Prognostic significance of FTO genotype in the development of obesity in Japanese: the J-SHIPP study.

OBJECTIVE: Susceptibility of fat mass and obesity-associated (FTO) gene polymorphisms to obesity has been reported in various populations. Polymorphisms in the melanocortin 4 receptor (MC4R) gene were recently explored as another susceptible locus. However, prognostic significance of these genetic variations has not been fully elucidated. Here, we investigated the involvement of FTO rs9939609 and MC4R rs17782313 polymorphisms in the development of obesity. Association with type 2 diabetes mellitus (T2DM) was also investigated. SUBJECTS: We analyzed 2806 community-dwelling middle-aged to elderly subjects (61+/-14 years). Clinical parameters were obtained from the subjects' personal health records, evaluated at their annual medical check-up. RESULTS: FTO genotype was significantly associated with current body mass index (BMI; TT 23.2+/-3.2, TA 23.7+/-3.2, AA 24.4+/-3.2 kg m(-2), P=2.5 x 10(-6)) and frequency of obesity (26.6, 32.0, 43.0% respectively, P=2.0 x 10(-4)). Age- and sex-adjusted odds ratio for obesity was 1.30 (P=0.004) in TA and 2.07 (P=0.002) in AA genotype. During the 9.4 years comprising the follow-up period, 214 new cases of obesity were diagnosed among 1718 subjects whose retrospective data were available. A allele frequency of the FTO genotype was significantly higher in subjects who developed obesity (22.2, 15.8%, P=0.001), Age-, sex- and initial BMI-adjusted odds ratio for the development of obesity was 1.46 (95% confidence interval, 1.04-2.04) (P=0.031). However, association studies and meta-analysis of T2DM did not actively support the involvement of FTO genotype. No significant differences were observed between the MC4R genotype and BMI (P=0.015), and the frequency of obesity (P=0.284). CONCLUSION: FTO genotype is an independent risk factor for future development of obesity.

Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids.

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.

Transcriptional activation of beta-tropomyosin mediated by serum response factor and a novel Barx homologue, Barx1b, in smooth muscle cells.

Tropomyosin (TM) is a regulatory protein of actomyosin system. Muscle type-specific expression of TM isoforms is generated from different genes and by alternative splicing. beta-TM isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a smooth muscle cell (SMC) phenotype-dependent change in beta-TM expression (Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato, K., and Sobue, K. (1997) J. Biol. Chem. 272, 15396-15404), and identified beta-TM as an SMC-differentiation marker. Here, we characterized the transcriptional machinery of the beta-TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor and its interaction with the CArG box sequence in the SMC-specific transcription of the beta-TM gene in differentiated SMCs. We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard. Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. Serum response factor and Barx1b bound each other directly, coordinately transactivated the beta-TM gene in differentiated SMCs and heterologous cells, and formed a ternary complex with a CArG probe. Taken together, these results suggest that SRF and Barx1b are coordinately involved in the SMC-specific transcription of the beta-TM gene in the upper digestive organs and their attached arteries.

Phenotype-dependent expression of cadherin 6B in vascular and visceral smooth muscle cells.

We used mRNA subtraction of differentiated and dedifferentiated smooth muscle cells (SMCs) to reveal the molecular mechanisms underlying the phenotypic modulation of SMCs. With this approach, we found that a 10 kb mRNA encoding a homotypic cell adhesion molecule, cadherin 6B, was strongly expressed in differentiated vascular and visceral SMCs, but not in the dedifferentiated SMCs derived from them. In vivo, cadherin 6B was expressed in vascular and visceral SMCs, in addition to brain, spinal cord, retina and kidney, at a late stage of chicken embryonic development. These results suggest that cadherin 6B is a novel molecular marker for vascular and visceral SMC phenotypes and is involved in the late differentiation of SMCs.

Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering.

Several scaffold proteins for neurotransmitter receptors have been identified as candidates for receptor targeting. However, the molecular mechanism underlying such receptor clustering and targeting to postsynaptic specializations remains unknown. PSD-Zip45 (also named Homer 1c/vesl-1L) consists of the NH(2) terminus containing the enabled/VASP homology 1 domain and the COOH terminus containing the leucine zipper. Here, we demonstrate immunohistochemically that metabotropic glutamate receptor 1alpha (mGluR1alpha) and PSD-Zip45/Homer 1c are colocalized to synapses in the cerebellar molecular layer but not in the hippocampus. In cultured hippocampal neurons, PSD-Zip45/Homer1c and N-methyl-D-aspartate receptors are preferentially colocalized to dendritic spines. Cotransfection of mGluR1alpha or mGluR5 and PSD-Zip45/Homer 1c into COS-7 cells results in mGluR clustering induced by PSD-Zip45/Homer 1c. An in vitro multimerization assay shows that the extreme COOH-terminal leucine zipper is involved in self-multimerization of PSD-Zip45/Homer 1c. A clustering assay of mGluRs in COS-7 cells also reveals a critical role of this leucine-zipper motif of PSD-Zip45/Homer 1c in mGluR clustering. These results suggest that the leucine zipper of subsynaptic scaffold protein is a candidate motif involved in neurotransmitter receptor clustering at the central synapse.

