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Activin B, a homodimer of the inhibin βB subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin βB gene and signalling pathways regulated by the canine inhibin βB. Using 5′‐ and 3′‐rapid amplification of cDNA end (RACE) and RT‐PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin βB gene. Immunoreactive inhibin βB molecules were detected at ~25 and ~14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin βB gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B‐stimulated transcriptions of reporter genes, CAGA‐luc and Hepcidin‐luc, regulated by the canonical activin/transforming growth factor‐β (TGF‐β) and bone morphogenetic protein (BMP) pathway, respectively. Activin B‐induced CAGA‐luc transcription was not detected in ALK7‐deficient MDCK canine‐derived cells; however, the forced expression of ALK7 resulted in the activin B‐dependent expression in MDCK cells. Unexpectedly, the activin B‐induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF‐β receptor activity. The present study identified an experimentally isolated long form of the canine inhibin βB gene producing activin B that transactivates BMP‐ and activin/TGF‐β‐regulated gene expression. [ABSTRACT FROM AUTHOR]

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1–4 weeks. Although intramuscular administration of iron increased iron‐related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver‐derived cells but not in bovine liver‐derived cells. A luciferase‐based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF‐β/activin‐mediated signaling. Induction of TGF‐β‐responsive genes was also lower after treatment with TGF‐β1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF‐β family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF‐β family signaling obtained in murine and human cells is not always applicable to bovine cells. [ABSTRACT FROM AUTHOR]

With longevity, the prevalence of osteoporosis, which occurs when the activity of osteoclast surpasses that of osteoblasts, has increased in dogs. However, limited information is available on canine osteoclastogenesis. We herein described culture conditions to induce osteoclasts from canine bone marrow cells, and identified factors affecting canine osteoclastogenesis. Tartrate-resistant acid phosphatase-positive multinucleated cells were efficiently formed in a culture of bone marrow mononuclear cells with macrophage colony-stimulating factor (M-CSF 25 ng/mL) for 3 days and a subsequent culture in the presence of M-CSF (25 ng/mL) and soluble receptor activator of NF-κB ligand (RANKL 50 ng/mL) for 4 days. We previously reported in a murine cell system that gene induction of the E isoform of microphthalmia-associated transcription factor (Mitf-E) was required and sufficient for osteoclastogenesis, while transforming growth factor-β (TGF-β) enhanced RANKL-induced Mitf-E expression and osteoclastogenesis. Mitf-E expression also increased during RANKL-induced osteoclastogenesis in canine cells; however, TGF-β down-regulated Mitf-E expression and osteoclastogenesis, indicating a species-dependent response. The results of the present study show that, consistent with murine cells, M-CSF and soluble RANKL enable canine bone marrow cells to differentiate into osteoclasts, and Mitf-E expression is induced during osteoclastogenesis. However, the role of TGF-β in osteoclast formation is distinct between murine and canine cells, suggesting the necessity of analyses using canine cells to examine the factors affecting canine osteoclastogenesis. [ABSTRACT FROM AUTHOR]

The liver is the organ that responds to nutritional disturbances including magnesium deficiency. The present study evaluated cellular responses to magnesium deficiency using model cells of the liver, namely, HepG2 cells as hepatocytes, RAW264.7 cells as Kupffer cells and human umbilical vein endothelial cells (HUVECs) as vascular endothelial cells; we examined effects of culture with magnesium deficient medium on cell responses in individual types of cells as well as interactive responses among cells. Metabolomic analyses indicated that magnesium deficiency differentially affected the cellular content of metabolites among HepG2 cells, RAW264.7 cells and HUVECs. The cellular content of the metabolites in HepG2 cells and HUVECs was also affected by the conditioned medium from RAW264.7 cells cultured with the magnesium-deficient media. The changes in HUVECs partly resembled those of the livers of magnesium-deficient rats previously described. RNA-seq analyses indicated that magnesium deficiency modulated the expression levels of molecules related to the ubiquitin-proteasome pathway and oxidative stress/antioxidant response in HepG2 cells and RAW264.7 cells, respectively. Furthermore, when HUVECs were co-cultured with RAW264.7 cells, lipopolysaccharide-induced expression of interleukin (IL)-1β and IL-6 was enhanced by magnesium deficiency, depending on the presence of RAW264.7 cells. The present study reveals that magnesium deficiency affects cellular metabolism in HepG2 liver cells, RAW264.7 macrophages and HUVECs, and that the modulation of cellular responses to extracellular magnesium deficiency in HUVECs depends on the presence of RAW264.7 cells. The complex responses in individual cells and through cell interactions partly explain the regulatory reaction to magnesium deficiency in the liver. [ABSTRACT FROM AUTHOR]

Iron is essential for a variety of physiological processes. Hepatic iron overload acts as a trigger for the progression of hepatic steatosis to nonalcoholic steatohepatitis and hepatocellular carcinoma. In the present study, we aimed to study the effects of iron overload on cellular responses in hepatocytes. Rat primary hepatocytes (RPH), mouse primary hepatocytes (MPH), HepG2 human hepatoma cells and Hepa1-6 mouse hepatoma cells were treated with FeCl3. Treatment with FeCl3 effectively increased iron accumulation in primary hepatocytes. Expression levels of molecules involved in cellular signaling such as AMPK pathway, TGF-β family pathway, and MAP kinase pathway were decreased by FeCl3 treatment in RPH. Cell viability in response to FeCl3 treatment was decreased in RPH but not in HepG2 and Hepa1-6 cells. Treatment with FeCl3 also decreased expression level of LC-3B, a marker of autophagy in RPH but not in liver-derived cell lines. Ultrastructural observations revealed that cell death resembling ferroptosis and necrosis was induced upon FeCl3 treatment in RPH. The expression level of genes involved in iron transport varied among different liver-derived cells- iron is thought to be efficiently incorporated as free Fe2+ in primary hepatocytes, whereas transferrin-iron is the main route for iron uptake in HepG2 cells. The present study reveals specific cellular responses in different liver-derived cells as a consequence of iron overload. [ABSTRACT FROM AUTHOR]

• Rosiglitazone and BMP7 induces brown adipogenesis in C2C12 myogenic cells. • Formed adipocytes are not always UCP1-positive. • The differentiation is not mediated by known pathways to induce brown adipogenesis. • FGF pathway may be partly involved in the brown adipogenesis. Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)- 3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes. [ABSTRACT FROM AUTHOR]

• UCP1 is a transporter responsible for nonshivering thermogenesis in mammalian brown/beige fat. • We identified four alternative splice variants of bovine Ucp1 (variant 1–4). • Bovine Ucp1 comprises a substantial proportion of variant 1/3. • The gene products from Ucp1 variant 2–4 are efficiently degraded by the proteasome system. [ABSTRACT FROM AUTHOR]