The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling.

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.

Ligand-dependent degradation of Smad3 by a ubiquitin ligase complex of ROC1 and associated proteins.

Smads are signal mediators for the members of the transforming growth factor-beta (TGF-beta) superfamily. Upon phosphorylation by the TGF-beta receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-beta is degraded by the ubiquitin-proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCF(Fbw1a) consisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed betaTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCF(Fbw1a) is then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-beta/Smad3 signaling is thus irreversibly terminated by the ubiquitin-proteasome pathway.

Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome.

Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock-gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR-based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferroni's corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59-38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation.

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.

Capacitative calcium entry is involved in steroidogenesis in bovine adrenocortical fasciculata cells.

Capacitative Ca(2+) entry into bovine adrenocortical fasciculata cells was investigated by using the mobilization of intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+)-induced steroidogenesis as the indicators. Bovine adrenocortical fasciculata cells on a glass coverslip were loaded with fura-2. The [Ca(2+)](i) mobilization was detected by a change of fura-2 fluorescence intensity. In the intracellular Ca(2+) store depleted cells, the addition of Ca(2+) to the incubation medium elicited a marked and sustained increase in [Ca(2+)](i). In the intracellular Ca(2+) store non-depleted cells, the addition of thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, in the absence of extracellular Ca(2+), induced a slight and transient increase in [Ca(2+)](i), but an extensive and sustained increase in [Ca(2+)](i) was obtained by adding Ca(2+) to the incubation medium after the thapsigargin treatment. The sustained increase induced by thapsigargin was not inhibited by nifedipine, but was inhibited by Zn(2+) and Cd(2+) in a concentration-dependent manner. The effect of Zn(2+) was more potent than that of Cd(2+). Thapsigargin stimulated steroidogenesis in the presence of extracellular Ca(2+). The steroidogenic effect of thapsigargin was inhibited by Zn(2+) and Cd(2+) but not by nifedipine. These results suggest that there is, in bovine adrenocortical fasciculata cells, a steroidogenesis-linked Ca(2+) entry process other than that involving voltage-operated Ca(2+) channels and that the process might be capacitative Ca(2+) entry.

cDNA cloning of a novel bHLH-PAS transcription factor superfamily gene, BMAL2: its mRNA expression, subcellular distribution, and chromosomal localization.

We isolated a human cDNA encoding a novel member of the bHLH-PAS transcription factor superfamily, BMAL2, which is highly similar to, but distinct from, BMAL1. The composite cDNA covered a 1720-bp sequence consisting of a putative 1653-bp open reading frame encoding a polypeptide of 551 amino acids. The deduced BMAL2 product contains a bHLH-PAS domain in its N-terminal region and a variable C-terminus. The overall identity of BMAL2 polypeptide to that of human BMAL1 is 49%. RNA analysis revealed that expression of BMAL2 transcripts was restricted to the fetal brain and to the adult liver in human, while human BMAL1 mRNA was expressed in the brain and skeletal muscle. The chromosomal localization of the human BMAL2 gene was determined by fluorescent in situ hybridization to be localized on chromosome 12 at region p12.2-p11.2. These results suggest that BMAL2 may play different roles from BMAL1 in the embryonic brain and in adult mammals.

P2-purinergic receptor antagonists reduce the minimum alveolar concentration of inhaled volatile anesthetics.

In the central nervous system (CNS), adenosine triphosphate (ATP) is reported to serve as a fast excitatory neurotransmitter via P2X receptor. To examine possible involvement of inhibition of ATP signal-transmission in anesthetic mechanism, the effect of intracerebroventricular (ICV) administration of P2 receptor antagonists on the minimum alveolar concentration (MAC) of sevoflurane and isoflurane was studied in rat. ICV administration of P2 receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), significantly reduced MAC of both anesthetics. The reduction of the MAC by both suramin and PPADS was dose-dependent and reached plateau at 150 microgram/rat. These results suggest that the inhibition of ATP-signal transmission may be involved in analgesic or anesthetic effect in brain.

Genetic polymorphisms of human melatonin 1b receptor gene in circadian rhythm sleep disorders and controls.

Recent studies suggest that melatonin 1b (Mel1b) receptor, as well as melatonin 1a (Mel1a) receptor, is involved in the modulation of circadian rhythms in mammals. Mutational analysis was performed in the entire coding region of the human Mel1b receptor gene using genomic DNA from sleep disorder subjects. We have identified two missense mutations, G24E and L66F. However, neither is likely to be associated with sleep disorders in our study population. One of the subjects with non-24-h sleep-wake syndrome carries missense mutations in both the Mel1a and Mel1b receptor genes.

[Molecular mechanism of biological clock: for chronobiological approach to stress].

Recent progress in molecular chronobiology revealed that the clock genes control intracellular feedback loops. CLOCK protein and BMAL1 protein, first discovered as components of the circadian clock in mammals, are known to function as transcriptional activators in the circadian feedback loop of drosophila. PERIOD and TIMELESS proteins work as inhibitors for these activators in drosophila and possibly in mammals. The clock genes described above are also expressed in peripheral tissues with circadian rhythmicity. Cultured rat-1 fibroblast shows circadian expression of clock genes after serum shock or forskolin stimulation. These results indicate that the clock genes function not only as components of the endogenous clock, but also as a coordinator of the circadian activity of peripheral tissues. It will be important to study stress from the aspect of circadian rhythm.

Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation.

Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-(&bgr;) superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.

Alleic variants of human melatonin 1a receptor: function and prevalence in subjects with circadian rhythm sleep disorders.

The human melatonin 1a (hMella) receptor gene was screened for mutations using genomic DNA samples from patients with circadian rhythm sleep disorders and control subjects by single strand conformational polymorphism analysis (SSCP). We found seven mutations, two of which predict amino acid changes R54W and A157V, respectively. The prevalence of the R54W variant and that of the A157V variant were several times more common in non-24-h sleep-wake syndrome subjects than among control subjects, although the incidence was not significant in our study group. When expressed in COS-7 cells, the R54W mutant receptor exhibited significantly reduced B(max) and slightly enhanced affinity (reduced K(d)) compared to the wild type receptor, while the A157V variant receptor showed similar binding characteristics to the wild type. The identification of variants in the hMella receptor will provide a useful tool for analyzing genetic predisposition toward various diseases related to melatonin function and to clarify the physiological role of melatonin receptors in humans.

Characterization of three splice variants and genomic organization of the mouse BMAL1 gene.

The BMAL1 gene encodes a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) family of transcription factors. It is a key regulator of circadian rhythms. Using sequence information from human BMAL1 (hBMAL1) cDNAs previously reported by our laboratory, we have isolated and characterized cDNAs encoding three splice variants of the mouse BMAL1 (mBMAL1) gene. Of the three splice variants, mBMAL1b extends for 1878 bp in the coding sequence, which is 91% identical to that of hBMAL1b; its deduced amino acid sequence is 626 residues long and is 98% identical to that of hBMAL1b, and sequence identities in the bHLH, PAS-A, and PAS-B regions are 98, 100, and 100%, respectively. mBMAL1b' arises from alternative usage of exon 2, which results in a 7-amino-acid insertion and alternative splice acceptor usage at the intron 9/exon 10 splice junction, which causes an alanine residue deletion. mBMAL1b' encodes 632 amino acids and contains the bHLH/PAS domains. mBMAL1g' is generated by alternative splice acceptor usage at the intron 6/exon 7 splice junction, which results in a 28-bp deletion adjacent to the 5' end of the PAS domain. Since the 28-bp deletion shifts the reading frame, mBMAL1g' is predicted to encode a product of only 222 amino acids that lacks the PAS domain. The tissue distributions of the three splice variants showed some variation. The variations in the tissue distributions and predicted amino acid sequences suggest that the three splice variants may have different functions. Direct sequencing of the genomic mBMAL1 clones indicated that the coding sequence of mBMAL1 spans 32 kb and includes 17 exons. An unusual exon/intron donor sequence was found in intron 14, which begins with GC at the 5' end. Comparison with the bHLH/PAS family genes revealed that the intron/exon splice pattern of mBMAL1 most closely matches that of the mAhr, which suggests that BMAL1 and Ahr belong to the same subclass and may be derived from a common primordial gene.

Region between alpha-helices 3 and 4 of the mad homology 2 domain of Smad4: functional roles in oligomer formation and transcriptional activation.

BACKGROUND: Smad4 has a unique region of 35 amino acids between alpha-helices 3 and 4 (termed H3/4 loop) of the Mad homology (MH) 2 domain. In order to elucidate the functional importance of the H3/4 loop, we prepared chimeric constructs of Smad4 containing the region corresponding to the alpha-helix 3, H3/4 loop and alpha-helix 4 of different Smads, including a chimera containing that of Smad2 (Smad4-HL2). RESULTS: Smad4-HL2 constitutively induced the transcriptional activation of p3TP-Lux, a TGF-beta-responsive reporter construct. However, co-transfection of Smad2 with Smad4-HL2 did not induce a further increase in the activation of p3TP-Lux. Smad4-HL2 did not induce the activation of pAR3-Lux, which contains FAST1-binding sites and is activated by a complex composed of FAST1, Smad2 and Smad4. Smad4-HL2 formed a homo-oligomer more efficiently than wild-type Smad4 in mammalian cells. Moreover, Smad4-HL2 bound to DNA containing the Smad-binding sites with a gretaer affinity than the wild-type Smad4. CONCLUSION: Smad4-HL2 spontaneously forms a homo-oligomer, which may bind to DNA with relatively high affinity and induce transcriptional activation of p3TP-Lux. The H3/4 loop of Smad4 may thus play a role in precluding the spontaneous oligomer formation of Smad4.

Urocortin, a newly identified corticotropin-releasing factor-related mammalian peptide, stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes.

