Activin A, a member of the TGF-β superfamily, is a pluripotent growth and differentiation factor. In this study, we report that murine Th cells produce activin A upon activation. Activin activity in the cultured CD4+ T cells was induced by anti-CD3 cross-linking. Activin βA mRNA level was increased in response to activation, indicating that activin production in CD4+ T cells is regulated at the mRNA level. Activin production was detected exclusively in CD4+CD25− T cells, but not in CD4+CD25+ regulatory T cells. When CD4+ T cells were differentiated into Th cell subsets, higher activin secretion was detected when cultured under Th2-skewing conditions. The mRNA level of activin βA was abundant in Th2, but not in Th1 cells. Furthermore, secretion of activin was significantly higher in activated Th2 clones than in Th1 clones. The activin βA-proximal promoter contains a binding site for c-Maf, a Th2-specific transcriptional factor, at close proximity with an NF-AT binding site. c-Maf was able to synergize with NF-AT to transactivate activin βA gene, and both factors are implicated in activin βA transcription in Th2 cells. Activin A induced macrophages to express arginase-1 (M-2 phenotype), whereas it inhibited inducible NO synthase expression (M-1 phenotype) induced by IFN-γ. Taken together, these observations suggest that activin A is a novel Th2 cytokine that promotes differentiation of macrophages toward the M-2 phenotype.

Components of the transforming growth factor-␤ and mitogen-activated protein kinase pathways interact in controlling cell growth and differentiation. We show that phosphorylation of Smad2, a mediator of the activin/transforming growth factor-␤ signal, by activated extracellular signal-regulated kinase 1 (ERK1) increases the amount of Smad2 protein and leads to enhanced transcriptional activity. Epidermal growth factor increased phosphorylation of Smad2 in COS7 cells, and Smad2-dependent transcription in a mink lung epithelial cell line, L17, was enhanced by co-transfection of a constitutively active MEK1. In addition, transfection of Smad2 mutants lacking ERK sites resulted in reduced transcription, whereas mutants that mimicked ERK phosphorylation stimulated transcription. The amount of Smad2 protein was increased by transfection with a constitutively active MEK1 and reduced by co-transfection with the ERK phosphatase, HVH2. The elevation of Smad2 protein levels was because of increased half-life and resulted in increased complex formation with Smad4. A site of ERK-dependent phosphorylation on Smad2 was located to Thr 8 , a site that overlaps with the calmodulin binding region. We show that calmodulin inhibits Smad2 phosphorylation by ERK1, and overexpressing calmodulin, or stimulating calmodulin activity with ionomycin, reduces Smad2 levels. These findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that negative regulation of Smad2 signaling by calmodulin is achieved in part by inhibiting this phosphorylation.Activin and transforming growth factor-␤ (TGF-␤) 1 are structurally related, multipotent growth and differentiation factors. These peptides are not only among the most potent known cellular growth inhibitors but also regulate other diverse biological processes including early embryonic patterning and cell fate determination (1). Signaling by these proteins is initiated by ligand-induced hetero-oligomerization of type I and type II receptor serine kinases. Transphosphorylation of a type I receptor serine kinase, ALK4 for activin and ALK5 for TGF-␤, by the type II receptor serine kinase activates the type I receptor, which then phosphorylates the C-terminal

The role of activin, a dimer of inhibin β subunit, in mouse peritoneal macrophages was evaluated. Activin activity in the cultured macrophages was augmented in response to activation by LPS. In Western blot analysis, immunoreactive activin A was detected in the culture medium only when the macrophages were stimulated by LPS. Although mRNA expression of βA subunit was detected, that of α and βB subunit was not found in macrophages by reverse RT-PCR. The activin βA mRNA level was increased in macrophages by LPS, suggesting that the activin production augmented by LPS is regulated at the mRNA level of the βA gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and ActRIIB) were also detected in the peritoneal macrophages, and the mRNA levels, except for ActRIB, were decreased during the LPS treatment. Exogenous activin A stimulated the mRNA expression and gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both the presence and the absence of LPS. In contrast, activin did not affect the production of MMP-9 in macrophages. These results suggested that 1) mouse peritoneal macrophages produced activin A; 2) expression of activin A was enhanced with activation of the macrophages; 3) the macrophages also expressed activin receptors; and 4) exogenous activin A stimulated MMP-2 expression and activity, implicating activin A as an positive regulator of MMP-2 expression. Considering that MMP-2 constitutes the rate-limiting proteinase governing the degradation of basement membrane collagens, activin A may be involved in migration and infiltration of macrophages through the basement membrane in an inflammatory state.