The unique 5-flanking region of the human basic calponin gene.

Calponin has been implicated in the regulation of smooth muscle contraction. Basic calponin, one of the calponin isoforms, is expressed exclusively in smooth muscle cell (SMC)-rich tissues, and is considered to be a phenotypic marker of differentiated SMC. To define the molecular mechanism of SMC-specific gene transcription in humans, we isolated and characterized the 5'-flanking region of this gene. Sequence analysis revealed that several putative cis-acting elements were clustered within a 500-bp sequence upstream of the transcription start site. However, the 1.9-kb promoter region obtained herein lacked a completely matched consensus sequence of the CArG box that is commonly identified in the promoter region of other SMC-specific genes. A luciferase assay demonstrated that the 1.9-kb promoter region was sufficient to drive a basal transcriptional activity not only in human vascular smooth muscle cells (VSMC) but also in HeLa cells. In particular, the sequence between positions -1,906 and -867 had a significantly higher transcriptional activity in VSMC than in HeLa cells. In contrast, the promoter activity was drastically decreased between positions -327 and -257 in both types of cells. These results indicate that the sequence spanning from position -327 to -257 contains an essential domain involved in the basal transcriptional activity of the human basic calponin gene, and that the distal region of the 1.9-kb 5'-flanking sequence presented herein may play a pivotal role in the phenotypic modulation of VSMC.

Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.

Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation.

Phenotypic modulation of smooth muscle cells (SMCs) plays an integral role in atherosclerosis, hypertension and leiomyogenic tumorigenicity. The morphological, functional, and biochemical characteristics of SMCs in different phenotypes such as differentiated and dedifferentiated states have been well studied. Recent researches have focused on the expressional regulation of SMC-specific marker genes in association with phenotypic modulation of SMCs. The SMC-specific marker genes are regulated at the levels of transcription and splicing. The caldesmon, smooth muscle myosin heavy chain, alpha-smooth muscle actin, calponin, SM22, alpha- and beta-tropomyosins, and alpha1 integrin genes are transcriptionally regulated; transcription of these genes except for the alpha-smooth muscle actin gene is upregulated in differentiated SMCs, but is downregulated in dedifferentiated SMCs. The expression pattern of alpha-smooth muscle actin is opposite in vascular and visceral SMCs. In almost all promoter regions of these genes, the CArG box and serum response factor (SRF) are involved in as the positive cis-element and the trans-acting factor, respectively. Isoform changes of caldesmon, alpha-tropomyosin, vinculin/metavinculin, and smooth muscle myosin heavy chain are regulated by alternative splicing in a SMC phenotype-dependent manner. Among them, isoform interconversions of caldesmon and alpha-tropomyosin are completely coordinated with phenotype of SMCs. The purpose of this paper is to summarize current knowledge of the expressional regulation of SMC-specific marker genes in different phenotypes of SMCs.

Isolation of PSD-Zip45, a novel Homer/vesl family protein containing leucine zipper motifs, from rat brain.

Using monoclonal antibody against the 45 kDa postsynaptic density protein, we isolated a novel isoform of Homer/vesl. The NH2-terminal region containing a PDZ domain of this protein is identical to that of Homer/vesl, and the COOH-terminal region containing unique leucine zippers shows self-multimerization. We named this protein PSD-Zip45. In addition to specific binding of PSD-Zip45 mediated by a PDZ domain to the metabotropic glutamate receptors 1alpha or 5, the distribution of PSD-Zip45 transcripts is highly consistent with that of metabotropic glutamate receptor transcripts. The PSD-Zip45 is, therefore, the first candidate as receptor anchoring proteins containing leucine zipper motifs in the central nervous system.

Molecular mechanism of phenotypic modulation of smooth muscle cells.

