Bradykinin deficiency causes high blood pressure in mice.

Bradykinin has a wide variety of physiological functions, including vasodilation and blood pressure reduction. However, the physiological roles of bradykinin are not fully understood. We used the CRISPR/Cas9 method to generate BKdelK1 and BKdelK2 mutant mice, targeting the BK portion of mouse kininogen1 and kininogen2 genes, respectively. The BKdelK1 and BKdelK2 mutant mice had about 50% reductions in plasma low molecular weight kininogen and trypsin-released BK, compared to wild mice. Both BKdelK1 and BKdelK2 mice had significantly elevated systolic blood pressure compared to WT mice. These results suggest that plasma LKNG is a source of KNG in the vascular kallikrein-kinin system and contributes to maintaining lower systolic blood pressure.

Sox6 suppression induces RA-dependent apoptosis mediated by BMP-4 expression during neuronal differentiation in P19 cells.

Sox6 is a transcription factor that induces neuronal differentiation in P19 cells; its suppression not only inhibits neuronal differentiation but also induces retinoic acid (RA)-dependent apoptosis of P19 cells. In the present study, we found that Sox6 suppression-induced apoptosis was mediated by activation of caspase 9 and 3. Moreover, we noted a weak leakage of cytochrome c into the cytoplasm from the mitochondria, indicating that apoptosis occurs through a mitochondrial pathway in Sox6-suppressed P19 (P19[anti-Sox6]) cells. Sox6 suppression in the presence of RA also induced the expression and secretion of bone morphogenetic protein 4 (BMP-4). Addition of an anti-BMP-4 antibody for neutralization increased cell viability and led to RA-dependent death of P19[anti-Sox6] cells. Our results indicate that Sox6 suppression induces RA-dependent cell death of P19 cells, mediated by BMP-4 expression and secretion. Normally, high Sox6 expression leads to RA-mediated neuronal differentiation in P19 cells; however, Sox6 deficiency induces production and secretion of BMP-4, which mediates selective cell death. Our findings suggest that Sox6 contributes to cell survival by suppressing BMP-4 transcription during neuronal differentiation.

Proteomic analysis of mouse choroid plexus cell line ECPC-4 treated with lipid A.

OBJECTIVES: Choroid plexus (CP) epithelial cells have multiple functions in the cerebral ventricles, including cerebrospinal fluid (CSF) production and forming part of the blood-CSF barrier. They are also responsible for producing inflammatory mediators involved in meningitis. The present study aimed to elucidate the functions of the CP epithelial cells during CNS inflammation. MATERIALS AND METHODS: We analyzed the proteome and phosphoproteome in lipid A-treated ECPC-4 mouse CP cells by gel electrophoresis and mass spectrometry. RESULTS: Levels of 10 proteins and seven phosphoproteins were significantly altered by lipid A in time-dependent manners, including V-type proton ATPase subunit B (ATP6V), protein 40 kD, elongation factor-1δ, coatomer subunit ε (COPE), vimentin (isoform CRA a), purine nucleoside phosphorylase, eukaryotic initiation factor-4F splicing variant, put. ß-actin, peroxiredoxin-6 isoform 1, and immunoglobulin heavy chain variable region. These proteins could be classified as having cytoskeleton/intermediate filament, protein-folding, signal-transduction, cell-growth, metabolism, and redox-regulation functions. The identified phosphoproteins were HSP 84, γ-actin, HSP 70 cognate, vimentin, tubulin ß-4B chain, protein disulfide-isomerase A6 precursor, and heterogenous nuclear ribonucleoprotein, which could be classified as having cytoskeleton/intermediate filament, protein-folding, and metabolism functions. CONCLUSIONS: These results indicate that lipid A can change the levels of proteins and phosphoproteins in ECPC-4 cells, suggesting that the identified proteins and phosphoproteins may play important roles in inflammation of the CP.

Nitric oxide promotes survival of cerebral cortex neurons with simultaneous addition of [Fe(II)(ß-citryl-L-glutamate)] complex in primary culture.

It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO). In present study, we found that NO significantly increased the number of surviving neurons via enhanced mitochondrial functions with simultaneous addition of the [Fe(II)(ß-citryl-L-glutamate; ß-CG)] complex. In vitro, a variety of aconitase-inhibitors, such as fluorocitrate, cyanide ion, ferricyanide ([Fe(CN)6]), and various oxidants including superoxide anion, inhibited the activity of m-aconitase even in the presence of Fe(II), whereas a NO-donor, nitroprusside (SNP) ([Fe(CN)5NO]), was the only agent that significantly increased activity of that enzyme. Therefore, it is reasonable to assume that NO released from SNP promotes Fe-dependent activation of aconitase. All other tested NO-donors, including 3-morpholino-sydnonimine (SIN), Deta NONOate (NOC18), and NaNO2, also promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II). The promoting effects of the NO-donors on activation disappeared with the addition of NO-scavengers. In intact mitochondria, all tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II), whereas that was not seen in its absence. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase. In mixed neuronal and glial cultures, NO-donors except for SNP enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(ß-CG)] complex significantly enhanced those activities and greatly increased the number of surviving neurons. Thus, NO can carry Fe ions into m-aconitase via the guide of the tag of ß-CG addressed to the enzyme.

