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Activin B is induced in response to inflammation in the liver and enhances hepcidin expression, but the source of activin B and the molecular mechanism underlying hepcidin induction are not clear yet. Lipopolysaccharide (LPS)-induced inflammation induced inhibin βB but not inhibin α or inhibin βA expression in the liver, implicating activin B induction. Immunoreactive inhibin βB was detected in endothelial cells and Kupffer cells in LPS-treated liver. Activin B, but not activin A or activin AB, directly increased hepcidin expression. Activin B induced phosphorylation and activation of Smad1/5/8, the BMP-regulated (BR)-Smads. The stimulation of hepcidin transcription by activin B was mediated by ALK2 and ActRIIA, receptors for the TGF-β family. Unexpectedly, activin B-induced hepcidin expression and BR-Smad phosphorylation were resistant to the effects of LDN-193189, an ALK2/3/6 inhibitor. ALK2 and ActRIIA complex formation in response to activin B may prevent the approach of LDN-193189 to ALK2 to inhibit its activity. Activin B also induced phosphorylation of Smad2/3, the TGF-β/activin-regulated (AR)-Smad, and increased expression of connective tissue growth factor, a gene related to liver fibrogenesis, through ALK4 and ActRIIA/B. Activin B-induced activation of the BR-Smad pathway was also detected in non-liver-derived cells. The present study reveals the broad signaling of activin B, which is induced in non-parenchymal cells in response to hepatic inflammation, in hepatocytes.

Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bone morphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in the regulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2 cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated this hepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)-1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expression of the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMP-mediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin expression is regulated by more diverse factors than had been previously considered., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We previously reported the presence of brown/beige adipocytes in the white fat depots of mature cattle. The present study examined the effects of dietary vitamin A on the expression of brown/beige adipocyte-related genes in the white fat depots of fattening cattle. No significant differences were observed in the expression of Ucp1 between vitamin A-deficient cattle and control cattle. However, the expression of the other brown/beige adipocyte-related genes was slightly higher in the mesenteric fat depots of vitamin A-deficient cattle. The present results suggest that a vitamin A deficiency does not markedly affect the expression of Ucp1 in white fat depots, but imply that it may stimulate the emergence of beige adipocytes in the mesenteric fat depots of fattening cattle.

Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin., (Copyright © 2014 Elsevier B.V. All rights reserved.)