There are two types of brown adipocytes: classical brown adipocytes that form the brown fat depots and beige adipocytes that emerge in the white fat depots. Beige adipocytes have a low level of uncoupling protein 1 (Ucp1) expression in the basal state, but Ucp1 expression is increased in response to β adrenergic receptor activation. The present study explored the factors responsible for the differentiation of 3T3-L1 white preadipocytes to beige adipocytes. Significant expression of Ucp1 was not detected under any tested conditions in the absence of isoproterenol (Iso), an agonist of β adrenergic receptor. Iso-induced Ucp1 expression was significantly higher in the cells treated with a mixture of triiodothyronine (T3) and 3-isobutyl-1-methylxanthine (IBMX) for days 0–8 than in the control cells. Chronic IBMX treatment was indispensable for the enhanced Iso-induced Ucp1 expression, and treatment with additional rosiglitazone (Rosi) for days 0–8 further increased the Ucp1 expression. Recently, genes were identified that are predominantly expressed in beige adipocytes, which were induced from stromal vascular cells in white fat depots. However, the expression levels of the beige adipocyte-selective genes in the adipocytes induced by the mixture of T3, IBMX and Rosi did not differ from those in the control adipocytes. The present study indicates that 3T3-L1 cells can differentiate to beige-like adipocytes by prolonged treatment with the mixture of T3, IBMX and Rosi and that the gene expression profile of the adipocytes is distinct from those previously induced from white fat depots.

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Brown adipocytes, which regulate non-shivering thermogenesis, have been believed to exist in a limited number of mammalian species, and only under limited physiological conditions. Recent discoveries indicate that adult humans possess a significant number of functional brown adipocytes. This study explores the regulatory emergence of brown adipocytes in white adipose tissue (WAT) depots of fattening cattle. RT-PCR analyses indicated significant expression of Ucp1, a brown adipocyte-specific gene, in the WAT of 31-month-old Japanese Black steers. Immunohistochemical analysis revealed that Ucp1-positive small adipocytes were dispersed in the subcutaneous WAT. Next, we examined expression level of Ucp1 and other brown adipocyte-selective genes such as Pgc1α, Cidea, Dio2, Cox1, Cox7a1 and Cox8b in WAT of 30-month-old steers fed either diet with low protein/energy content (roughage diet) or that with high protein/energy content (concentrate diet) for 20months. Ucp1 expression in the subcutaneous WAT was significantly higher in the concentrate diet group than in the roughage diet group. Furthermore, the higher Ucp1 expression levels were limited to the subcutaneous WAT, and no differences between groups were detected in the mesenteric, perirenal, intermuscular or intramuscular WAT. Expression of Dio2, Cox1 and Cox8b was higher in the subcutaneous WAT but not in the mesenteric WAT of the concentrate diet group. Furthermore, expression of Prdm16, a positive regulator of differentiation toward brown adipocyte-lineage cells, and expression of leptin, a molecule that enhances activity of brown adipocytes, were significantly higher in the subcutaneous WAT of the concentrate diet group. This study demonstrates the presence of brown adipocytes in WAT depots of fattening cattle, and suggests the diet-related modulation of expression of genes predominantly expressed in brown adipocytes.

Highlights d Hepatic overexpression of activin E (Alb-ActE) activates thermogenesis d Alb-ActE induces emergence of beige adipocytes and improved insulin sensitivity d Knockout of activin E gene inhibits cold-induced Ucp1 in inguinal fat depot d Activin E directly stimulates expression of Ucp1 in cultured brown adipocytes

Hepcidin is a liver-derived hormone that negatively regulates serum iron levels and is mainly regulated at the transcriptional level. Previous studies have clarified that in addition to hepatic iron levels, inflammation also efficiently increases hepatic hepcidin expression. The principle regions responsible for efficient transcription are bone morphogenetic protein-responsive elements (BMP-REs) 1 and 2 as well as the signal transducer and activator of transcription 3-binding site (STAT-BS). Here, we show that the proinflammatory cytokine interleukin-1β (IL-1β) efficiently increases expression in human HepG2 liver-derived cells and primary mouse hepatocytes. The primary region responsible for IL-1β-mediated transcription was the putative CCAAT enhancer-binding protein (C/EBP)-binding site (C/EBP-BS) at the hepcidin promoter spanning nucleotides -329 to -320. IL-1β induces the expression of C/EBPδ but neither C/EBPα nor C/EBPβ in hepatocytes, and C/EBPδ bound to the C/EBP-BS in an IL-1β-dependent manner. Lipopolysaccharide (LPS) induced the expression of IL-1β in Kupffer cells and hepatocytes in the mouse liver; furthermore, the culture supernatants from the macrophage-like cell line RAW264.7 treated with LPS potentiated the stimulation of expression in hepatocytes. The present study reveals that: 1) inflammation induces IL-1β production in Kupffer cells and hepatocytes; 2) IL-1β increases C/EBPδ expression in hepatocytes; and 3) induction of C/EBPδ activates transcription via the C/EBP-BS that has been uncharacterized yet. In cooperation with the other pathways activated by inflammation, IL-1β pathway stimulation leads to excess production of hepcidin, which could be causative to anemia of inflammation.