Phenotypic modulation of smooth muscle cells is closely associated with vasculogenesis, enterogenesis and some diseases such as atherosclerosis, hypertension and leiomyogenic tumorigenicity. During phenotypic modulation, smooth muscle cells change their morphology, cell function and biochemical characteristics. Recent studies have focused on the regulation mechanism of smooth muscle cell-specific genes at the levels of transcription and/or alternative splicing in a phenotype-dependent manner. Typical examples of such genes include caldesmon, alpha-tropomyosin, myosin heavy chain, SM22, calponin and alpha 1 integrin. Cell adhesion molecules and growth factors/cytokines also play a critical role for controlling phenotype of smooth muscle cells via signal transduction pathways such as phosphoinositide 3-kinase and mitogen-activated protein kinases.

Smooth muscle cell phenotype-dependent transcriptional regulation of the alpha1 integrin gene.

The expressional regulation of chicken alpha1 integrin in smooth muscle cells was studied. The alpha1 integrin mRNA was expressed developmentally and was distributed dominantly in vascular and visceral smooth muscles in chick embryos. In a primary culture of smooth muscle cells, alpha1 integrin expression was dramatically down-regulated during serum-induced dedifferentiation. Promoter analyses revealed that the 5'-upstream region (-516 to +281) was sufficient for transcriptional activation in differentiated smooth muscle cells but not in dedifferentiated smooth muscle cells or chick embryo fibroblasts. Like other alpha integrin promoters, the promoter region of the alpha1 integrin gene lacks TATA and CCAAT boxes and contains binding sites for AP1 and AP2. The essential difference from other alpha integrin promoters is the presence of a CArG box-like motif. Deletion and site-directed mutation analyses revealed that the CArG box-like motif was an essential cis-element for transcriptional activation in differentiated smooth muscle cells, whereas the binding sites for AP1 and AP2 were not. Using specific antibodies, a nuclear protein factor specifically bound to the CArG box-like motif was identified as serum response factor. These results indicate that alpha1 integrin expression in smooth muscle cells is regulated transcriptionally in a phenotype-dependent manner and that serum response factor binding plays a crucial role in this regulation.

Coordinate expression of alpha-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells.

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha-tropomyosin (alpha-TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of beta-TM-SM, alpha-TM-SM converted to alpha-TM-F1 and alpha-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high Mr type to low Mr type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express alpha-TM-SM, beta-TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha-TM-SM and high Mr caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.

Near-death asthmatic reaction induced by disodium cromoglycate.

A near-death asthmatic reaction was induced by disodium cromoglycate (DSCG) as evidenced by positive skin and inhalation provocation tests. The patient's history revealed an episode of exacerbation by inhalation of DSCG. In spite of such an experience, he inhaled DSCG for relief of asthmatic attack, resulting in near-death exacerbation. This patient emphasizes the need to re-recognize that DSCG is not a reliever and the DSCG could cause fatal asthma.

[A very old man with Crush syndrome].

A 90-year-old man fell into a marsh and was rescued 18 hours later. When he was admitted to our emergency room, physical examination revealed no remarkable findings except for many abrasions on his skin. Laboratory examination revealed a serum CPK level of 46, 904 IU/L, which had further increased to 84,678 IU/L by the following day. Oliguria developed on the second day, along with an increase in serum creatinine to 5.5 mg/dt. Hemodialysis was considered for the treatment of acute renal failure, but his renal function recovered soon by the continuation of conservative fluid therapy. Fluid therapy may be an effective and easy treatment for acute renal failure due to the crush syndrome, even in very old patients.

Angiotensin-converting enzyme gene polymorphism in essential hypertensive patients in Japanese population.

Association between angiotensin-converting enzyme (ACE) gene polymorphism and essential hypertension in a Japanese population with the same socioeconomic background was investigated. Insertion-deletion (I/D) polymorphism of the ACE gene located on intron 16 was detected by polymerase chain reaction. Association between ACE gene polymorphism and family history of essential hypertension as well as the development of vascular damage in eye fundi were also investigated. Variation at ACE loci did not contribute to essential hypertension and the vascular damages in eye fundi. These results suggest that the ACE gene was not directly responsible for essential hypertension in this particular Japanese population with the same socioeconomic background.

[A case of idiopathic orthostatic hypotension manifesting sick sinus syndrome due to sympathetic nervous dysfunction].

A 67-year-old woman with idiopathic orthostatic hypotension was presented. The patient started to experience faintness on standing since 1993. During a physical examination, her systolic blood pressure fell from 148 to 50 mmHg on standing. Blood pressure responses to the mental arithmetic test and hyperventilation stress were normal. However, cold pressor test failed to increase blood pressure. These observations, with the finding that phase IV response on Valsalva's maneuver was absent, indicate afferent sympathetic nervous dysfunction. Peripheral neuropathy including diabetes mellitus and involvement of central nervous system such as multiple system atrophy were excluded. Holter ECG examination revealed a 3.9 second sinus arrest and bradycardia (total beats 88901/day). the blunted responses of the heart rate to atropine as well as isoproterenol further suggested the presence of sick sinus syndrome. Amezinium administration significantly improved her orthostatic hypotension and eliminated sinus arrest. These findings indicate that sympathetic nervous dysfunction could account for at least a part of the sick sinus syndrome in this patient.

