Baby G. Tholanikunnel

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 Mr 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 α and β 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 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 AMP∶ATP 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.

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. (Mol Cancer Res 2009;7(8):1354–66)

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.

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.

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.

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.

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 G0/G1 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 G0/G1 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.

Loss of caveolin-1 from bronchial epithelial cells and monocytes in human subjects with asthma.

Caveolin‐1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation.

The 3′-untranslated region length and AU-rich RNA location modulate RNA–protein interaction and translational control of β2-adrenergic receptor mRNA

Posttranscriptional controls play a major role in β2-adrenergic receptor (β2-AR) expression. We recently reported that β2-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 β2-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 β2-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 β2-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 β2-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.