Yoshii Nishino

Receptor expression modulates the specificity of transforming growth factor-β signaling pathways

In current models of transforming growth factor‐β (TGF‐β) family signaling, type II receptors activate specific activin receptor‐like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand‐dependent receptor regulated (R)‐Smad by phosphorylating carboxy‐terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R‐Smad subclasses, activin/TGF‐β‐specific (AR‐Smad) and bone morphogenetic protein (BMP)‐specific (BR‐Smad). Co‐expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R‐Smad subclasses in a ligand‐independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF‐β1 and BMP‐2 induced phosphorylation of both R‐Smad subclasses, and TGF‐β1 up‐regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP‐2 up‐regulated plasminogen activator inhibitor‐1 (PAI‐1), which is an AR‐Smad‐regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF‐β1 and BMP‐2 induced phosphorylation of only the expected R‐Smad. These results help to explain the diverse effects of this ligand family.

Detection of anti-Borna Disease Virus (BDV) antibodies from patients with schizophrenia and mood disorders in Japan

The relationship between infection with the Borna Disease Virus (BDV) and the clinical symptoms of schizophrenia and mood disorders (DMS-IV) was investigated. Western blotting techniques were used to examine anti-p10-BDV antibodies in serum from 32 patients with schizophrenia and 33 patients with mood disorders in Japan. The results showed that 1 out of 25 controls (4.0%), 7 out of 32 patients with schizophrenia (21.9%) and 9 out of 33 patients with mood disorders (27.3%) were positive for anti-BDV-p10 antibodies. Compared with levels of anti-BDV-p10 antibodies in controls, the production of anti-BDV-p10 antibodies failed to show a statistically significant relationship with schizophrenia but did show a significant relationship with mood disorder. The subgroup of schizophrenia patients with positive syndromes had a non-significantly higher frequency of anti-BDV-p10 antibodies than the subgroup of patients with negative syndromes. Similarly, the production of anti-BDV-p10 antibodies was non-significantly higher among patients with the unipolar subtype of mood disorder than in those with the bipolar subtype.

Gene expression of the TGF-β family in rat brain infected with Borna disease virus

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)-beta 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-beta1 expression in the brain, (2) the expressions of signal receptors for TGF-beta1 are also increased, (3) the expression of brain inhibin/activin betaE is up-regulated by BDV infection, and (4) the expression of brain inhibin/activin betaC tends to be higher in rats exhibiting severe Borna disease. These results indicate that members of the TGF-beta family are involved in neuronal disorders induced by BDV infection in a ligand-dependent manner. In particular, up-regulation of inhibin/activin betaC may be a key event responsible for induction of the stronger pathogenesis of the CRNP5 variant of BDV.

Regulatory expression of genes related to metastasis by TGF-β and activin A in B16 murine melanoma cells

TGF-β induces epithelial-mesenchymal transition, which occurs during tumor cell invasiveness in pathological state, in limited cells. As a first step to understand the role of TGF-β and the structurally related activin during melanoma metastasis, expression of metastasis-related genes was examined in murine melanoma cells. Treatment with TGF-β1 or activin A down-regulated E-cadherin in B16 cells in a dose-dependent manner. In epithelial cells, TGF-β-induced high mobility group A2 (HMGA2) gene product is suggested to down-regulate E-cadherin through up-regulation of zinc-finger transcription factors Slug and Snail, and basic helix-loop-helix transcription factor Twist. Unlike the regulation in epithelial cells, TGF-β1 treatment rather decreased mRNA expression of HMGA2, indicating a distinct mechanism on TGF-β/activin-induced down-regulation. Transfection of double-stranded interfering RNA (dsRNAi) for activin receptor-like kinase (ALK) type I receptors revealed that ALK5, a prototype of TGF-β receptor, mainly transmits TGF-β signals on the E-cadherin down-regulation at the mRNA level, and that a prototype receptor ALK4 elicited the activin effect. TGF-β/activin potentiated down-regulation of E-cadherin and HMGA2 also in B16 sublines that are susceptible to metastasis. However, the extent of down-regulation tended to be smaller, and less Smad2, a signal mediator for TGF-β/activin, was phosphorylated in response to the ligand, resulting from less expression of type I receptors in the B16 sublines. These results suggest that the receptor expression level determines strength of the signals for TGF-β/activin through phosphorylation of Smad2, which explains pluripotency of the ligand family partly.

Adaptive expression of uncoupling protein 1 in the carp liver and kidney in response to changes in ambient temperature.

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.

Expression of Activin Receptor-like Kinase 7 in Adipose Tissues

The tissue distribution of activin receptor-like kinase 7 (Alk7) expression, the signaling ability of Alk7 variants, and Alk7 expression in response to β3-adrenergic receptor activation were examined. Expression levels of Alk7 varied greatly among tissues but were highest in white adipose tissue and brown adipose tissue. In addition to full-length Alk7 (Alk7-v1), Alk7-v3, an Alk7 variant, was expressed in adipose tissues, brain, and ovary. Nodal transmits signals via Alk7 in cooperation with its coreceptor, Cripto. Evaluation of the ability of Alk7 variants to confer Nodal signaling using luciferase-based reporter assays showed that Alk7-v3 does not transmit Nodal-Cripto-mediated signals. Expression of Alk7 was down-regulated in brown but not in white adipose tissue treated with CL316,243, a β3-adrenergic receptor agonist. These results suggest involvement of Alk7 in modulation of metabolism in the adipose tissues in response to β3-adrenergic receptor activation.

Expression and role of the TGF-β family in glial cells infected with Borna disease virus.

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.

Changes in Borna disease virus genome with adaptation to host.

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.

A Study on Borna Disease Virus Infection in Domestic Cats in Japan

ABSTRACT Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats.

Nucleotide sequence of canine Smad3.

The whole genome sequence of a Boxer dog suggested that the amino acid sequence of the carboxyl terminus of a putative Smad3 is SSVF-COOH, not SSVS-COOH as in all Smad3 sequences identified in many species. Because phosphorylation of the last two serines at the carboxyl terminus is generally indispensable for Smad3-mediated signaling, the role of Smad3 may be unique in dogs. The present study determines the nucleotide sequence of the coding region of canine Smad3 and deduces the carboxyl terminal amino acids of Smad3 in several breeds. Except for the Boxer, the deduced amino acid sequence was SSVS-COOH in all dogs examined. In addition, the nucleotide at position 1204 in the Boxer was different from that of the other dogs. Furthermore, there was a SNP at nt 240. The present study indicates that the carboxyl terminal amino acid of canine Smad3 is not unique, although it is unknown in the Boxer breed.