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Abstract: Factors affecting struvite, a magnesium–ammonium–phosphate complex (MgNH4PO4 6H2O), in feline urine were evaluated. Incubation of just “urine mineral (UM)” solution, in which mineral concentrations are compatible with those in feline urine, for 4 h at 37 °C did not induce the formation of crystals. Similarly, incubation of urine alone did not produce crystals. However, struvite crystals were formed by the addition of urine to UM solution. Mg, NH3 and P were all required for urine-induced struvite crystallization. The lower molecular weight (LMW) fraction of urine was essential for struvite crystal formation, and the higher molecular weight (HMW) fraction enhanced formation of LMW-induced struvite crystals. The effects of urine proteins further fractionated by column chromatography were examined. A protein at >250 kDa and cauxin, a major urine protein recently identified as a regulator of felinine production, potentiated struvite crystal formation induced by the LMW fraction. In contrast, Tamm–Horsfall glycoprotein, a urine protein thought to promote struvite crystallization, did not have this activity. The present study reveals a novel mechanism of feline struvite crystallization. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β (TGF-β) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-β-mediated cell responses in BMMCs. Treating BMMCs with TGF-β induced the phosphorylation of p38 within 2 h and persisted for 24 h. The involvement of p38 in TGF-β-induced cell responses depended upon mast cell functions; it was necessary for up-regulation of mmcp-1 and migration, but not for up-regulation of mmcp-7 and inhibition of metabolic activity. New protein synthesis was required for the up-regulation of mmcp-1 but not mmcp-7 in response to TGF-β treatment, and stabilization of mRNA was partially responsible for the increase in gene transcript of mmcp-1. The decrease in metabolic activity in response to TGF-β treatment was smaller in Smad3-deficient BMMCs compared to wild-type BMMCs. Maximal migration was detected at a TGF-β concentration of 40 fM in wild-type BMMCs, whereas TGF-β-induced migration was absent in Smad3-deficient BMMCs. Thus, the roles of p38 and Smad3 are different among TGF-β-mediated cell responses in BMMCs. [Copyright &y& Elsevier]

Abstract: Previous studies have demonstrated that treatment with activin A and TGF-β1, members of the TGF-β family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-β1-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-β family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-β1 at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 μM SB431542, an inhibitor of activin and TGF-β type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-β1, whereas <0.5 μM SB431542 effectively reduced TGF-β1-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-β-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-β pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-β occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-β signaling. [Copyright &y& Elsevier]

Abstract: Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-β (TGF-β) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-β. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt −126 to −122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-β pathway. In addition, a region from nt −104 to −98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-β signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-β. Treatment with TGF-β in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-β stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-β pathway. [Copyright &y& Elsevier]

Previous studies have revealed that members of the transforming growth factor-β (TGF-β) including TGF-β1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-β1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-β1 or activin A, which was regulated at the transcription level. Reporter assays showed that Smad3, a signal mediator of the TGF-β/activin pathway, was responsible for the transcription. The TGF-β response element is located at −153 bp relative to the transcription initiation site, CAGA. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, the heart and skeletal muscle, also stimulated the transcription of mMCP-6. The region at −166 bp, GACCTG, was responsible for MITF-induced transcription. Mutations of the CAGA motif and the MITF responsive site indicated that the MITF site of mMCP-6 promoter is indispensable for the transcriptional activation by a constitutively active TGF-β receptor (ALK5-TD), whereas the CAGA motif is dispensable for transcription by MITF. Transcriptional activation of mMCP-6 by the TGF-β pathway was differently interacted with that by MITF isoform; ALK5-TD further enhanced MITF-E-induced transcription, whereas MITF-M-induced transcription abolished responsiveness to ALK5-TD. The positive regulation of mMCP-6 by the TGF-β/activin pathway and the differential regulation by the MITF isoform suggest a rigorous regulation of mast cell function as effector cells of immune response. [Copyright &y& Elsevier]

