Cytokine and estrogen stimulation of endothelial cells augments activation of the prekallikrein-high molecular weight kininogen complex: Implications for hereditary angioedema.

BACKGROUND: When the prekallikrein-high molecular weight kininogen complex is bound to endothelial cells, prekallikrein is stoichiometrically converted to kallikrein because of release of heat shock protein-90 (Hsp90). Although bradykinin formation is typically initiated by factor XII autoactivation, it is also possible to activate factor XII either by kallikrein, thus formed, or by plasmin. OBJECTIVE: Because attacks of hereditary angioedema can be related to infection and/or exposure to estrogen, we questioned whether estrogen or cytokine stimulation of endothelial cells could augment release of Hsp90 and prekallikrein activation. We also tested release of profibrinolytic enzymes, urokinase, and tissue plasminogen activator (TPA) as a source for plasmin formation. METHODS: Cells were stimulated with agonists, and secretion of Hsp90, urokinase, and TPA was measured in the culture supernatants by ELISA. Activation of the prekallikrein-HK complex was measured by using pro-phe-arg-p-nitroanilide reflecting kallikrein formation. RESULTS: Hsp90 release was stimulated with optimal doses of estradiol, IL-1, and TNF-α (10 ng/mL) from 15 minutes to 120 minutes. TPA release was not augmented by any of the agonists tested but urokinase was released by IL-1, TNF-α, and thrombin (positive control), but not estrogen. Augmented activation of the prekallikrein-HK complex to generate kallikrein was seen with each agonist that releases Hsp90. Addition of 0.1% factor XII relative to prekallikrein-HK leads to rapid formation of kallikrein; factor XII alone does not autoactivate. CONCLUSIONS: IL-1, TNF-α, and estrogen stimulate release of Hsp90 and augment activation of the prekallikrein-HK complex to generate kallikrein and bradykinin. IL-1 and TNF-α stimulate release of urokinase, which can convert plasminogen to plasmin and represents a possible source for plasmin generation in all types of hereditary angioedema, but particularly hereditary angioedema with normal C1 inhibitor with a factor XII mutation. Both kallikrein and plasmin activate factor XII; kallikrein is 20 times more potent on a molar basis.

Deficiency of plasminogen activator inhibitor 2 in plasma of patients with hereditary angioedema with normal C1 inhibitor levels.

BACKGROUND: Hereditary angioedema with normal C1 inhibitor levels (HAE-N) is associated with a Factor XII mutation in 30% of subjects; however, the role of this mutation in the pathogenesis of angioedema is unclear. OBJECTIVE: We sought evidence of abnormalities in the pathways of bradykinin formation and bradykinin degradation in the plasma of patients with HAE-N both with and without the mutation. METHODS: Bradykinin was added to plasma, and its rate of degradation was measured by using ELISA. Plasma autoactivation was assessed by using a chromogenic assay of kallikrein formation. Plasminogen activator inhibitors (PAIs) 1 and 2 were also measured by means of ELISA. RESULTS: PAI-1 levels varied from 0.1 to 4.5 ng/mL (mean, 2.4 ng/mL) in 23 control subjects, from 0.0 to 2 ng/mL (mean, 0.54 ng/mL) in patients with HAE-N with a Factor XII mutation (12 samples), and from 0.0 to 3.7 ng/mL (mean, 1.03 ng/mL) in patients with HAE-N without a Factor XII mutation (11 samples). PAI-2 levels varied from 25 to 87 ng/mL (mean, 53.8 ng/mL) in control subjects and were 0 to 25 ng/mL (mean, 4.3 ng/mL) in patients with HAE-N with or without the Factor XII mutation. Autoactivation at a 1:2 dilution was abnormally high in 8 of 17 patients with HAE-N (4 in each subcategory) and could be corrected by supplemental C1 inhibitor in 4 of them. Bradykinin degradation was markedly abnormal in 1 of 23 patients with HAE-N and normal in the remaining 22 patients. CONCLUSIONS: Bradykinin degradation was normal in all but 1 of 23 patients with HAE-N studied. By contrast, there was a marked abnormality in PAI-2 levels in patients with HAE-N that is not seen in patients with C1 inhibitor deficiency. PAI-1 levels varied considerably, but a statistically significant difference was not seen. A link between excessive fibrinolysis and bradykinin generation that is estrogen dependent is suggested.

Regulation of nucleolin expression by miR-194, miR-206, and HuR.

