The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice.

A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells.

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.

A possible association between aldosterone response to vasopressin and circadian change of aldosterone in the patients with aldosterone-producing adenoma.

Vasopressin was reported to stimulate secretion of both cortisol and aldosterone through eutopic V1a receptors in adrenal gland. Recently, adrenal hyper-responsiveness of plasma cortisol to vasopressin with eutopic overexpession of V1a receptors has been reported in Cushing's syndrome, such as a majority of cases of ACTH-independent macronodular adrenal hyperplasia and some cases of Cushing's adenomas. There were a few reports regarding the aldosterone response to vasopressin in aldosterone-producing adenoma. The aim of our study was to investigate the aldosterone response to vasopressin and its pathophysiological roles in the patients with aldosterone-producing adenoma. Vasopressin-loading test was performed in 10 patients with aldosterone-producing adenoma, and in 16 patients with non-functioning adrenal tumors. The roles of the aldosterone response to vasopressin were analyzed in terms of hormonal secretion and the expression of V1a receptor mRNA on the operated adrenal gland in aldosterone-producing adenoma. We found that (1) a varying aldosterone response to vasopressin was observed, (2) absolute response of plasma aldosterone in aldosterone-producing adenoma was significantly higher than that in non-functioning tumor, (3) aldosterone response rate to vasopressin was significantly and negatively correlated with the decline rate (%) in plasma aldosterone from morning to evening in aldosterone-producing adenoma, (4) V1a receptor mRNA was expressed at various values in aldosterone-producing adenoma, and (5) surgical removal of aldosterone-producing adenoma eliminated the aldosterone response to vasopressin observed in patients with aldosterone-producing adenoma. These findings indicated that vasopressin might be involved in the coordination of aldosterone secretion through eutopic expression of V1a receptor in aldosterone-producing adenoma.

Molecular cloning and characterization of rat brain endothelial cell derived gene-1 (tumor suppressor candidate 5) expressing abundantly in adipose tissues.

We report the cloning and expressional analysis of rat brain endothelial cell derived gene-1 (BEC-1), detected as a gene dominantly expressed in rat brain endothelial cells by the use of suppression subtractive hybridization technique. The complementary deoxyribonucleic acid sequence of BEC-1 messenger ribonucleic acid was completely determined with a full length of 3410 bp. The open reading frame within the sequence consisted of 522 bp, and the predicted protein sequence was 173 amino acid residues. BEC-1 gene was thought to be rat tumor suppressor candidate 5 (TUSC5), since BEC-1 had considerable homology with both mouse TUSC5 and human located at 17-p-13 point three 1 (LOST1) categorized as human TUSC5 (identities of 97% and 85%, respectively), which were recently identified as a novel tumor suppressor gene candidate. Expressional analyses for BEC-1 mRNA with real-time PCR and of BEC-1 protein by Western blotting demonstrated that both were dominantly expressed in the adipose tissues of Sprague-Dawley (SD) rats. We analyzed and compared the differential expressions of BEC-1 (TUSC5) mRNA and protein in fat tissues between obese homozygous (fa/fa) and lean wild-type (+/+) Zucker rats. Both expressions in the epididymal white adipose tissue (WAT) were highest, followed by those in the interscapular brown adipose tissue (BAT), subcutaneous, and mesenteric WATs, respectively. Interestingly, both expressions in epididymal WAT of obese Zucker rats were significantly lower than those in lean rats. Although cold exposure at 4 degrees C for 6 h significantly stimulated uncoupling protein-1 (UCP-1) mRNA expression, it significantly inhibited BEC-1 (TUSC5) mRNA expression in the interscapular BAT. These data indicated that rat BEC-1 (TUSC5) was abundantly expressed in adipose tissues, and that it might be involved in their regulation independently of UCP-1.

Isoprenoid-independent pathway is involved in apoptosis induced by risedronate, a bisphosphonate, in which Bim plays a critical role in breast cancer cell line MCF-7.

Bisphosphonates cause apoptosis to various types of cancer cells including breast cancer. Inhibition of the mevalonate pathway was reported to be involved in the apoptosis induced by bisphosphonates, but its precise mechanism has not been unveiled. In the present study, we investigated the molecular mechanism of risedronate, a bisphosphonate, in the apoptosis of the breast cancer cell line MCF-7 in comparison with that of cerivastatin, an HMG CoA reductase inhibitor (statin), since statin has been known to induce apoptosis through an isoprenoid-dependent pathway in these cells. We found that i) risedronate induced MCF-7 cells into apoptosis in a manner similar to cerivastatin with the activation of caspase-9 followed by caspase-6 and -7, that ii) bisphosphonate-induced apoptosis was significantly, but not fully, recovered by the addition of GGOH, an isoprenoid, which completely rescued in case of cerivastatin-induced apoptosis, that iii) risedronate induced G2 arrest with the induction of Bim (BH3-only protein), but that statin induced G1 arrest without it, and that iv) the down-regulation of Bim protein by siRNA significantly attenuated the risedronate-induced apoptosis. These data clearly indicate that both isoprenoid-dependent and -independent pathways might be involved in the apoptosis induced by bisphosphonate, and Bim might be a critical component for the isoprenoid-independent apoptotic pathway.

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1.

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.

Analysis of genes dominantly expressed in rat cerebral endothelial cells using suppression subtractive hybridization.

