A vasodilating ß1 blocker celiprolol inhibits muscular release of uric acid precursor in patients with essential hypertension.

Although nonvasodilating ß1 blockers increase the levels of uric acid in serum, it is not known whether vasodilating ß1 blockers have a similar effect. In the present study, we evaluated the effect of celiprolol on the release of hypoxanthine, a uric acid precursor, from muscles after an exercise. We used the semi-ischemic forearm test to examine the release of lactate (ΔLAC), ammonia (ΔAmm), and hypoxanthine (ΔHX) before and 4, 10, and 60 min after an exercise in 18 hypertensive patients as well as 4 normotensive subjects. Before celiprolol treatment, all the levels of ΔHX and ΔAmm, and ΔLAC were increased by semi-ischemic exercise in hypertensive patients, and the increases were remarkably larger than those in normotensive subjects. Celiprolol decreased both systolic and diastolic pressure. It also decreased the levels of ΔHX and ΔAmm without changes in ΔLAC after an exercise. These findings also were confirmed by summation of each metabolite (ΣΔMetabolites). Celiprolol caused a marginal decrease of serum uric acid, but the difference was not statistically significant. On the other hand, nonvasodilating ß1 blockers did not suppress the levels of ΔHX and ΔAmm, whereas they significantly increased ΔLAC after an exercise. Celiprolol improved energy metabolism in skeletal muscles. It suppressed HX production and consequently did not adversely affect serum uric acid levels.

Development of spontaneous optic neuropathy in NF-kappaBetap50-deficient mice: requirement for NF-kappaBetap50 in ganglion cell survival.

Although the transcription factor NF-kappaBeta is known to regulate cell death and survival, its precise role in cell death within the central nervous system remains unknown. The purpose of this study was to investigate the role of NF-kappaBetap50 in the age-related survival of retinal ganglion cells (RGCs). Eyes of mice with a deleted NF-kappaBetap50 gene and its wild-type mice at each of age were studied by histopathological studies. The number of RGCs was counted using retrograde labelling methods. Mice were subjected to intravitreous injection of N-methyl-D aspartate (NMDA) to induce RGC death. In p50-deficient mice, the number of RGCs significantly decreased with age in total independence of intraocular pressure measurement. Optic nerves of p50-deficient mice showed hypertrophy astrocytes and enlargement of the axons, together with a decreased number of axons. Immunohistochemistry showed a strong expression of glial fibrillary acidic protein. The histological results show obvious excavation of the optic nerve head in p50-deficient mice at 10 months of age. Intravitreal injection of NMDA in young p50-deficient mice damaged RGCs more intensively than in control animals. We further noticed that autoantibodies against RGCs were produced in p50-deficient mice. Our results show that p50 deficiency induced age-related RGC death, indicating a new insight into the role of p50 in the pathophysiology of neuropathy, and further experiments with p50-deficient mice may provide new targets for therapeutic intervention for human glaucoma.

Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region.

Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.

Ca2+-Sensitization of contraction in the h1 calponin-deficient smooth muscle.

Role of h1 calponin on Ca2+-sensitivity of smooth muscle contraction was investigated using h1 calponin gene-deficient mice (CP -/-) and wild type mice (CP +/+). PGF2. induced a comparable force in intact aorta of CP +/+ and CP -/-. DPB showed similar effects to PGF2alpha. In membrane-permeabilized ileal smooth muscle, PDBu enhanced Ca2+-sensitivity of contraction comparably in CP +/+ and CP -/-. GTPgamma-S showed similar effects. Our results suggest that h1 calponin does not regulate Ca2+-sensitivity in the contractile mechanism of smooth muscle.

Hormone-dependent regulation of intercellular adhesion molecule-1 gene expression: cloning and analysis of 5'-regulatory region of rat intercellular adhesion molecule-1 gene in FRTL-5 rat thyroid cells.