Nucleolin is a proliferation-associated protein that is overexpressed in multiple types of cancer. The mechanisms leading to overexpression of nucleolin in specific cancers are not fully understood. This study found that nucleolin is notably elevated in breast cancer cell lines MCF-7 and MDA-231 compared to nonmalignant breast epithelial MCF-10A cells. In silico analyses revealed the presence of putative binding sites for microRNAs miR-194 and miR-206 in the 3'-untranslated region (3'-UTR) of Ncl mRNA. Transfection of the three cell lines with pre-miR-194 or pre-miR-206 specifically decreased the Ncl mRNA and protein expression. Treatments of the cells with antagomiR-194 or antagomiR-206 upregulated nucleolin expression ~2- to 3-fold. Co-transfection of cells with a reporter vector containing the Ncl 3'-UTR downstream from the Renilla luciferase gene and pre-miR-194 or pre-miR-206 led to a ~3-fold decrease in Renilla/firefly luciferase activity. Cytoplasmic levels of the RNA-binding protein HuR were higher in MCF-7 and MDA-231 cells than those in MCF-10A cells. RNA immunoprecipitation assays demonstrated that HuR binds to Ncl mRNA in all the three cell types. ShRNA-mediated knock-down of HuR induced a decrease in nucleolin expression, while exogenous expression of HuR led to upregulation of nucleolin expression. Analysis of the polysome-monosome distribution of Ncl mRNA in HuR knock-down cells demonstrated that HuR enhances the translation efficiency of Ncl mRNA. These findings demonstrate that nucleolin expression is down-regulated by miR-194 and miR-206 and upregulated by HuR.

The Pim protein kinases regulate energy metabolism and cell growth.

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMPATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5'-cap-dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

Factor XII-independent activation of the bradykinin-forming cascade: Implications for the pathogenesis of hereditary angioedema types I and II.

BACKGROUND: We have previously reported that prekallikrein expresses an active site when it is bound to high-molecular-weight kininogen (HK) and can digest HK to produce bradykinin. The reaction is stoichiometric and inhibited by C1 inhibitor (C1-INH) or corn trypsin inhibitor. Addition of heat shock protein 90 leads to conversion of prekallikrein to kallikrein in a zinc-dependent reaction. OBJECTIVE: Our goal was to determine whether these reactions are demonstrable in plasma and distinguish them from activation through factor XII. METHODS: Plasma was incubated in polystyrene plates and assayed for kallikrein formation. C1-INH was removed from factor XII-deficient plasma by means of immunoadsorption. RESULTS: We demonstrate that prekallikrein-HK will activate to kallikrein in phosphate-containing buffers and that the rate is further accelerated on addition of heat shock protein 90. Prolonged incubation of plasma deficient in both factor XII and C1-INH led to conversion of prekallikrein to kallikrein and cleavage of HK, as was seen in plasma from patients with hereditary angioedema but not plasma from healthy subjects. CONCLUSIONS: These results indicate that C1-INH stabilizes the prekallikrein-HK complex to prevent HK cleavage either by prekallikrein or by prekallikrein-HK autoactivation to generate kallikrein. In patients with hereditary angioedema, kallikrein and bradykinin formation can occur without invoking factor XII activation, although the kallikrein formed can rapidly activate factor XII if it is surface bound.

Neutral sphingomyelinase-2 mediates growth arrest by retinoic acid through modulation of ribosomal S6 kinase.

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.

Novel mechanisms in the regulation of G protein-coupled receptor trafficking to the plasma membrane.

ß(2)-adrenergic receptors (ß(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated ß(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced ß(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. ß(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, ß(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between ß(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

The 3'-untranslated region length and AU-rich RNA location modulate RNA-protein interaction and translational control of ß2-adrenergic receptor mRNA.

Posttranscriptional controls play a major role in ß(2)-adrenergic receptor (ß(2)-AR) expression. We recently reported that ß(2)-AR mRNA translation is suppressed by elements in its 3'-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with ß(2)-AR mRNA 3'-UTR. In this study, we identified a poly(U) region at the distal end of the 3'-UTR as critical for TIAR binding to ß(2)-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3'-UTR are important determinants that control the translation of ß(2)-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3'-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3'-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3'-UTR length and the ARE RNA location within the 3'-UTR, as key determinants of RNA/protein interactions and translational control of ß(2)-AR mRNA. Further, the importance of 3'-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

Factor XII-independent cleavage of high-molecular-weight kininogen by prekallikrein and inhibition by C1 inhibitor.

