Rapid assessment of G protein signaling of four opioid receptors using a real-time fluorescence-based membrane potential assay.

Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.

Potentiation of P2X3 receptor mediated currents by endothelin-1 in rat dorsal root ganglion neurons.

Endothelin-1 (ET-1), an endogenous vasoconstrictor, has been known as a pro-nociceptive agent involved in multitude of pain. ET-1 acts on endothelin receptors on vascular endothelial cells, sensitizes release of ATP, which then acts on P2X3 receptors on nociceptors and results in mechanical hyperalgesia. Both endothelin receptors and P2X3 receptors are present in primary sensory neuron, where it remains unclear whether there is an interaction between them. Herein, we reported that ET-1 potentiated the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. ET-1 concentration-dependently increased α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents, which were mediated by P2X3 receptors. ET-1 shifted the α,ß-meATP concentration-response curve upwards, with an increase of 34.38 ± 4.72% in the maximal current response to α,ß-meATP in the presence of ET-1. ET-1 potentiation of α,ß-meATP-evoked currents was voltage-independent. ET-1 potentiated P2X3 receptor-mediated currents through endothelin-A receptors (ETAR), but not endothelin-B receptors (ETBR). ET-1 potentiation was supressed by blockade of intracellular G-protein or protein kinase C (PKC) signaling. Moreover, there is a synergistic effect on mechanical allodynia induced by intraplantar injection of ET-1 and α,ß-meATP in rats. Pharmacological blockade of P2X3 receptors also alleviated ET-1-induced mechanical allodynia. These results suggested that ET-1 sensitized P2X3 receptors in primary sensory neurons via an ETAR and PKC signaling pathway. Our data provide evidence that cutaneous ET-1 induced mechanical allodynia not only by increasing the release of ATP from vascular endothelial cells, but also by sensitizing P2X3 receptors on nociceptive DRG neurons.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

A Precision Strategy to Cure Renal Cell Carcinoma by Targeting Transglutaminase 2.

In a recent report, no significance of transglutaminase 2 (TGase 2) was noted in the analyses of expression differences between normal and clear cell renal cell carcinoma (ccRCC), although we found that knock down of TGase 2 induced significant p53-mediated cell death in ccRCC. Generally, to find effective therapeutic targets, we need to identify targets that belong specifically to a cancer phenotype that can be differentiated from a normal phenotype. Here, we offer precise reasons why TGase 2 may be the first therapeutic target for ccRCC, according to several lines of evidence. TGase 2 is negatively regulated by von Hippel-Lindau tumor suppressor protein (pVHL) and positively regulated by hypoxia-inducible factor 1-α (HIF-1α in renal cell carcinoma (RCC). Therefore, most of ccRCC presents high level expression of TGase 2 because over 90% of ccRCC showed VHL inactivity through mutation and methylation. Cell death, angiogenesis and drug resistance were specifically regulated by TGase 2 through p53 depletion in ccRCC because over 90% of ccRCC express wild type p53, which is a cell death inducer as well as a HIF-1α suppressor. Although there have been no detailed studies of the physiological role of TGase 2 in multi-omics analyses of ccRCC, a life-long study of the physiological roles of TGase 2 led to the discovery of the first target as well as the first therapeutic treatment for ccRCC in the clinical field.

Statins and bonehealth: a mini review / Estatinas y salud ósea: una minirevisión

Statins are a widely prescribed class of medications that inhibit similar pathways as the anti-resorptive bisphosphonate drugs. Statins target the mevalonate pathway by blocking HMG-CoA reductase. Several recent meta-analyses concluded statins are osteoprotective in the general population. Here we present current literature exploring the mechanisms underlying the putative osteoprotective effects of statins. We also review recent clinical studies, ranging from observational cohort studies to randomized clinical trials, testing the effect of statins on bone health in various populations. (AU)

Las estatinas son un grupo de drogas prescriptas en forma habitual, con la capacidad de bloquear vías de señalización similares a las inhibidas por los amino-bisfosfonatos. Las estatinas inhiben la vía del mevalonato, a través del bloqueo de diferentes enzimas. Varios metaanálisis recientes llevaron a la conclusión de que las estatinas tienen capacidad osteoprotectora en la población general. En esta revisión presentamos la literatura actual describiendo los mecanismos que subyacen en el potencial efecto osteoprotector de las estatinas, como así también estudios observacionales y clínicos aleatorizados sobre el efecto de estatinas en la salud ósea en diversas poblaciones. (AU)

Transglutaminase is a Critical Link Between Inflammation and Hypertension.

BACKGROUND: The pathogenesis of essential hypertension is multifactorial with different underlying mechanisms contributing to disease. We have recently shown that TNF superfamily member 14 LIGHT (an acronym for homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, also known as TNFSF14) induces hypertension when injected into mice. Research reported here was undertaken to examine the role of transglutaminase (TGase) in LIGHT-induced hypertension. METHODS AND RESULTS: Initial experiments showed that plasma and kidney TGase activity was induced by LIGHT infusion (13.91±2.92 versus 6.75±1.92 mU/mL and 19.86±3.55 versus 12.00±0.97 mU/10 µg) and was accompanied with hypertension (169±7.16 versus 117.17±11.57 mm Hg at day 14) and renal impairment (proteinuria, 61.33±23.21 versus 20.38±9.01 µg/mg; osmolality, 879.57±93.02 versus 1407.2±308.04 mmol/kg). The increase in renal TGase activity corresponded to an increase in RNA for the tissue TGase isoform, termed TG2. Pharmacologically, we showed that LIGHT-induced hypertension and renal impairment did not occur in the presence of cystamine, a well-known competitive inhibitor of TGase activity. Genetically, we showed that LIGHT-mediated induction of TGase, along with hypertension and renal impairment, was dependent on interleukin-6 and endothelial hypoxia inducible factor-1α. We also demonstrated that interleukin-6, endothelial hypoxia inducible factor-1α, and TGase are required for LIGHT-induced production of angiotensin receptor agonistic autoantibodies. CONCLUSIONS: Thus, LIGHT-induced hypertension, renal impairment, and production of angiotensin receptor agonistic autoantibodies require TGase, most likely the TG2 isoform. Our findings establish TGase as a critical link between inflammation, hypertension, and autoimmunity.

DHEA modulates the effect of cortisol on RACK1 expression via interference with the splicing of the glucocorticoid receptor.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). EXPERIMENTAL APPROACH: The THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific elisa. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRß. KEY RESULTS: DHEA induced a dose-related up-regulation of GRß and GRß knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. CONCLUSIONS AND IMPLICATIONS: These data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.

BACKGROUND: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. METHODS: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. RESULTS: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the µ and δ receptor subtypes revealed µ-receptor selectivity. CONCLUSION: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

Role of pertussis toxin-sensitive G-protein, K+ channels, and voltage-gated Ca2+ channels in the antinociceptive effect of inosine.

Inosine is the first metabolite of adenosine. It exerts an antinociceptive effect by activating the adenosine A(1) and A(2A) receptors. We have previously demonstrated that inosine exhibits antinociceptive properties in acute and chronic mice models of nociception. The aim of this study was to investigate the involvement of pertussis toxin-sensitive G-protein-coupled receptors, as well as K(+) and Ca(2+) channels, in the antinociception promoted by inosine in the formalin test. Mice were pretreated with pertussis toxin (2.5 µg/site, i.t., an inactivator of G(i/0) protein); after 7 days, they received inosine (10 mg/kg, i.p.) or morphine (2.5 mg/kg, s.c., used as positive control) immediately before the formalin test. Another group of animals received tetraethylammonium (TEA) or 4-aminopyridine (4-AP) (1 µg/site, i.t., a non-specific voltage-gated K(+) channel blockers), apamin (50 ng/site, i.t., a small conductance Ca(2+)-activated K(+) channel blocker), charybdotoxin (250 pg/site, i.t., a large-conductance Ca(2+)-activated K(+) channel blocker), glibenclamide (100 µg/site, i.t., an ATP-sensitive K(+) channel blocker) or CaCl(2) (200 nmol/site, i.t.). Afterwards, the mice received inosine (10 mg/kg, i.p.), diclofenac (10 mg/kg, i.p., a positive control), or morphine (2.5 mg/kg, s.c., a positive control) immediately before the formalin test. The antinociceptive effect of inosine was reversed by the pre-administration of pertussis toxin (2.5 µg/site, i.t.), TEA, 4-aminopyridine, charybdotoxin, glibenclamide, and CaCl(2), but not apamin. Further, all K(+) channel blockers and CaCl(2) reversed the antinociception induced by diclofenac and morphine, respectively. Taken together, these data suggest that the antinociceptive effect of inosine is mediated, in part, by pertussis toxin-sensitive G-protein coupled receptors and the subsequent activation of voltage gated K(+) channel, large conductance Ca(2+)-activated and ATP-sensitive K(+) channels or inactivation of voltage-gated Ca(2+) channels. Finally, small conductance Ca(2+)-activated K(+) channels are not involved in the antinociceptive effect of inosine.

