Increased stability of heterogeneous ribonucleoproteins by a deacetylase inhibitor.

Splicing factors are often influenced by various signaling pathways, contributing to the dynamic changes of protein isoforms in cells. Heterogeneous ribonucleoproteins (hnRNPs) regulate many steps of RNA metabolism including pre-mRNA splicing but their control by cell signaling particularly through acetylation and ubiquitination pathways remains largely unknown. Here we show that TSA, a deacetylase inhibitor, reduced the ratio of Bcl-x splice variants Bcl-xL/xS in MDA-MB-231 breast cancer cells. This TSA effect was independent of TGFß1; however, only in the presence of TGFß1 was TSA able to change the splicing regulators hnRNP F/H by slightly reducing their mRNA transcripts but strongly preventing protein degradation. The latter was also efficiently prevented by lactacystin, a proteasome inhibitor, suggesting their protein stability control by both acetylation and ubiquitination pathways. Three lysines K87, K98 and K224 of hnRNP F are potential targets of the mutually exclusive acetylation or ubiquitination (K(Ac/Ub)) in the protein modification database PhosphoSitePlus. Mutating each of them but not a control non-K(Ac/Ub) (K68) specifically abolished the TSA enhancement of protein stability. Moreover, mutating K98 (K98R) and K224 (K224R) also abolished the TSA regulation of alternative splicing of a Bcl-x mini-gene. Furthermore, about 86% (30 of 35) of the multi-functional hnRNP proteins in the database contain lysines that are potential sites for acetylation/ubiquitination. We demonstrate that the degradation of three of them (A1, I and L) are also prevented by TSA. Thus, the deacetylase inhibitor TSA enhances hnRNP F stability through the K(Ac/Ub) lysines, with some of them essential for its regulation of alternative splicing. Such a regulation of protein stability is perhaps common for a group of hnRNPs and RNA metabolism.

Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation.

Regulation between protein kinases is critical for the establishment of signaling pathways/networks to 'orchestrate' cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells has barely been explored. Here we examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca²âº/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKKß1 variant proteins were found to be kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKKß1 was found to be essential for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (-E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter one enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks.

Effect of PON1 polymorphism on HDL antioxidant potential is blunted with aging.

Paraoxonase1 is a HDL-associated enzyme, which is responsible for their antioxidant property. This study was aimed to investigate the effect of PON1 [Q192R] and [L55M] genotypes on susceptibility of LDL and HDL to lipid peroxidation and on antioxidant activity of HDL as a function of aging. Seventy-eight healthy subjects distributed in two age groups, young (20-30 years) and elderly (60-89 years) were recruited. PON1 activities and genotype polymorphisms were determined for each subject. LDL and HDL susceptibility to lipid peroxidation was evaluated by the measure of lag-phase (LP) for conjugated diene formation. HDL antioxidant property was evaluated by the measure of their capacity to protect LDL against lipid peroxidation. Our results show that LP for LDL and HDL peroxidation decreased with age of donors. Moreover, PON1 genotypes affect significantly the susceptibility of LDL and HDL to lipid peroxidation. Furthermore, basal- and salt-stimulated paraoxonase as well arylesterase activities were significantly reduced in elderly compared to young subjects. These results show a beneficial effect of PON1 towards susceptibility of HDL to oxidation as well to their antioxidant effect. However, this PON1 protective effect seems to be blunted with advancing age. Altogether our results suggest that the decrease in the PON1 protective effect with aging may contribute to the acceleration of the atherosclerosis process in elderly.

Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 µM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation.

Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis.

Effect of interferon-γ on inflammatory cytokine-induced bradykinin B1 receptor expression in human vascular cells.

The expression of the bradykinin B(1) receptor is strongly regulated in vascular tissue following injury, with little or no expression in healthy tissues. The present work aimed to verify whether primary human vascular cells (umbilical vein endothelial cells, umbilical artery smooth muscle cells) respond to tumor necrosis factor (TNF)-α and interferon (IFN)-γ by an upregulation of B(1) receptors and whether these pathways interact. B(1) receptor expression was quantified using a [(3)H]Lys-des-Arg(9)-bradykinin binding assay (cell surface protein) and RT-PCR (mRNA). A pharmacological approach exploiting several inhibitory drugs related to cytokine signaling was applied. The combined treatment with TNF-α and IFN-γ had a synergistic effect on B(1) receptor expression in both cell types, increasing primarily receptor abundance in both cell types (16 h) and mRNA concentration (4h) in endothelial cells. The synergistic effect of the IFN-γ-TNF-α combination was abated by drugs targeted at the signaling of either cytokine (for TNF-α: etanercept or the IκB kinase 2 inhibitor TPCA-1; for IFN-γ: neutralizing antibodies to IFN-γ, a pan-Jak inhibitor but not the Jak2 inhibitor AG490). Thus, Jak2 signaling may not be recruited by the IFN-γ receptors in vascular cells; however, Stat1 phosphorylation was correlated as expected to the effect of IFN-γ on B(1) receptor expression. Random migration was inhibited by the B(1) receptor agonist Lys-des-Arg(9)-bradykinin only in smooth muscle cells pretreated with the cytokine combination. The amplificatory effect of IFN-γ on TNF-α-induced bradykinin B(1) receptor expression is relevant to vasculopathies associated with T helper 1 cytokines.