Intercellular adhesion molecule-1 (ICAM-1) has been suggested to play an important role in the perpetuation of autoimmune thyroid disease. To clarify the regulation of ICAM-1 gene in thyroid cells, we investigated ICAM-1 expression in the FRTL-5 thyroid cell model and defined several elements in the 5'-regulatory region that are important for transcriptional regulation of the rat ICAM-1 gene. Cells maintained in medium with 5% serum but without hydrocortisone, insulin, and thyrotropin (TSH) express the highest levels of ICAM-1 RNA. TSH/forskolin downregulate ICAM-1 RNA levels independent of the presence or absence of hydrocortisone or insulin. Moreover, TSH/forskolin decrease ICAM-1 RNA levels that are maximally induced by two cytokines: 100 ng/mL tumor necrosis factor-alpha (TNF-alpha) or 100 U/ml interferon-gamma (IFN-gamma). The effect of TSH/forskolin, as well as TNF-alpha and IFN-gamma, on ICAM-1 RNA levels is transcriptional. Thus, we cloned a 1.8-kb fragment of the 5'-flanking region of the rat ICAM-1 gene, upstream of the translational start site, and showed that TNF-alpha or IFN-gamma caused a 3.5- and greater than 12-fold increase respectively, in its promoter activity, when linked to a luciferase reporter gene and stably transfected into FRTL-5 cells. TSH or forskolin, in contrast, halved the activity of the full length chimera within 24 hours and significantly suppressed the TNF-alpha and IFN-gamma-induced increase (>50%; p < 0.02). Using 5'-deletion mutants, we located the element important for the TNF-alpha effect between -431 and -175 bp; we additionally show that deletion of a NF-kappaB core element within this region, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-alpha inducibility. The effect of IFN-gamma could be localized between -175 bp and -97 bp from the start of translation. This region contains 2 regulatory elements known to be involved in IFN-gamma action in other eukaryotic cells, an IFN-gamma activated site (GAS), -138 to -128 bp, and Spl site, -112 to -108 bp. Deletion of the 10 bp GAS sequence resulted in the complete loss of IFN-gamma induction of pCAM-175 promoter activity. TSH and forskolin action was also mapped between -175 bp and -97 bp from the start of translation. The mutant construct, pCAM-175delGAS mutl, which has no GAS sequence, exhibited no TSH-mediated suppression of promoter activity. We thus show that TSH/cAMP can downregulate ICAM-1 gene expression and inhibit the activity of cytokines (TNF-alpha and IFN-gamma) to increase ICAM-1 gene expression in FRTL-5 thyroid cells. We also localized elements on the 5'-flanking region of ICAM-1 important for these actions. We propose that this TSH/cyclic adenosine monophosphate (cAMP) action is a component of the mechanism to preserve self-tolerance of the thyroid during hormone-induced growth and function of the gland, and it may attenuate cytokine action during inflammatory reactions.

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.

Iodide suppression of major histocompatibility class I gene expression in thyroid cells involves enhancer A and the transcription factor NF-kappa B.

High concentrations of iodide can induce transient, clinical improvement in patients with autoimmune Graves' disease. Previous work has related this iodide action to the autoregulatory effect of iodide on the growth and function of the thyroid; more recently, we additionally related this to the ability of iodide to suppress major histocompatibility (MHC) class I RNA levels and antigen expression on thyrocytes. In this report, we describe a transcriptional mechanism involved in iodide suppression of class I gene expression, which is potentially relevant to the autoregulatory action of iodide. Transfection experiments in FRTL-5 cells show that iodide decreases class I promoter activity and that this effect can be ascribed to the ability of iodide to modulate the formation of two specific protein/DNA complexes with enhancer A, -180 to -170 bp, of the class 1 5'-flanking region. Thus, iodide decreases the formation of Mod-1, an enhancer A complex involving the p50 subunit of NF-kappa B and a c-fos family member, fra-2, which was previously shown to be important in the suppression of class I levels by hydrocortisone. Unlike hydrocortisone, iodide also increases the formation of a complex with enhancer A, which we show, in antibody shift experiments, is a heterodimer of the p50 and p65 subunits of NF-kappa B. The changes in these complexes are not duplicated by chloride and are related to the action of iodide on class I RNA levels by the following observations. First, FRTL-5 thyroid cells with an aged phenotype coincidentally lose the ability of iodide to decrease MHC class I RNA levels and to induce changes in either complex. Second, the effect of iodide on class I RNA levels and on enhancer A complex formation with Mod-1 and the p50/p65 heterodimer is inhibited by agents that block the inositol phosphate, Ca++, phospholipase A2, arachidonate signal transduction pathway: acetylsalicylate, indomethacin, and 5,8,11,14-eicosatetraynoic acid. Interestingly, iodide can also decrease formation of the Mod-1 complex and increase formation of the complex with the p50/p65 subunits of NF-kappa B when the NF-kappa B enhancer sequence from the Ig kappa light chain, rather than enhancer A, is used as probe; and both actions mimic the action of a phorbol ester. This suggests that iodide may regulate complex formation with NF-kappa B regulatory elements on multiple genes associated with growth and function, providing a potential mechanism relating the autoregulatory action of iodide on thyroid cells and its action on class I gene expression.