The upregulation of transglutaminase-2 by cyclosporin a in human gingival fibroblasts is augmented by oxidative stress.

BACKGROUND: Transglutaminase-2 (TGM-2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM-2 via ROS, and this regulation may have a role in the pathogenesis of CsA-induced gingival overgrowth. METHODS: Cytotoxicity, 2',7'-dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal-regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3-kinase inhibitor LY294002, glutathione precursor N-acetyl-L-cysteine (NAC), curcumin, epigallocatechin-3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. RESULTS: Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM-2 protein induced by CsA was found in HGFs in a dose- and time-dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM-2 expression induced by CsA (P <0.05). CONCLUSIONS: These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM-2 expression in HGFs. In addition, TGM-2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.

Novel suppressive effects of cardamonin on the activity and expression of transglutaminase-2 lead to blocking the migration and invasion of cancer cells.

AIMS: Alpinia katsumadai was recently found in our previous study to have anti-migratory and anti-invasion activities against HT-1080 cells. However, the study did not demonstrate the exact component of Alpinia katsumadai with anti-migratory and anti-invasive activities. We tested the effects and relevant mechanism of cardamonin (CDN) on the migration and invasion of cancer cells. MAIN METHODS: Migration and invasion of cancer cells were measured using multi-well chambers. Zymography and Western blots were used to examine the effects of CDN on the activities of matrix metalloproteinases (MMPs) and expression of transglutaminase-2 (Tgase-2). KEY FINDINGS: CDN, but not alpinetin, dose-dependently suppressed the migration and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasion of HT-1080 sarcoma cells. CDN suppressed the expression of Tgase-2, MMP-2, NF-κB and MMP-9 in HT-1080 cells, and suppressed MMP-2 and MMP-9 activities. Gene silencing of Tgase-2 suppressed the migration and invasion of HT-1080 cells and suppressed the activities of MMP-2 and MMP-9. Migration of various cancer cells having high levels of Tgase-2 were also inhibited by CDN. CDN and Alpinia katsumadai extracts also directly inhibited the activity of Tgase-2. SIGNIFICANCE: CDN inhibits migration of several cancer cell lines expressing Tgase-2 via suppression of Tgase-2 expression and inhibition of Tgase-2 activity. The finding that CDN has Tgase-2 inhibitory activity will give us a new scaffold or clue of pharmacophore for the development of more effective Tgase-2 inhibitors.

Apple polyphenols extract (APE) improves colon damage in a rat model of colitis.

BACKGROUND AND AIM: Searching for alternative therapies that are effective, safe and less expensive of those currently used for ulcerative colitis, we investigated the efficacy of a polyphenol extract from apple in rat colitis. METHODS: Rats with trinitrobenzensulphonic acid-induced colitis were treated daily with rectal administration of apple polyphenols 10(-4) M for 14 days. COX-2, TNF-α, tissue transglutaminase and calpain in colon mucosa samples were assessed by reverse transcription-polymerase chain reaction and western blot analyses. To ascertain the role of tissue transglutaminase in mucosal healing, wounded rat fibroblasts were incubated with cystamine (a tissue transglutaminase activity inhibitor). RESULTS: Colitis was associated with increased COX-2, TNF-α, calpain, and tissue transglutaminase mRNA. The protein expression of COX-2, TNF-α and calpain was increased whilst tissue transglutaminase was decreased. Apple extract treatment reduced the severity of colitis (p<0.05) and restored all the considered biomarkers at the baseline level. Apple polyphenols reduced the degradation of tissue transglutaminase protein occurring through calpain action. Apple polyphenols-treated wounded fibroblast recovered within 24h showing intense immunoreactivity for tissue transglutaminase. CONCLUSION: The efficacy of apple extract is mediated by its effects on COX-2 and TNF-α. The unbalance between calpain and tissue transglutaminase may play a role in colonic damage and future therapeutic interventions in ulcerative colitis can target this mechanisms.

