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Hepcidin plays a central role in systemic iron metabolism. The bone morphogenetic protein (BMP) pathway regulates expression of hepcidin through transcriptional activation via BMP-responsive elements (REs) 1 and 2 on the promoter. Previous studies also revealed that the BMP pathway stimulates transcription of its target genes via GC-rich sequences on the promoter. A search for GC-rich sequences on the hepcidin promoter indicated 13 regions across the distal (A to F), middle (G to I), and proximal (J to M) areas; among them, mutations of the GC-rich element found in regions B to D exhibited decreased responsiveness to ALK3(QD) expression in the presence of BMP-RE1 mutations, indicating necessity of the elements for full expression of hepcidin by the BMP pathway., (© 2017 Federation of European Biochemical Societies.)

Systemic iron balance is governed by the liver-derived peptide hormone hepcidin. The transcription of hepcidin is primarily regulated by the bone morphogenetic protein (BMP) and inflammatory cytokine pathways through the BMP-response element (BMP-RE) and STAT-binding site, respectively. In addition to these elements, we previously identified a TPA-responsive element (TRE) in the hepcidin promoter and showed that it mediated the transcriptional activation of hepcidin through activator protein (AP)-1 induced by serum. In the present study, we examined the role of TRE in the BMP-induced transcription of hepcidin in HepG2 liver cells. The serum treatment increased the basal transcription of hepcidin; however, responsiveness to the expression of ALK3(QD), a constitutively active BMP type I receptor, was unaffected. Consistent with these results, mutations in TRE in the hepcidin promoter decreased basal transcription, whereas responsiveness to the expression of ALK3(QD) remained unchanged. HepG2 cells significantly expressed AP-1 components in the basal state, whereas BMP did not up-regulate the expression of these components. The expression of c-fos enhanced the basal transcription of hepcidin as well as ALK3(QD)-mediated hepcidin transcription, whereas that of dominant-negative c-fos decreased hepcidin transcription. The results of the present study suggested that the cis-elements of the hepcidin promoter, BMP-RE and TRE, individually transmitted BMP-mediated and AP-1-mediated signals, respectively, whereas transcription was synergistically increased by the stimulation of BMP-RE and TRE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-β (TGF-β), representative members of the TGF-β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-β family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-β-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-β family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis., (Copyright © 2010 Wiley-Liss, Inc.)

Previous studies revealed that bone morphogenetic protein (BMP) induces commitment to the adipocyte lineage in pluripotent stem cells. The present study explored the role of endogenous BMP activity in 3T3-L1 preadipocytes. The expression of phospho-Smad1/5/8 was monitored because BMP transmits its signal through Smad1/5/8 phosphorylation. Phosphorylated Smad1/5/8 was higher in proliferating preadipocytes, and lower in differentiating adipocytes after removing differentiation inducers. Reporter assays revealed that dorsomorphin predominantly inhibits the BMP pathway but not the structurally related TGF-beta/activin pathway. The addition of dorsomorphin to the culture medium prior to treatment with differentiation inducers impaired lipid accumulation in 3T3-L1 cells. The present study indicated that activation of BMP signaling in preadipocytes is required for these cells to initiate the adipogenic program.