BACKGROUND: Bradykinin formation typically requires interaction of Factor XII, prekallikrein (PK), and high-molecular-weight kininogen (HK) with negatively charged exogenous initiators or cell-surface proteins. Approximately 85% of plasma PK circulates as a complex with HK. Nonenzymatic cell-derived initiators, such as heat shock protein 90, can activate the HK-PK complex to generate kallikrein, bradykinin, and cleaved HK, even in the absence of Factor XII. OBJECTIVE: We sought to determine whether PK, without activation to kallikrein, can digest HK to release bradykinin. METHODS: Kallikrein was measured by using a chromogenic assay, and bradykinin levels were determined by ELISA. Cleavage of PK and HK were assessed by SDS-PAGE and Western blot analysis. RESULTS: Cleavage of HK by PK is demonstrated without any conversion of PK to kallikrein. HK cleavage by PK is distinguished from that of kallikrein by the following: (1) stoichiometric activation of HK by PK with release of bradykinin proportional to the PK input; (2) inhibition of PK cleavage of HK by corn trypsin inhibitor, which has no effect on kallikrein; and (3) inhibition of PK cleavage of HK by a peptide derived from HK, which inhibits binding of PK to HK. The same peptide has no effect on kallikrein activation of HK. C1 inhibitor (C1INH), the major control protein of the plasma bradykinin-forming cascade, inhibits PK cleavage of HK. CONCLUSION: PK is an enzyme that can cleave HK to release bradykinin, and this reaction is inhibited by C1INH. This might account, in part, for circulating bradykinin levels and initiation of kinin formation in C1INH deficiency.

Interaction of high molecular weight kininogen binding proteins on endothelial cells.

Cell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endo-thelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular com-plexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

Activation of the bradykinin-forming cascade on endothelial cells: a role for heat shock protein 90.

Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyper-reactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of Factor XII, prekallikrein and high molecular weight kininogen (HK). The mechanism by which initiation occurs is uncertain. Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of Factor XII. We demonstrate herein that during a 2-h incubation time, plasma deficient in either Factor XII or high molecular weight kininogen (HK) fail to activate kinin-forming cascade as compared to normal plasma. With more prolonged incubation, Factor XII deficient plasma gradually activates and HK deficient plasma does not. Our data support both Factor XII-dependent (rapid) and Factor XII-independent (slow) mechanisms; the latter may require a cell-derived protein (possibly protease) to activate prekallikrein in the presence of zinc ion and HK. To further define this cellular factor, we demonstrated that both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein in the presence of HK and zinc ion. We purified this factor from cytosol by affinity chromatography employing corn trypsin inhibitor (CTI) as ligand. The fractions with peak activity were subjected to SDS-PAGE analysis, ligand blotted with biotinylated CTI, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as one of the proteins. Zinc-dependent activation of the prekallikrein-HK complex on endothelial cells was inhibited upon the addition of polyclonal antibody to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not yet clear, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.

Regulatory mechanism of G protein-coupled receptor trafficking to the plasma membrane: a role for mRNA localization.

Trafficking and localization of G protein-coupled receptors (GPCRs) to the plasma membrane and its retention in the agonist-naive state are critically important for signaling by these receptors. Agonist-induced desensitization of activated GPCRs and their removal from the cell surface have been studied and reviewed extensively. However, less attention has been given to the regulatory mechanisms and different steps that control the trafficking of newly synthesized receptors to the plasma membrane. It is generally believed that the mRNAs encoding GPCRs are targeted to the endoplasmic reticulum by a cotranslational, signal-sequence recognition particle-dependent pathway that results in protein translation and translocation to the plasma membrane. In this chapter, we discuss the importance of cis-targeting elements and trans-recognition factors in GPCR mRNA translational silencing, trafficking, and localization within the cell and its importance in receptor trafficking to the plasma membrane. Knockdown of the critical trans-recognition factors (RNA-binding proteins) resulted in translation of GPCR mRNAs in the perinuclear region and the receptors failed to traffic to the plasma membrane. Thus, a new paradigm is emerging in GPCR trafficking that suggests a fundamental role for mRNA partitioning to specific cytoplasmic regions for efficient plasma membrane localization of the receptors.

Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells.

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.

Translational control of beta2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein.

Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.

Heat shock protein 90 catalyzes activation of the prekallikrein-kininogen complex in the absence of factor XII.

Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyperreactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of factor XII, prekallikrein, and high M(r) kininogen (HK). Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of factor XII. We sought to further define this factor XII-independent mechanism of kinin formation. Both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein, and activation depended on the presence of HK and zinc ion. We fractionated the cytosol by ion exchange chromatography and affinity chromatography by using corn trypsin inhibitor as ligand. The fractions with peak activity were subjected to SDS gel electrophoresis and ligand blot with biotinylated corn trypsin inhibitor, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as the protein responsible for zinc-dependent prekallikrein activation in the presence of HK. Zinc-dependent activation of the prekallikrein-HK complex also depended on addition of either alpha and beta isoforms of Hsp90 and the activation on endothelial cells was inhibited on addition of polyclonal Ab to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not understood, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.

The 3'-untranslated region of the beta2-adrenergic receptor mRNA regulates receptor synthesis.

beta(2)-Adrenergic receptors (beta(2)-ARs) are low abundance integral membrane proteins that mediate the effects of catecholamines at the cell surface. Post-transcriptional regulation of beta(2)-AR is dependent, in part, on sequences within the 5'- and 3'-untranslated regions (UTRs) of the receptor mRNA. In this work, we demonstrate that 3'-UTR sequences regulate the translation of the receptor mRNA. Deletion of the 3'-UTR sequences resulted in 2-2.5-fold increases in receptor expression. The steadystate levels of beta(2)-AR mRNA did not change significantly in the presence or absence of the 3'-UTR, suggesting that the translation of the receptor mRNA is suppressed by 3'-UTR sequences. Introduction of the receptor 3'-UTR sequences into the 3'-UTR of a heterologous reporter gene (luciferase) resulted in a 70% decrease in reporter gene expression without significant changes in luciferase mRNA levels. Sucrose density gradient fractionation of cytoplasmic extracts from Chinese hamster ovary cells transfected with full-length receptor cDNA demonstrated that the receptor transcripts were distributed between polysomal and non-polysomal fractions. Deletion of 3'-UTR sequences from the receptor cDNA resulted in a clear shift in the distribution of receptor mRNA toward the polysomal fractions, favoring increased translation. The 3'-UTR sequences of the receptor mRNA were sufficient to shift the distribution of luciferase mRNA from predominantly polysomal fractions toward non-polysomal fractions in cells transfected with the chimeric luciferase construct. Taken together, our results provide the first evidence for translational control of beta(2)-AR expression by 3'-UTR sequences. Presumably, this occurs by affecting the receptor mRNA localization.

Potential importance of glomerular citrate synthase activity in remnant nephropathy.

BACKGROUND: Aldosterone fosters progressive renal injury, but the mechanism is unknown. Both Wistar-Furth rats, which are resistant to aldosterone actions, and adrenalectomized Sprague-Dawley rats, which lack aldosterone, are characterized by resistance to remnant nephropathy and by reduced whole kidney citrate synthase activity. Increase in citrate synthase activity is a well-characterized, specific renal response to aldosterone. Therefore, we performed experiments to test the hypothesis that enhanced citrate synthase activity contributes to remnant nephropathy. METHODS: Rat models included Wistar (control for Wistar-Furth), Wistar-Furth (resistant to aldosterone), Sprague-Dawley (normal), adrenalectomy (lacking aldosterone), and 5/6 nephrectomy (renal injury). Glomeruli were obtained by differential sieving. Citrate synthase activity was determined spectrophotometrically. Binding characteristics of cytosolic mineralocorticoid receptors were determined by equilibrium competition binding between tritiated and unlabeled aldosterone. Gene sequencing was performed with reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent dye terminators. RESULTS: In glomeruli isolated from adrenalectomized Wistar rats with intact renal mass, aldosterone stimulated a threefold increase in citrate synthase activity; this stimulation was not observed in glomeruli from Wistar-Furth rats. Similarly, citrate synthase activity in glomeruli isolated from adrenally intact Sprague-Dawley rats was 65% greater than that from adrenalectomized Sprague-Dawley rats. Compared to sham surgery, subtotal nephrectomy resulted in 100% greater glomerular citrate synthase activity in Sprague-Dawley rats. In Wistar-Furth rats, mineralocorticoid receptor binding was not reduced, and mutations in the mineralocorticoid receptor DNA binding segment were not found. CONCLUSION: Citrate synthase activity is elevated in remnant glomeruli, and experimental models characterized by reduced glomerular citrate synthase activity (Wistar-Furth rats, adrenalectomized Sprague-Dawley rats) are protected from remnant nephropathy.

Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation.

We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.