Previous studies have revealed that activin A and transforming growth factor-β[sub 1] (TGF-β[sub 1]) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-β[sub 1] in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-β pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominantnegative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-β signaling in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) recognize and signal the presence of bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PG) as a part of innate immunity. Our previous studies revealed that mast cells function as effector cells in the protection of mice against lethal enterobacterial infections. In this study, we examined both the gene expression of molecules involved in TLR signaling and the effects of LPS and PG in bone marrow-derived cultured mast cells (BMCMCs). The mRNA expression of TLR2, TLR4 and TLR6 was detected in BMCMCs. CD14, MD-2 and MyD88, which are also involved in TLR pathway, were also expressed. Neither LPS nor PG affected degranulation in BMCMCs, but release of tumor necrosis factor increased slightly in response to LPS and PG. Both LPS and PG enhanced expression of pro-matrix metalloproteinase 9 (pro-MMP-9) in a dose-dependent manner, and DNA fragmentation was induced by LPS, but not by PG. These results suggest that mast cells are the targets of LPS and PG, and that the functions of these molecules produced exclusively by bacteria partly overlap, but are distinct. [Copyright &y& Elsevier]

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin βA. The steady-state mRNA of inhibin/activin βA was also induced by increasing cytosolic Ca2+ concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin βA transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin βA induction was also partially blocked by preincubation with c-Jun NH2-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin βA gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells. [Copyright &y& Elsevier]

Presents a study on bone growth and myofibrillar protein degradation, after early weaning in calves. Experimental methods use; Detrimental effects on nitrogen and calcium retention.

A metabolomic study was performed on the kidneys and skeletal muscles of rats fed diets containing varying contents of Mg for 4 weeks. The kidneys are divided into two parts, the aerobic cortex and the anaerobic medulla, that differ in metabolism. The relative contents of 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvic acid increased with Mg restriction in both renal regions. In contrast, pyruvic acid content decreased with Mg restriction in the diets, suggesting an inhibitory conversion of phosphoenolpyruvic acid to pyruvic acid. The lactic acid content increased in both regions of the kidneys of Mg-restricted rats, implying changes towards a more glycolytic metabolism, possibly resulting from the impairment of mitochondrial function. There are two types of muscle fibers: glycolytic fast and oxidative slow muscle fibers. The soleus muscle consists of slow muscle fibers, whereas the gastrocnemius muscle consists of a combination of fast and slow muscle fibers. Similar to the changes in the kidneys, the contents of 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid increased in the soleus and gastrocnemius muscles with dietary Mg restriction. Unlike in the kidney, pyruvic acid content increased in the soleus muscle in response to Mg restriction. Severe Mg restriction decreased contents of carnosine and its constituent β-alanine and increased the levels of purine derivatives such as xanthine and uric acid in the gastrocnemius muscle. The present study suggests a region-dependent sensitivity to dietary restriction of Mg, which may lead to the onset of various metabolic disorders. [ABSTRACT FROM AUTHOR]

A previous study revealed that the expression of the Borna disease virus (BDV)-encoding phosphoprotein in glial cells was sufficient to induce neurobehavioral abnormalities resembling Borna disease. To evaluate the involvement of the TGF-β family in BDV-induced changes in cell responses by C6 glial cells, we examined the expression levels of the TGF-β family and effects of inhibiting the TGF-β family pathway in BDV-infected C6 (C6BV) cells. The expression of activin βA and BMP7 was markedly increased in BDV-infected cells. Expression of Smad7, a TGF-β family-inducible gene, was increased by BDV infection, and the expression was decreased by treatment with A-83-01 or LDN-193189, inhibitors of the TGF-β/activin or BMP pathway, respectively. These results suggest autocrine effects of activin A and BMP7 in C6BV cells. IGFBP-3 expression was also induced by BDV infection; it was below the detection limit in C6 cells. The expression level of IGFBP-3 was decreased by LDN-193189 in C6BV cells, suggesting that endogenous BMP activity is responsible for IGFBP-3 gene induction. Our results reveal the regulatory expression of genes related to the TGF-β family, and the role of the enhanced BMP pathway in modulating cell responses in BDV-infected glial cells. [ABSTRACT FROM AUTHOR]