Hepcidin negatively regulates the circulating iron levels by inhibiting the intestinal absorption of iron as well as iron release from macrophages. Hepcidin activity is largely determined by its expression, which is regulated at the transcriptional level. Hepcidin transcription is induced not only by the iron status‐related bone morphogenetic protein (BMP)‐2/6, but also by inflammatory cytokines, such as interleukin (IL)‐1β and IL‐6. The present study reveals that the microphthalmia (MiT)/transcription factor E (TFE) family members are novel regulators of hepcidin transcription. Melanocyte‐inducing transcription factor (MITF)‐A, a member of the MiT/TFE family, was identified as a positive regulator of hepcidin transcription via stimulus screening for transcription regulators. An E‐box (5′‐CATGTG‐3′) spanning nt‐645 to nt‐640 of the murine hepcidin promoter was identified as an MITF‐A‐responsive element. Responsiveness to MITF‐A on hepcidin transcription decreased when the cells were stimulated with BMP2 or IL‐1β. These results suggest a functional interaction between the MITF pathway and BMP‐ or IL‐1β‐mediated signaling. TFEB and TFE3, members of the MiT/TFE family, also stimulated hepcidin transcription, but the main region responsible for hepcidin transcription was distinct from that induced by MITF‐A. The region spanning nt‐581 to nt‐526 was involved in TFEB/TFE3‐mediated hepcidin transcription. Considering that members of the MiT/TFE family act as regulators of starvation‐induced lysosomal biogenesis, hepcidin expression may be controlled by additional pathways apart from those identified so far. Significance statement: Systemic iron levels are negatively regulated by hepatic transcription of hepcidin. Hepcidin transcription has been shown to be mainly regulated by iron levels and inflammation, but recent evidence suggested the involvement of the other factors in hepcidin transcription. The present study identified novel inducers of hepcidin transcription; melanocyte‐inducing transcription factor (MITF)‐A, TFEB, and TFE3, members of the MiT/TFE family, stimulated hepcidin transcription. MITF‐A modified the bone morphogenetic protein‐ or interleukin‐1β‐mediated hepcidin transcription. Furthermore, region responsible for MITF‐mediated hepcidin transcription was distinct from that induced by TFEB/TFE3. Considering that members of the microphthalmia/transcription factor E (TFE) family act as regulators of starvation‐induced lysosomal biogenesis, hepcidin expression may be controlled by additional pathways apart from those identified so far. [ABSTRACT FROM AUTHOR]

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]

Uncoupling protein 1 (UCP1) is responsible for non‐shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all‐trans retinoic acid (RA). The transforming growth factor‐β (TGF‐β)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A‐83‐01, an inhibitor of the TGF‐β/activin pathway, stimulated myogenesis in these cells. A‐83‐01 significantly increased the expression of some brown fat signature genes such as Pgc‐1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN‐193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN‐193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte‐related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. Significance of the study: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte‐selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF‐β activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte‐selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized. [ABSTRACT FROM AUTHOR]

Mg deficiency accelerates Fe accumulation in the liver, which may induce various metabolic disturbances. In the present study, we examined the gene expression of Hepcidin, a peptide hormone produced in the liver to regulate intestinal Fe absorption negatively, in Mg-deficient rats. Although liver Fe concentration was significantly higher in rats fed an Mg-deficient diet for 4 weeks than in rats fed a control diet, Hepcidin expression in the liver was comparable between the dietary groups. Previous studies revealed that Fe overload up-regulated Hepcidin expression through transcriptional activation by Fe-induced bone morphogenetic protein (Bmp) 6, a growth/differentiation factor belonging to the transforming growth factor-β family, in the liver. Mg deficiency up-regulated the expression of Bmp6 but did not affect the expression of inhibition of DNA binding 1, a sensitive Bmp-responsive gene. In addition, the expression of Bmp receptors such as activin receptor-like kinase 2 (Alk2), activin receptor type IIA (Actr2a), activin receptor type IIB (Actr2b) and Bmp type II receptor (Bmpr2) was lower in the liver of Mg-deficient rats than in that of control rats. The present study indicates that accumulation of hepatic Fe by Mg deficiency is a stimulant inducing Bmp6 expression but not Hepcidin expression by blunting Bmp signalling possibly resulting from down-regulation of the receptor expression. Unresponsive Hepcidin expression may have a role in Mg deficiency-induced changes related to increased liver Fe. [ABSTRACT FROM PUBLISHER]

• 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]