In mammals, a fully developed, highly branched vascular system specialized for each particular organ or tissue is essential for obtaining metabolic nutrients supply. The formation of a blood-brain barrier that protects against environmental insults is a distinguishing feature of the brain's vascular system. Since this is accomplished by cerebral endothelial cells (CECs), we analyzed the genes specifically and/or dominantly expressed in rat CECs using Suppression Subtractive Hybridization (SSH). We found 39 genes specifically and/or dominantly expressed in CECs. 24 genes of known function (thrombospondin-2, vimentin, etc.), 13 genes of known sequence but unknown function including 7 of ESTs (SNERG1, rat GPCR, etc.), and 2 novel genes. The physiological significance of these genes in CECs has been under investigation. SSH is useful for identifying genes regulated in an organ-specific manner in cells such as CECs to obtain clarification of their physiological roles.

Effect of fenofibrate on uric acid metabolism in Japanese hyperlipidemic patients.

Forty Type IIb or IV hyperlipidemic patients (serum triglyceride concentrations were higher than 150 mg/dl) were treated with fenofibrate (300 mg/day) for 12 weeks. Lipid profile and uric acid metabolism were evaluated before and after the treatment; the serum concentrations of total cholesterol and triglyceride respectively decreased from 224 +/- 41.9 mg/dl to 199 +/- 35.2 mg/dl and from 205 +/- 71.7 mg/dl to 134 +/- 67.5 mg/dl (p < 0.001). The uric acid concentrations in the serum also significantly decreased from 7.0 +/- 1.58 mg/dl to 5.2 +/- 1.57 mg/dl (p < 0.001). Fenofibrate treatment did not cause any change in the serum xanthine and hypoxanthine concentrations. Instead the urinary concentrations of uric acid decreased from 7.0 +/- 1.58 mg/dl to 5.2 +/- 1.57 mg/dl (p < 0.01), while the clearance ratio of uric acid and creatinin increased from 6.1 +/- 2.56 to 9.9 +/- 3.87 (p = 0.02) by the fenofibrate treatment. Fenofibrate decreases uric acid concentrations in the serum not as a result of inhibition of uric acid production but by increasing the urinary excretion of uric acid.

Bilateral symmetrical pallidal lesions following severe anemia associated with gastrointestinal hemorrhage: report of two cases.

We herein report the cases of two patients with bilateral symmetrical pallidal lesions mimicking hypoxic encephalopathy following severe anemia associated with gastrointestinal hemorrhage despite a lack of carbon monoxide intoxication. Although severe anemia can theoretically result in anemic hypoxia, vulnerable pallidal lesions have rarely been described in anemic patients. Interestingly, both patients shared common conditions associated with atherosclerosis, including heavy smoking, hypertension, type 2 diabetes mellitus and a history of coronary artery bypass grafting for ischemic heart disease. Anemic hypoxia may cause pallidal involvement in atherosclerotic patients with multiple risk factors.

Tumor suppressor candidate 5 (TUSC5) is expressed in brown adipocytes.

Rat brain endothelial cell derived gene-1 (BEC-1) had considerable homology with tumor suppressor candidate 5 (TUSC5). TUSC5 was expressed abundantly, and its mRNA was inhibited by cold exposure in rat brown adipose tissue (BAT). In the present study, we investigated its regulatory mechanism using primary cultured rat brown preadipocytes (RBPA) and Zucker lean rats (ZL). We found that: (1) TUSC5 mRNA began to increase in a manner similar to C/EBP-alpha, PPAR-gamma, and adiponectin during differentiation in RBPA; (2) neither beta3-adrenoceptor agonist BRL 37344 nor dexamethasone affected TUSC5 mRNA in RBPA; (3) propranolol did not block the decrease of TUSC5 mRNA by cold exposure in ZL; (4) BRL 37344 did not influence TUSC5 mRNA in ZL; and (5) dexamethasone inhibited TUSC5 mRNA in a dose-dependent manner similar to UCP-1 in ZL. These data suggested that TUSC5 is involved in the differentiation, and its expression is regulated independently of the beta-adrenergic pathway in BAT.

Regulation of geranylgeranyl pyrophosphate synthase in the proliferation of rat FRTL-5 cells: involvement of both cAMP-PKA and PI3-AKT pathways.

We have reported that geranylgeranyl pyrophosphate (GGPP), one of the isoprenoids in the mevalonate pathway, plays an essential role for cell growth through the geranylgeranylation of Rho small GTPases, which control the degradation of P27Kip1 at G1/S transition in rat thyroid FRTL-5 cells. Since GGPP is synthesized from isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) by GGPP synthase, we analyzed the regulatory roles of GGPP synthase in the proliferation of FRTL-5 cells stimulated by thyrotropin and insulin in the presence of 5% calf serum (TSH+Ins). We found that: (1) GGPP synthase was activated at G1/S transition with increasing mRNA accumulation followed by protein expression, (2) pravastatin, an inhibitor of HMG-CoA reductase, did not suppress the increasing activity of GGPP synthase with its protein expression although it inhibits proliferation in growth-stimulated FRTL-5 cells, (3) forskolin stimulated proliferation with activation of GGPP synthase in FRTL-5 cells, and (4) LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited proliferation with the decreasing activity of GGPP synthase in growth-stimulated FRTL-5 cells. These data indicated that growth stimulation by TSH+Ins increased the activity of GGPP synthase with its increasing protein expression from G1/S transition, in which both cAMP-PKA and PI3-kinase pathways are involved in the proliferation of FRTL-5 cells.