Major histocompatibility class II HLA-DR alpha gene expression in thyrocytes: counter regulation by the class II transactivator and the thyroid Y box protein.

Aberrant expression of major histocompatibility complex (MHC) class II proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by gamma-interferon (gamma-IFN). To define elements and factors that regulate class II gene expression in thyrocytes and that might be involved in aberrant expression, we have studied gamma-IFN-induced HLA-DR alpha gene expression in rat FRTL-5 thyroid cells. The present report shows that class II expression in FRTL-5 thyrocytes is positively regulated by the class II transactivator (CIITA), and that CIITA mimics the action of gamma-IFN. Thus, as is the case for gamma-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DR alpha gene, the S, X1, X2, and Y boxes between -137 to -65 bp, are required for class II gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and gamma-IFN do not cause additive increases in HLA-DR alpha gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the gamma-IFN pathway to increased class II gene expression. Additionally, gamma-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of gamma-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both gamma-IFN and CIITA to increase class II gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC class I gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC class II expression in human glioma cells. We conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the gamma-IFN-regulated transduction system which, respectively, increase or decrease class II gene expression in thyrocytes and may, therefore, be involved in aberrant class II expression associated with autoimmune thyroid disease.

Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocytes: opposite effects of interferon and methimazole.

Aberrant expression of major histocompatibility complex (MHC) class II antigens is associated with autoimmune thyroid disease; aberrant expression duplicating the autoimmune state can be induced by interferon-gamma (IFNgamma). We have studied IFNgamma-induced human leukocyte antigen (HLA)-DR alpha gene expression in rat FRTL-5 thyroid cells to identify the elements and factors important for aberrant expression. Using an HLA-DR alpha 5'-flanking region construct from -176 to +45 bp coupled to the chloramphenicol acetyltransferase reporter gene, we show that there is no basal class II gene expression in FRTL-5 thyroid cells, that IFNgamma can induce expression, and, as is the case for antigen-presenting cells from the immune system, that IFNgamma-induced expression requires several highly conserved elements on the 5'-flanking region, which, from 5' to 3', are the S, X1, X2, and Y boxes. Methimazole (MMI), a drug used to treat patients with Graves' disease and experimental thyroiditis in rats or mice, can suppress the IFNgamma-induced increase in HLA-DR alpha gene expression as a function of time and concentration; MMI simultaneously decreases IFNgamma-induced endogenous antigen presentation by the cell. Using gel shift assays and the HLA-DR alpha 5'-flanking region from -176 or -137 to +45 bp as radiolabeled probes, we observed the formation of a major protein-DNA complex with extracts from FRTL-5 cells untreated with IFNgamma, termed the basal or constitutive complex, and formation of an additional complex with a slightly faster mobility in extracts from cells treated with IFNgamma. MMI treatment of cells prevents IFNgamma from increasing the formation of this faster migrating complex. Formation of both complexes is specific, as evidenced in competition studies with unlabeled fragments between -137 and -38 bp from the start of transcription; nevertheless, they can be distinguished in such studies. Thus, high concentrations of double stranded oligonucleotides containing the sequence of the Y box, but not S, X1, or X2 box sequences, can prevent formation of the IFNgamma-increased faster migrating complex, but not the basal complex. Both complexes involve multiple proteins and can be distinguished by differences in their protein composition. Thus, using specific antisera, we show that two cAMP response element-binding proteins, activating transcription factor-1 and/or -2, are dominant proteins in the upper or basal complex. The upper or basal complex also includes c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes the IFNgamma-increased lower complex is CREB-binding protein (CBP), a coactivator of cAMP response element-binding proteins. We, therefore, show that aberrant expression of MHC class II in thyrocytes, induced by IFNgamma, is associated with the induction or increased formation of a novel protein-DNA complex and that its formation as well as aberrant class II expression are suppressed by MMI, a drug used to treat human and experimental autoimmune thyroid disease. Its component proteins differ from those in a major, basal, or constitutive protein-DNA complex formed with the class II 5'-flanking region in cells that are not treated with IFNgamma and that do not express the class II gene.