The BMP pathway positively regulates murine brown adipogenesis. We herein examined the mRNA levels of BMPs and activin βB as well as receptors for the BMP pathway in the adipose tissues of cattle fed diets with a differential ratio of concentrate to roughage or a vitamin A-deficient diet. The expression of activin βB was significantly increased in the subcutaneous fat depot of animals fed the concentrate diet, while the vitamin A-deficient diet significantly increased the expression of BMP4 in the mesenteric fat depot. The expression of receptors for the BMP pathway, ALK2, ALK3, ActRIIA, and BMPR2, showed a similar pattern to that of BMP4 and activin βB in response to the dietary treatments. The results of the present study demonstrated that diet modulated the expression of components of the BMP pathway and may be responsible for the regulatory expression of brown/beige adipocyte-related genes in the adipose tissues of cattle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Highlights: [•] Endogenous Bmp activity is higher in adipogenic cells before differentiation. [•] Higher Bmp4 in preadipocytes is indispensable for adipocyte differentiation. [•] Repression of Interleukin-11 expression may play a role in Bmp4-induced adipogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Background: Our previous study revealed the indispensable activity of endogenous bone morphogenetic protein (Bmp) prior to differentiation induction of C2C12 myoblasts for myogenesis. Here we investigated the Bmp isoform responsible for endogenous Bmp activity during differentiation and its role in myogenesis. Methods: Gene expression of Bmp4 during myogenesis was evaluated in C2C12 cells. Effects of inhibition of the Bmp pathway on myogenesis were examined. Cells expressing Bmp4 and regulation of Bmp4 expression in myoblasts were explored. Results: The expression of Bmp4 increased with the progression of myogenesis, although the extent of the increase after differentiation induction was smaller than that before the induction. Down-regulation of Bmp signal components including Bmp4, Bmpr2, and Alk2/3 inhibited the emergence of positive cells for myosin heavy chain II. The treatments also decreased the Myogenin expression. Treatment with cytosine arabinoside decreased the expression of Bmp4. Also, Bmp4 expression was also lower in isolated myotubes than in residual cells. Expression of Rgm c was higher in the myotube fraction. Transcription of Bmp4 was repressed by the conditioned medium of mixed cells consisting of myoblasts and myotubes. Conclusion: Bmp4 expressed in myoblasts has a positive role in myotube formation/maturation through myogenin expression. The presence of myotubes inhibits Bmp4 expression in proliferating myoblasts through transcriptional regulation, although the expression is intrinsically increased with time of culture. General significance: Taken previous results on involvement of Bmp in the commitment of osteoblasts and adipocytes with the present results together, Bmp may act as a general promoter of mesenchymal cell differentiation. [Copyright &y& Elsevier]

Expression of hepcidin, a hormone produced by hepatocytes which negatively regulates the circulating iron levels, is known to be positively regulated by BMP6, a member of transforming growth factor (TGF)-β family. Previous studies have shown that iron status is sensed by sinusoidal endothelial cells of hepatic lamina, leading to the modulation of BMP6 expression. ISOS-1, HUVEC, F-2, and SK-HEP1 endothelial cells were treated with either iron or 2,2′-dipyridyl (2DP), a cell-permeable iron-chelator, and expression level of Bmp6 was examined. To identify factors affecting Bmp6 transcription, stimulus screening for regulator of transcription (SSRT) was developed. Treatment with iron slightly increased the expression levels of Bmp6 , while 2DP unexpectedly increased Bmp6 expression in a dose-dependent manner. 2DP-induced Bmp6 expression was resistant to co-treatment with iron. 2DP-induced Bmp6 expression was also detected in HUVEC, F-2 cells, and SK-HEP1 cells. Luciferase-based reporter assays indicated that forced expression of JunB increased the transcription of Bmp6. 2DP induced phosphorylation of JunB; co-treatment with SP600125 blocked the 2DP-induced Bmp6 expression partially. JunB-induced Bmp6 transcription was not affected by mutations of putative JunB-responsive elements. Some endoplasmic reticulum stress inducers increased the expression of Bmp6. SSRT revealed pathways regulating Bmp6 transcription positively and negatively. Hepa1–6 liver cells and C2C12 myogenic cells were prone to 2DP induced Bmp6 expression. The present study reveals non‑iron-regulated Bmp6 expression in endothelial cells. Regulatory expression of Bmp6 may be important as a key step for fine tuning of BMP activity. • An iron-chelator 2,2′-dipyridyl (2DP) induces Bmp6 expression in endothelial cells. • 2DP-induced Bmp6 expression is iron-independent. • 2DP stimulates JunB phosphorylation, leading to Bmp6 expression. • A novel screening regulator of transcription names as SSRT is developed. • 2DP-induced Bmp6 expression is also detected in non-endothelial cells. [ABSTRACT FROM AUTHOR]