The effect of urocortin (UCN), a recently characterized mammalian member of corticotropin-releasing factor (CRF)-related peptide and a putative endogenous ligand for CRF type 2 beta receptor in the regulation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) release, was investigated using cultured neonatal rat cardiomyocytes. Treatment with UCN (10(-10)-10(-6)M) resulted in significant increase in ANP and BNP secretions, and the effect of UCN on ANP and BNP secretions was more potent than that of CRF on an equimolar basis. The effect of UCN (10(-7)M) was completely blocked by alpha-helical CRF (9-41), a specific CRF type 2 receptor antagonist. The effect of UCN (10(-7)M) was not only blunted by cAMP-dependent protein kinase A (PKA) inhibitor, H-89 (10(-5)M), but also diltiazem (10(-7)M), a voltage-dependent Ca2+ channel blocker. Further, UCN stimulated cAMP production in cardiomyocytes. Also, UCN (10(-7)M) itself stimulated [3H]leucine uptake into neonatal rat cardiomyocytes and potentiated endothelin-1-induced increase of [3H]leucine uptake. These results suggest that activation of CRF type 2 receptor, especially type 2 beta receptor, with UCN induces ANP and BNP secretions, at least in part, via PKA pathway during cardiac hypertrophy.

[Multiple overlapping Wiktor stenting for the treatment of diffuse lesions of small coronary arteries].

This study evaluated the usefulness of multiple overlapping stenting for the treatment of a diffuse lesion in a single and small coronary artery. We studied 48 consecutive patients with a lesion of a vessel with reference diameter < 3.0 mm who received Wiktor stent implants due to suboptimal result or threatened closure after plain old balloon angioplasty (POBA). The patients were divided into 2 groups: Group D, 18 patients with a diffuse lesion with length > or = 15.0 mm who were treated with 2 overlapping Wiktor stents; and Group S, 30 patients with a discrete lesion who were treated with a single Wiktor stent. Two patients with a discrete lesion were not included in Group S because of failure of stenting. Stenting was successful in all patients in Group D. One patient in Group S developed subacute thrombosis after stenting. Follow-up angiography was performed after 6 months. Before treatment, the lesion lengths in Groups D and S were 24.2 +/- 10.0 and 9.2 +/- 4.4 mm, the reference diameters were 2.56 +/- 0.32 and 2.69 +/- 0.33 mm, and the minimal lumen diameters were 0.42 +/- 0.34 and 0.53 +/- 0.40 mm, respectively, with no significant difference in the latter 2 parameters. Restenosis occurred in 72% and 30% (p = 0.01) and revascularization of the target lesion was successful in 67% and in 30% (p = 0.03), in Groups D and S, respectively, both significantly higher in Group D. Follow-up study revealed that the minimal lumen diameter and the net gain were significantly greater in the overlapping portion of the 2 stents than in the proximal and distal portions in Group D, with no significant difference in restenosis rate between these 3 portions. Multiple overlapping stenting using Wiktor stents is useful for diffuse lesions with unfavorable results after POBA even if the affected vessel is smaller than 3.0 mm. However, patients should be carefully observed because restenosis occurs frequently.

Cloning of a melatonin-related receptor from human pituitary.

We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.

Complementary DNAs for two arylamine N-acetyltransferases with identical 5' non-coding regions from rat pineal gland.

A cDNA library was constructed from the pineal gland of rats injected with isoproterenol and screened with 32P-labeled cDNAs encoding arylamine N-acetyltransferases from rabbit and human liver. Two types of cDNAs for arylamine N-acetyltransferases (A-type and B-type) were isolated. Expression of the cDNAs in Chinese hamster ovary cells indicated that A-type N-acetyltransferase acetylates both arylamines and arylalkylamines, while the B-type enzyme acetylates only arylamines. Therefore, neither the A-type nor the B-type of enzyme seems to be the arylalkylamine N-acetyltransferase involved in melatonin synthesis in the pineal gland. Nucleotide sequence analysis revealed that both A-type and B-type cDNAs code for 290 amino acids, and that they showed 82.8% similarity in the coding region. However, the nucleotide sequence in the 5' non-coding region was identical in the A-type and B-type cDNAs. In addition, the 5' non-coding region contained another possible open reading frame for 79 amino acids. Data base research revealed that the complementary sequence of the 5' non-coding region has high similarity with the coding regions of cDNAs for high-mobility-group proteins (HMG) 1 and 2, which are thought to regulate mRNA transcription.

Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses.

The pineal hormone melatonin regulates seasonal reproductive function and modulates circadian rhythms in mammals. We now report the cloning and characterization of a high affinity receptor for melatonin from the sheep and human. The receptor cDNAs encode proteins that are members of a newly discovered group within the G protein-coupled receptor family. Expression of the sheep and human receptors in COS-7 cells results in high affinity 2-[125I]iodomelatonin binding and pharmacological characteristics similar to endogenous high affinity receptors. Functional studies of NIH 3T3 cells stably expressing the sheep receptor show that the mammalian melatonin receptor is coupled to inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism. In situ hybridization studies of melatonin receptor mRNA in several mammals reveal hybridization signals in the hypophyseal pars tuberalis and hypothalamic suprachiasmatic nucleus. The cloned high affinity receptor likely mediates the reproductive and circadian actions of melatonin in mammals.

Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.