Altered cardiac bradykinin metabolism in experimental diabetes caused by the variations of angiotensin-converting enzyme and other peptidases.

The peptidases angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) mediate most of the kinin catabolism in normal cardiac tissue and are the molecular targets of inhibitory drugs that favorably influence diabetic complications. We studied the variations of those kininases in the myocardium of rats in experimental diabetes. ACE and NEP activities were significantly decreased in heart membranes 4-8weeks post-streptozotocin (STZ) injection. However, insulin-dependent diabetes did not modify significantly bradykinin (BK) half-life (t(1/2)) while the effect of both ACE (enalaprilat) and ACE and NEP (omapatrilat) inhibitors on BK degradation progressively decreased, which may be explained by the upregulation of other unidentified metallopeptidase(s). In vivo insulin treatment restored the activities of both ACE and NEP. ACE and NEP activities were significantly higher in hearts of young Zucker rats than in those of Sprague-Dawley rats. BK t(1/2) and the effects of peptidase inhibitors on t(1/2) varied accordingly. It is concluded that kininase activities are subjected to large and opposite variations in rat cardiac tissue in type I and II diabetes models. A number of tissue or molecular factors may determine these variations, such as remodeling of cardiac tissue, ectoenzyme shedding to the extracellular fluid and the pathologic regulation of peptidase gene expression.

A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells.

Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [(3)H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K(i) 525 nM) among known substrates to displace [(3)H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-alpha repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24h (approximately 12-fold dynamic range by [(3)H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-alpha was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [(3)H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes.

Vascular smooth muscle contractility assays for inflammatory and immunological mediators.

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.

Receptor tyrosine kinases as mediators of injury-induced bradykinin B1 receptor expression in rabbit aortic smooth muscle.

Prolonged in vitro incubation of rabbit aortic rings allows recording contractile responses mediated by the inducible bradykinin B(1) receptors; addition of interleukin (IL)-1 or epidermal growth factor (EGF) to the bathing fluid increases the rate of sensitization, a process partially inhibited by the nonspecific Tyr-kinase inhibitor genistein. The recent development of specific inhibitors for receptor associated Tyr-kinase activities (tyrphostin AG 1478 for EGF receptor, sunitinib for VEGF receptor and others) allows assessing the role of such signaling molecules in this process. AG 1478 reduced the potentiating effects of exogenous EGF, and also the spontaneous sensitization to the agonist des-Arg(9)-bradykinin. Sunitinib or GM 6001, a wide spectrum inhibitor of metalloproteinases, had no effect on these responses. In rabbit aortic smooth muscle cells, the cytokines IL-1beta and EGF increased the density of binding sites for [(3)H]Lys-des-Arg(9)-bradykinin in 4 h; AG 1478 reduced only the effect of exogenous EGF. IL-1 receptor antagonist decreased both the effect of IL-1beta and of EGF in rabbit smooth muscle cells. EGF was weakly and slowly coupled to nuclear factor-kappaB nuclear translocation in these cells, as compared to the effect of IL-1beta. EGF-induced EGF receptor autophosphorylation and ERK1/2 phosphorylation was selectively inhibited by AG 1478 in smooth muscle cells; Lys-des-Arg(9)-bradykinin did not transactivate EGF receptor in these cells. While the Tyr-kinase activity sensitive to AG 1478 is recruited by tissue damage and exogenous EGF to upregulate bradykinin B(1) receptors in freshly isolated aortas, this signaling system interacts with others (e.g., IL-1) for the optimal expression of B(1) receptors.

Antiatherogenic activity of extracts of Argania spinosa L. pericarp: beneficial effects on lipid peroxidation and cholesterol homeostasis.

Prevention of lipoprotein oxidation by natural compounds may prevent atherosclerosis via reducing early atherogenesis. In this study, we investigated for the first time the beneficial properties of methanolic extract of argania pericarp (MEAP) towards atherogenesis by protecting human low-density lipoprotein (LDL) against oxidation while promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. By measuring the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase and the progression rate of lipid peroxidation, the MEAP was found to possess an inhibitory effect. In addition, MEAP reduced the rate of disappearance of alpha-tocopherol as well as the apoB electrophoretic mobility in a dose-dependent manner. These effects are related to the free radical scavenging and copper-chelating effects of MEAP. In terms of cell viability, MEAP has shown a cytotoxic effect (0-40 microg/mL). Incubation of 3H-cholesterol-loaded J774 macrophages with HDL in the presence of increasing concentrations of MEAP enhanced HDL-mediated cholesterol efflux independently of ABCA1 receptor pathways. Our findings suggest that argania seed pericarp provides a source of natural antioxidants that inhibit LDL oxidation and enhance cholesterol efflux and thus can prevent development of cardiovascular diseases.