Abstract: Background: The roles of microphthalmia-associated transcription factor (Mitf) in the skeletal muscle and during myogenesis are unclear. Methods: Expression of Mitf in mouse tissues and during myogenesis was evaluated. Effects of Mitf knockdown on myogenesis and gene expression related to myogenesis were subsequently explored. Furthermore, effects of p21, a cyclin-dependent kinase inhibitor, and integrin α9 (Itga9) were examined. Results: Mitf was highly expressed in the skeletal muscle; Mitf-A and -J were expressed. Mitf expression increased after differentiation stimulation in C2C12 myogenic cells. Down-regulation of Mitf expression by transfection of siRNA for common Mitf inhibited myotube formation, which was reproduced by Mitf-A knockdown. Morphometric analyses indicated that both multinucleated cell number and the proportion of myotubes with more than 6 nuclei were decreased in Mitf-knockdown cells, suggesting that Mitf is required for not only the formation of nascent myotubes but also their maturation. Searching for genes positively regulated by Mitf revealed p21 and Itga9; decreasing Mitf expression inhibited up-regulation of p21 expression after differentiation stimulation and blocked the induction of Itga9 expression in response to differentiation. Knockdown of p21 decreased the number of multinucleated cells, whereas Itga9 knockdown did not affect the myotube number. Both p21 knockdown and Itga9 knockdown decreased the proportion of myotubes with more than 6 nuclei. General significance: Mitf positively regulates skeletal muscle formation; Mitf is significantly expressed during myogenesis, and is required for efficient myotube formation through expression of p21 and Itga9. [Copyright &y& Elsevier]

Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels. Results from competitive RT-PCR analysis correlated closely with those from real-time RT-PCR analysis. When the differences in mRNA level between samples are relatively smaller, however, the correlation tended to be weaker. Real-time RT-PCR analysis has higher reliability, but is expensive. In contrast, competitive RT-PCR analysis is inexpensive, but is weaker at detecting smaller differences in gene transcript level between samples. Therefore, the most appropriate analytical method to measure mRNA levels should be chosen, depending on the experimental conditions. [Copyright &y& Elsevier]

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]

Although the insulin-mediated cell signaling pathway has been extensively examined, changes in the cellular content of metabolites currently remain unclear. We herein examined metabolite contents in 3T3-L1 adipocytes treated with insulin using a metabolomic analysis. Fifty-four compounds were detected, and the contents of metabolites from the citric acid cycle increased in response to the insulin treatment for 4 h, which was sensitive to U0126 and LY294002, inhibitors for mitogen-activated protein kinase kinase-1 and phosphoinositide 3-kinase, respectively. The cellular contents of fumaric acid and malic acid were increased more by insulin than those of citric acid and succinic acid. Time-course changes in metabolites from the citric acid cycle exhibited oscillations with a 2-h cycle. A metabolic pathway analysis also indicated that insulin affected the metabolism of alanine, aspartate and glutamate, as well as that of arginine and proline. The contents of free amino acids were slightly decreased by the insulin treatment, while the co-treatment with U0126 and LY294002 abrogated these insulin-mediated decreases. The present study revealed the unexpected accumulation of citric acid cycle metabolites in adipocytes by insulin. Our results indicate the usefulness of metabolomic analyses for obtaining a more comprehensive understanding of the regulation of metabolic pathways in cell-culture systems. [ABSTRACT FROM AUTHOR]

The expression of uncoupling protein (UCP1) is up-regulated in mammalian brown adipocytes during cold exposure. However, a previous study revealed that UCP1 was highly expressed in the liver of common carps, and that the hepatic expression of UCP1 was down-regulated during cold exposure. The present study examined the effects of temperature on the recovery of UCP1 expression levels and the expression of genes involved in UCP1 transcription in the livers and kidneys of common carps. The hepatic and renal expressions of UCP1 were decreased by acclimation from 22 °C to 8 °C, and a subsequent increase in the water temperature from 8 °C to 28 °C recovered the renal, but not hepatic expression of UCP1. Changes in the expression of peroxisome proliferator-activator receptor (PPAR) γ, retinoid X receptor (RXR) α and PPARγ co-activator (PGC)-1α, genes that are involved in the expression of UCP1 in mammals, with ambient temperature indicated that the expressions of PPARγ and RXRα, but not expression of PGC-1α was decreased in response to cold exposure; the hepatic and renal expressions of PPARγ and RXRα recovered to basal levels with the cessation of cold exposure, although this was not complete for hepatic expression of PPARγ. The results of the present study indicate that a unique regulatory mechanism is responsible for the hepatic and renal expressions of carp UCP1 during cold exposure and subsequent reacclimation, and is distinct from that in murine brown adipocytes. [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]

• 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]

Abstract: Background: Brown adipocytes generate heat through the expression of mitochondrial Ucp1. Compared with the information on the regulatory differentiation of white preadipocytes, the factors affecting brown adipogenesis are not as well understood. The present study examined the roles of the Tgf-β family members Bmp, Tgf-β and Activin during differentiation of HB2 brown preadipocytes. Methods: Endogenous Bmp activity and effects of exogenous Tgf-β family members were examined. Role of Srebp1c in brown adipogenesis was further explored. Results: Although Bmp7 has been suggested to be a potent stimulator of brown adipogenesis, it affected neither the expression of brown adipocyte-selective genes nor Ucp1 induction in response to a β adrenergic receptor agonist. Unlike in 3T3-L1 white preadipocytes, endogenous Bmp activity was not required for brown adipogenesis; treatment with inhibitors of the Bmp pathway did not affect differentiation of preadipocytes. Administration of Tgf-β1 or Activin A efficiently decreased the insulin-induced expression of brown adipocyte-selective genes. Tgf-β1 and Activin A decreased the expression of Pparγ2 and C/ebpα, suggesting the inhibition of adipogenesis. The Tgf-β- and Activin-induced inhibition of brown adipogenesis was mediated by the repression of Srebp1c expression; Tgf-β1 and Activin A blocked Srebp1c gene induction in response to the differentiation induction, and knock-down of Srebp1 expression inhibited brown adipogenesis. Conclusion: Endogenous Bmp is dispensable for brown adipogenesis, and Srebp1c is indispensable, which is negatively regulated by Tgf-β and Activin. General significance: Control of activity of the Tgf-β family is potentially useful for maintenance of energy homeostasis through manipulation of brown adipogenesis. [Copyright &y& Elsevier]

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]

Abstract: CRNP5, a variant of Borna disease virus (BDV), has stronger pathogenesis in rats than the related variant CRP3, although only 4 amino acids in the whole genome are different. As a first step to clarify the differential pathogenesis between the variants, the present study focused on examining the expression of the transforming growth factor (TGF)-β family in the brain of rats infected with BDV. The main results were as follows. (1) BDV infection, irrespective of the variant, up-regulates TGF-β1 expression in the brain, (2) the expressions of signal receptors for TGF-β1 are also increased, (3) the expression of brain inhibin/activin βE is up-regulated by BDV infection, and (4) the expression of brain inhibin/activin βC tends to be higher in rats exhibiting severe Borna disease. These results indicate that members of the TGF-β family are involved in neuronal disorders induced by BDV infection in a ligand-dependent manner. In particular, up-regulation of inhibin/activin βC may be a key event responsible for induction of the stronger pathogenesis of the CRNP5 variant of BDV. [Copyright &y& Elsevier]

Abstract: The CRNP5 variant of Borna disease virus (BDV) has stronger pathogenesis than the CRP3 variant in which only 4 nucleotides in the whole genome are different. The CRP3 is produced by 3 passages in rat brains of BDV, whereas the CRNP5 is produced by 5 passages in mouse brains after 2 passages in rat brains of the BDV. Thymidylic acids at nt 3608 and 3673 were replaced by cytidylic acids during 3 passages in mice. Three passages in mice caused replacement of adenylic acid at nt 7936 by guanylic acid. No replacement at nt 8742 occurred during passages in mice. [Copyright &y& Elsevier]

Abstract: Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-β1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix–loop–helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-β1 did not increase PAI-1 mRNA levels in mast cells from mi/mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-β1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-β1, whereas transcriptional activation by TGF-β1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-β family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-β1 and activin A treatment was also detected in B16 melanocytes; TGF-β1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-β1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner. [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]