Toll-like receptor 3-initiated antiviral responses in mouse male germ cells in vitro.

The testis is an immunoprivileged site where local cell-initiated innate immunity plays a crucial role in antimicrobial responses. Toll-like receptors (TLRs) mediate innate immune responses in testicular somatic cells. Although several TLRs are expressed in some stages of male germ cells, the potential role of TLRs in triggering antimicrobial responses in the germ cells has yet to be exclusively studied. The current study demonstrates that TLR3 is constitutively expressed in spermatogonia and spermatocytes and can be activated by a synthetic double-strained RNA analog, polyinosinic-polycytidylic acid. TLR3 activation in these male germ cells up-regulates the expression of proinflammatory cytokines, such as interleukin IL1B, IL6, and tumor necrosis factor alpha, through activation of nuclear factor kappa B; it also induces production of type 1 interferons (IFNA and IFNB) through the activation of IFN regulatory factor 3. In addition, TLR3 activation increases the production of two major antiviral proteins, namely, double-stranded RNA-activated protein kinase and MX1 protein, by germ cells. Data in this article describe an antiviral response of male germ cells through the activation of TLR3 in vitro.

Receptor for activated protein kinase C1 regulates cell proliferation by modulating calcium signaling.

Receptor for activated protein kinase C1 (RACK1) is an intracellular scaffolding protein known to interact with the inositol-1,4,5-trisphosphate receptor and thereby enhance calcium release from the sarcoplasmic reticulum. Because calcium signaling may affect vascular smooth muscle cell proliferation, we investigated whether RACK1 regulates proliferation of rat preglomerular microvascular smooth muscle cells. Western blot analysis indicated that preglomerular microvascular smooth muscle cells robustly express RACK1 protein, and coimmunoprecipitation experiments demonstrated that RACK1 binds the inositol-1,4,5-trisphosphate receptor. RACK1 small interfering RNA (siRNA) decreased RACK1 mRNA and protein expression, significantly (P=0.0225) reduced steady-state basal levels of intracellular calcium (6712±156 versus 7408±248, arbitrary fluorescence units in RACK1 siRNA-treated versus control cells, respectively) and significantly (P<0.0001) decreased cell proliferation by ≈50%. Xestospongin C and 2-aminoethoxydiphenyl borate (antagonists of inositol-1,4,5-trisphosphate receptors), cyclopiazonic acid (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor), and calmidazolium (calmodulin inhibitor) mimicked the effects of RACK1 siRNA on proliferation, and RACK1 siRNA had no additional effects on proliferation in the presence of these agents. RACK1 siRNA did not affect the expression of cyclin D1/2 or phosphorylation of retinoblastoma protein (progrowth cell cycle regulators), yet it caused compensatory decreases in the expression of p21(Cip1/Waf1) and p27(Kip1) (antigrowth cell cycle regulators). Like preglomerular microvascular smooth muscle cells, glomerular mesangial cells also expressed high levels of RACK1, and RACK1 siRNA inhibited their proliferation. In conclusion, RACK1 modulates proliferation of preglomerular microvascular smooth muscle cells and glomerular mesangial cells, likely via the inositol-1,4,5-trisphosphate receptor/calcium/calmodulin pathway. RACK1 may represent a novel druggable target for treating renal diseases, such as glomerulosclerosis.

Neonatal dexamethasone treatment leads to alterations in cell signaling cascades controlling hepatic and cardiac function in adulthood.