Mice lacking smooth muscle calponin display increased bone formation that is associated with enhancement of bone morphogenetic protein responses.

BACKGROUND: Calponin is a calmodulin-and actin-binding protein expressed in smooth muscle. It promotes actin polymerization and inhibits actin-activated myosin ATPase activity. Despite the molecular and functional characterization of calponin in vitro, the physiological role of calponin in vivo has not been clarified. RESULTS: We investigated the in vivo function of smooth muscle calponin (also called basic calponin or calponin h1) by generating mice carrying a targeted mutation in both alleles of the calponin gene. Mice lacking basic calponin expression displayed enhanced ectopic bone formation in vivo, induced by recombinant human bone morphogenetic protein-2 (rhBMP-2), and an augmentation of the degree of osteoblastic differentiation of embryonic mesenchymal cells when they were stimulated by rhBMP-2. Basic calponin messenger RNA was shown to be expressed in developing and healing bone tissues, and in undifferentiated MC3T3-E1 osteoblasts. An examination of the skeletons of mutated mice showed an early onset of cartilage formation and ossification, and increased postnatal bone formation characterized by an increase in the number of activated periosteal osteoblasts. Bone fracture healing was accelerated in mutated mice. CONCLUSION: This is the first demonstration of animals with enhanced BMP responsiveness in host cells, suggesting that endogenous basic calponin may play a negative role in an osteogenic programme.

Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence.

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, -107 to -100 base pairs, is a critical component of a previously unrecognized silencer, -127 to -90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.

Hormonal modulation of major histocompatibility complex class I gene expression involves an enhancer A-binding complex consisting of Fra-2 and the p50 subunit of NF-kappa B.

Hydrocortisone decreases major histocompatibility complex (MHC) class I gene expression in rat thyroid cells and counteracts increases induced by interferons. Using FRTL-5 cells transfected with class I promoter-reporter gene chimeras, we show that hydrocortisone action is transcriptional and mediated by an element located between 180 and 170 base pairs upstream of the start of transcription. Gel shift assays reveal that hydrocortisone causes the decrease of a specific protein-DNA complex; this same complex, referred to as Mod-1, is increased by interferon. Oligonucleotide competition assays reveal that the Mod-1 complex is associated with enhancer A of the class I gene, -180 to -170 base pairs (5'-GGGGAGTCCCC-3'), immediately upstream of the interferon response element. Antibodies to fra-2, a fos family member, and to the p50, but not the p65, subunit of NF-kappa B supershift the Mod-1 complex. We suggest that hydrocortisone decreases MHC class I gene expression by reducing the formation of Mod-1, which contains both p50 and fra-2; interferon reverses the hydrocortisone effect and increases Mod-1 formation. These observations are relevant to the molecular basis of hydrocortisone therapy in autoimmune thyroid disease and to the actions of interferon to exacerbate or induce autoimmune disease.