signaling as well as canonical Smad2/3 signaling in hepatocytes.
Aims
We examine the following three points in the present study: (1) which cell-types in the liver are responsible for the up-regulation of activin B by inflammatory stimuli, (2) which receptor combination is responsible for the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B, and (3) whether activin B induces uncanonical Smad1/5/8 signaling in extrahepatic cells.
Methods
A male Sprague-Dawley rat was intraperitoneally injected with LPS (5 mg/kg). After 2.5 h of injection, hepatocytes and non-parenchymal cells were collected from the liver by collagenase-perfusion. Sinusoidal endothelial and Kupffer cell-rich fraction was isolated from non-parenchymal cells by a two-step density gradient centrifugation. The expression of activin B was measured by RT-qPCR.Rat primary hepatocytes, HepG2 human hepatoma cells, C2C12 myoblasts, 3T3-L1 pre-adipocytes, and RAW264.7 murine macrophage-like cells were stimulated with 2 nM of activin B. Phosphorylation of Smad1/5/8 and mRNA level of hepcidin or Id1 were examined.HepG2 cells were transfected with siRNAs of type I or type II receptors and luciferase reporter of murine hepcidin promoter. After transfection, cells were treated with activin B. Transcriptional activity of hepcidin was assayed.
Results
Significant expression of activin B was detected in sinusoidal endothelial and Kupffer cell-enriched fraction isolated from the liver of a rat injected with LPS, while expression of activin B in hepatocyte-enriched fraction was almost negligible, suggesting that sinusoidal endothelial cells or Kupffer cells are the major sources of hepatic activin B under inflammation. Knockdown of the BMP type I receptor, ALK2 or ALK3, indicated that ALK2, but not ALK3, is involved in the responsiveness of the hepcidin promoter to activin B through the activation of Smad1/5/8 signaling in HepG2 cells. Knockdown of the type II receptor ActRIIA inhibited activin B-mediated Smad1/5/8 phosphorylation and hepcidin transcription. These results suggest that upon the binding of activin B, the receptor complex, ALK2 and ActRIIA, stimulates Smad1/5/8 signaling, followed by induction of hepcidin. Unexpectedly, the induction of Smad1/5/8 phosphorylation and hepcidin expression by activin B was resistant to LDN-193189, an inhibitor of ALK2 and ALK3, whereas those actions of BMP2 was sensitive to the inhibitor. These results implicate that unknown mechanisms distinct from BMP signaling are involved in the modulation of Smad1/5/8 signaling by activin B. We also showed that activin B increased phosphorylation of Smad1/5/8 and mRNA level of Id1, a representative BMP response gene, in C2C12 cells, 3T3-L1 cells and RAW264.7 cells, implicating that activin B-induced uncanonical Smad1/5/8 signaling is independent of cell-type.
Conclusion
Upon inflammatory stimuli, sinusoidal endothelial cells and/or Kupffer cells up-regulate activin B expression in the liver, followed by induction of Smad1/5/8 phosphorylation and hepcidin expression in hepatocytes via the receptor complex of ALK2 and ActRIIA. In addition, activin B-induced Smad1/5/8 signaling is not limited to hepatocytes. The present study provide novel insights into the molecular mechanisms underlying induction of hepcidin by inflammation and the possible roles of activin B-mediated Smad1/5/8 signaling in several tissues.
Session topic: E-poster
Keyword(s): Anemia, Hepcidin, Inflammation
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