Increasing evidence indicates that early-life glucocorticoid exposure, either involving stress or the therapy of preterm labor, contributes to metabolic and cardiovascular disorders in adulthood. We investigated cellular mechanisms underlying these effects by administering dexamethasone (DEX) to neonatal rats on postnatal (PN) days 1-3 or 7-9, using doses spanning the threshold for somatic growth impairment: 0.05, 0.2 and 0.8 mg/kg. In adulthood, we assessed the effects on hepatic and cardiac cell function mediated through the adenylyl cyclase (AC) signaling cascade, which controls neuronal and hormonal inputs that regulate hepatic glucose metabolism and cardiac contractility. Treatment on PN1-3 produced heterologous sensitization of hepatic signaling, with upregulation of AC itself leading to parallel increases in the responses to beta-adrenergic or glucagon receptor stimulation, or to activation of G-proteins by fluoride. The effects were seen at the lowest dose but increasing DEX past the point of somatic growth impairment led to loss of the effect in females. Nonmonotonic effects were also present in the heart, where males showed AC sensitization at the lowest dose, with decreasing effects as the dose was raised; females showed progressive deficits of cardiac AC activity. Shifting the exposure to PN7-9 still elicited AC sensitization but with a greater offsetting contribution at the higher doses. Our findings show that, in contrast to growth restriction, the glucocorticoids associated with stress or the therapy of preterm labor are more sensitive and more important contributors to the cellular abnormalities underlying subsequent metabolic and cardiovascular dysfunction.

Increased transglutaminase 2 and GLUT-1 expression in breast tumors not susceptible to chemoprevention with antioxidants.

GOALS: Expression of GLUT-1 and transglutaminase 2 is increased in aggressive breast cancer, whereas claudin-1, which is expressed in normal tissues, is absent in such tumors. This experimental study was undertaken to establish the aggressiveness and prognosis of DMBA-induced mammary tumors in female Wistar rats based on the assessment of these markers. MATERIALS AND METHODS: The rats were divided into two groups, a control group (n = 70) and a chemoprevention group (n = 70). Breast tumors were induced in both groups by administration of 7,12-dimethylbenz[a] anthracene (DMBA). The chemoprevention group also received alpha-tocopherol and a solution of micronutrients containing ascorbic acid and selenium. Neoplastic lesions of both groups were randomly selected for immunohistochemical assessment of the expression of GLUT-1, transglutaminase 2 and claudin-1. RESULTS: A higher proportion of mammary tumors expressed GLUT-1 and transglutaminase 2 in the chemoprevention group. Claudin-1 expression was absent in all tumors of both groups. CONCLUSIONS: These results are suggestive of increased aggressiveness of tumors not susceptible to chemoprevention by the agents used in this study.

Transglutaminase-2 regulation by arecoline in gingival fibroblasts.

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P=0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

T3 administration in adult hypothyroid mice modulates expression of proteins involved in striatal synaptic plasticity and improves motor behavior.

Adult-onset hypothyroidism is associated with neurological changes such as cognitive dysfunction and impaired learning, which may be related to alterations of synaptic plasticity. We investigate the consequence of adult-onset hypothyroidism on thyroid-mediated transcription events in striatal synaptic plasticity, and the effect of triiodothyronine (T3) replacement. We used hypothyroid mice, treated with propylthiouracil (PTU) and methimazole (MMI), with or without subsequent administration of T3. We evaluated the amount of T3 nuclear receptors (TRalpha1, TRbeta) and striatal plasticity indicators: neurogranin (RC3), Ras homolog enriched in striatum (Rhes), Ca2+/calmodulin-dependent protein kinase (CaMKII), and dopamine- and cAMP-regulated phosphoprotein (DARPP-32). In addition, we assessed hypothyroid mice motor behavior as related to striatum synaptic functions. Hypothyroid mice exhibited significantly reduced TRbeta, RC3 and Rhes expression. T3 administration reversed the expression of TRbeta, RC3, and up-regulated CaMKII levels as well as motor behavior, and decreased DARPP-32 protein phosphorylation. We suggest that thyroid hormone modulation had a major impact on striatal synaptic plasticity of adult mice which produced in turn motor behavior modifications.

TGFbeta mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation.

Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFbeta, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFbeta-mediated activation of TGase2. These results indicate that TGFbeta mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFbeta-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.

Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRalpha, mPRbeta, and mPRgamma have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRalpha and mPRbeta but not mPRgamma, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRalpha and mPRbeta proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRalpha mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [(3)H]progesterone binding to T- and Jurkat cell membranes (K(d) 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (G(i)), suggesting that mPRs are coupled to G(i) in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.