Molecular cloning and gene mapping of human basic and acidic calponins.

The nucleotide and deduced amino acid sequences of human basic and acidic calponins were determined. The basic calponin cDNA from human aorta (1496 bp) contained a single open reading frame (ORF) which encodes 297 amino acids (33,169 Da). The acidic calponin cDNA from human kidney (1607 bp) contained a single ORF which encodes 329 amino acids (36,412 Da). Basic calponin mRNA was expressed in only smooth muscle tissues, but acidic calponin mRNA was expressed in non-smooth muscle tissues as well as smooth muscle tissues. Fluorescent in situ hybridization revealed that basic and acidic calponin genes localize in 19p13.1-13.2 and 1p21-22 of human chromosomes, respectively.

Autonomic nervous function in non-dipper essential hypertensive subjects. Evaluation by power spectral analysis of heart rate variability.

Autonomic nervous function was evaluated by means of power spectral analysis of heart rate variability in hospitalized dipper (n = 31) and non-dipper (n = 31) essential hypertensive subjects. Twenty-four-hour blood pressure (BP) measurement was performed by the cuff-oscillometric method to evaluate the nocturnal decrease of BP. The non-dipper subjects were defined as those whose nocturnal decrease of systolic BP was < 10% of daytime BP. Power spectral analysis of RR interval was performed from Holter ECG every 10 minutes by the maximum entropy method to obtain the low-frequency band (LFB, 0.04 to 0.15 Hz), which is an index of both parasympathetic and sympathetic nervous activities, and the high frequency band (HFB, 0.15 to 0.4 Hz), which reflects parasympathetic nervous activity. LFB and HFB were averaged every hour to obtain hourly LFB and HFB values. Total LFB and total HFB were calculated as the mean values of 24 hourly averaged LFBs and HFBs. Both LFB and HFB were significantly lower in non-dipper hypertensives than in dipper subjects throughout the day. In dipper hypertensives, LFB showed a nocturnal decrease, whereas HFB was significantly increased during the nighttime. However, these diurnal changes in LFB and HFB were significantly blunted in non-dipper subjects. These findings indicate that non-dipper hypertensive subjects were characterized with a decreased physiological circadian fluctuation on autonomic functions compared with dipper subjects. This alteration in the autonomic nervous function may explain the non-dipper phenomenon in essential hypertension.

cDNA cloning and mRNA expression of calponin and SM22 in rat aorta smooth muscle cells.

We cloned and sequenced cDNAs encoding calponin (Calp) and SM22 (smooth muscle-specific 22-kDa protein) from rat aorta (RaA) smooth muscle (Smu) cells. The 1504-bp calp cDNA contains a single open reading frame (ORF) which encodes 297 amino acids (aa) (M(r) 33,342). The 1186-bp SM22 cDNA contains a single ORF which encodes 201 aa (M(r) 22,601). There were 43% identical aa in a 181-aa overlap between RaA Calp and SM22. Especially for the C-terminal region of SM22 and for the first repeat motif of Calp, 70% identity was observed. Northern blot analysis revealed that the calp and SM22 mRNAs were expressed in RaA Smu, but not in rat cardiac and skeletal muscles. SM22 mRNA was much more abundant than calp mRNA in RaA (3- to 4-fold). The expression levels of the calp and SM22 mRNAs in RaA showed a significant increase for 5 to 15 week old rats (1.5- to 3-fold) with vascular development and blood pressure elevation. No significant differences were observed in the expression of the RaA calp and SM22 mRNAs between normotensive (Wistar Kyoto) and spontaneously hypertensive rats (SHR).

Anti-caldesmon monoclonal antibody reverses the inhibition of actomyosin Mg(2+)-ATPase activity by caldesmon.

Five mouse monoclonal antibodies, CaD 1-5, against chicken gizzard caldesmon were prepared. One of them (CaD4) was characterized by means of immunoblotting and its effect on actomyosin Mg(2+)-ATPase activity. CaD4 recognized the tropomyosin-binding site of caldesmon. CaD4 reversed the caldesmon-induced inhibition of actomyosin Mg(2+)-ATPase activity in a dose-dependent manner. These results suggest that the epitope recognized by CaD4 is an important domain for the function of caldesmon on the actinmyosin interaction in the smooth muscle contraction-relaxation system.