ß-Citryl-L-glutamate acts as an iron carrier to activate aconitase activity.

The compound ß-citryl-L-glutamate (ß-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(ß-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxamine displayed such an effect. ß-CG and [Fe(II)(ß-CG)] both bound to the fourth labile Fe atom (Fe(a)) in the [4Fe-4S] cluster of aconitase. Furthermore, [Fe(II)(ß-CG)] reactivated aconitase damaged by ammonium peroxodisulfate (APS), while ß-CG and citrate had no effect. These findings suggest that [Fe(II)(ß-CG)] can transfer Fe to aconitase disassembled by APS. In intact mitochondria, both ß-CG and [Fe(II)(ß-CG)] bound to Fe(a) of aconitase, whereas only [Fe(II)(ß-CG)] reactivated the enzyme disassembled by APS. In cultured neuronal cells, ß-CG significantly enhanced cell viability by accelerating mitochondrial activity in primary cultures of neurons from newborn mouse cerebrum tissues. Thus, the ß-CG plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. Taken together, these findings suggest that ß-CG is an endogenous low molecular weight Fe chaperone for aconitase.

Rimklb mutation causes male infertility in mice.

Rimklb is a mammalian homologue of the E. coli enzyme RimK, which catalyzes addition of glutamic acid to the ribosomal protein S6. To date, no previous studies have shown any physiological role for Rimklb in mammals. In this study, using Western blotting, we found that Rimklb is distributed and expressed in mouse testis and heart. Rimklb was subsequently localized to the testicular Leydig cells using immunohistochemistry with an anti-Rimklb antibody. We generated a Rimklb mutant mouse in which a three-base deletion results in deletion of Ala 29 and substitution of Leu 30 with Val, which we named the RimklbA29del, L30V mutant mouse. RimklbA29del, L30V mutant mice show a decrease in testicular size and weight, and in vitro fertilization demonstrates complete male infertility. Furthermore, we found that a key factor in the mammalian target of the rapamycin/ribosomal protein S6 transcriptional pathway is hyperphosphorylated in the seminiferous tubules of the mutant testis. We conclude that Rimklb has important roles that include spermatogenesis in seminiferous tubules. In summary, male RimklbA29del, L30V mice are infertile.

Superoxide scavenging and xanthine oxidase inhibiting activities of copper-ß-citryl-L-glutamate complex.

ß-Citryl-L-glutamate (ß-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation of neurons in developing animals, and then decreases with maturation. However, its functional roles remain unclear. The stability constant obtained in our previous pH titration studies showed that ß-CG forms relatively strong complexes with copper. Reactive oxygen species (ROS) and nitric oxide (NO) have been suggested to act as mediators of the cell death that occurs in neurons during development of the nervous system. However, regulation of ROS and NO formation by Cu in the developing brain remains poorly understood. The activity of superoxide dismutase (SOD), a key superoxide scavenging enzyme, is low in the developing brain. Furthermore, xanthine oxidase (XO) has been implicated in diverse pathological situations due to its capability of generating both ROS and NO. Therefore, we examined the effects of ß-CG and its Cu-complex on SOD and XO activities. We found that the [Cu(II)(ß-CG)] complex had SOD activity and a strong competitive inhibition of XO, while reduced glutathione caused concentration-dependent decreases of the XO inhibitory activities in the [Cu(II)(ß-CG)] complex.

beta-Citryl-L-glutamate is an endogenous iron chelator that occurs naturally in the developing brain.

The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.

Lipopolysaccharide activates the kallikrein-kinin system in mouse choroid plexus cell line ECPC4.

Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.

Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis.

The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast-like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6-suppressed P19 (P19[anti-Sox6]) cells by transfection of antisense-Sox6 cDNA. Most of the P19[anti-Sox6] cells showed no neurites and were not stained by the anti-MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA-dependent apoptosis by P19[anti-Sox6] cells: RA-treated P19[anti-Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase-3-like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid.

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

cDNA cloning of the Sry-related gene Sox6 from rat with tissue-specific expression.

A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively. The leucine-zipper and HMG-box motif were almost completely conserved between these homologues. The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR. rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting. Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined. The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary.