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The article focuses on a study which explored the role of endogenous bone morphogenetic protein (BMP) activity in 3T3-L1 preadipocytes. Because BMP transmits its signal through Smad 1/5/8 phosphorylation, the expression of phospho-Smad 1/5/8 was monitored. Dorsomorphin predominantly inhibits the BMP pathway but not the structurally related transforming growth factor (TGF)-β/activin pathway as revealed by reported assays. Results indicated that activation of BMP signaling in preadipocytes is required for these cells to initiate the adipogenic program.

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: The involvement of the TGF-β family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-β and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-β pathway by anti-TGF-β neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-β and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-β-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion. [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]

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]

Inhibin-A is a glycoprotein composed of an a subunit containing a glycosylation site and a βA subunit, whereas activin-A is a homodimer of two inhibin βA subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the α or βA; subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the α subunit [α(l–19) and α(lα26)], and four antisera to the βA subunit [βA(l–10), βA(70–79), βA(87–99), and βA(94–105)], was generated. On Western blot analysis, the anti-βA(87–99) and βA(94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-βA(87–99) and βA(94–105) sera. In addition, when activin-A bound to a nitrocellulose membrane was p re-incubated with recombinant human follistatin, the recognition of activin-A by the anti-βA(87–99) and βA(94–105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87–105 of the inhibin 0A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87–105 of the inhibin/activin βA subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the α subunit, (2) the region responsible for follistatin binding within the activin βA subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation. [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: 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: 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]