Gilad Rimon - Academia.edu (original) (raw)

Papers by Gilad Rimon

Journal of Biological Chemistry

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of... more The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may...

European Journal of Cancer Supplements, 2004

We employed in vivo phage display in human prostate tumor-bearing mice to identify peptides that ... more We employed in vivo phage display in human prostate tumor-bearing mice to identify peptides that extravasate the vasculature and specifically target the tumor cells, not just the surrounding vasculature.

The Journal of biological chemistry, Jan 7, 2014

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the pr... more The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequen...

Regulatory Peptides, 1998

Corticotropin releasing factor (CRF) is a predominant regulator of the neuroendocrine, autonomic ... more Corticotropin releasing factor (CRF) is a predominant regulator of the neuroendocrine, autonomic and behavioral responses to stress. In addition, numerous studies support autocrine / paracrine roles for this peptide at peripheral sites. CRF and CRF binding sites have been identified in different regions of the central nervous system as well as in the heart, spleen, adrenal and testis, and high levels of CRF were detected in inflamed fibroblasts. However, the precise physiological or pathophysiological role of peripheral CRF cannot yet be discerned. Here we show that CRF, through interaction with specific membrane receptors, blocks the interleukin-1a (IL-1a)-stimulated prostaglandin 125 (PG) synthesis in fibroblasts. Binding of [ I]-labeled CRF in fibroblasts was saturable and fitted a two sites model. K for the D higher-affinity class of receptors was 2062.2 pM, and B 1.9560.22 fmol / mg protein. For the lower-affinity class of receptors K was max D 160617 nM, and B 2.3860.27 fmol / mg protein. CRF blocked the effect of IL-1a on PGE synthesis, and this was antagonised by max 2 D-PheCRF . In addition, the CRF receptor antagonists a helical CRF and D-PheCRF at high concentrations inhibited the 12 -41 9 -41 12 -41

Proceedings of the National Academy of Sciences, 1984

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the la... more Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135TC. Subsequently, the lac carrier protein was extracted into octyl f-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl 13-D-glucopyranoside extract

Proceedings of the National Academy of Sciences, 2006

Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin ... more Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin H2 in the committed step of prostanoid biosynthesis. These enzymes are pharmacological targets of nonsteroidal antiinflammatory drugs and cyclooxygenase (COX) 2 inhibitors. Although PGHSs function as homodimers and each monomer has its own COX and peroxidase active sites, the question of whether there is cross-talk between monomers has remained unresolved. Here we describe two heterodimers in which a native subunit of human PGHS-2 has been coupled to a subunit having a defect within the COX active site at some distance from the dimer interface. Native͞G533A PGHS-2, a heterodimer with a COX-inactive subunit, had the same specific COX activity as the native homodimer. Native͞R120Q PGHS-2, a heterodimer in which both subunits can oxygenate arachidonic acid but in which the R120Q subunit cannot bind the COX inhibitor flurbiprofen, was inhibited by flurbiprofen to about the same extent as native PGHS-2. These results imply that native PGHS-2 exhibits half-ofsites reactivity. Isothermal titration calorimetry established that only one monomer of the native PGHS-2 homodimer binds flurbiprofen tightly. In short, binding of ligand to the COX site of one monomer alters its companion monomer so that it is unable to bind substrate or inhibitor. We conclude that PGHS monomers comprising a dimer, although identical in the resting enzyme, differ from one another during catalysis. The nonfunctioning subunit may provide structural support enabling its partner monomer to catalyze the COX reaction. This subunit complementarity may prove to be characteristic of other dimeric enzymes having tightly associated monomers.

Proceedings of the National Academy of Sciences, 1983

Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escheric... more Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-

Proceedings of the National Academy of Sciences, 2010

Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) ... more Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A 2 formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs. arachidonic acid | adrenic acid | nonsteroidal antiinflammatory drugs | platelet | prostaglandin

Pharmacological Research, 1995

Neuroscience, 1985

The fluorescent dye Lucifer Yellow CH was intracellularly injected into neurons in slices of guin... more The fluorescent dye Lucifer Yellow CH was intracellularly injected into neurons in slices of guinea-pig visual neocortex which had been prepared by sectioning either in a plane normal to the pial surface (radial slices) or in a plane parallel to the pial surface (tangential slices). In radial slices 44.3% of the injections resulted in dye-coupling and the number of cells coupled to the impaled neuron per injection followed a Poisson distribution. In contrast dye-coupling was not observed in tangential slices. Incidence of dye-coupling in slices that had been sectioned in both the radial and tangential planes was the same as in intact radial slices, indicating that slicing in the radial plane induced the formation of pathways for dye movement between neurons. The results suggest that formation and/or strengthening of direct intercellular junctions between neocortical neurons may occur as a specific neuronal response to partial dendrotomy.

European Journal of Pharmacology, 2001

Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with ... more Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E(2) and prostaglandin F2alpha. Although prostaglandin H synthase-1 activity was not altered by these concentrations of the prostaglandin H synthase-2 inhibitors, it was found that exposure to these agents prior to aspirin or indomethacin (irreversible prostaglandin H synthase inhibitors) significantly attenuated the inhibition obtained by the latter inhibitors. On the other hand, the same concentrations of the prostaglandin H synthase-2 inhibitors did not interfere with prostaglandin H synthase-1 inhibition that was induced by naproxen or ibuprofen (competitive prostaglandin H synthase inhibitors). Attenuation of the indomethacin inhibition of prostaglandin H synthase-1 by prostaglandin H synthase-2 inhibitors was observed only when the microsomes were pre-exposed to DuP-697 or NS-398 in the absence, but not in the presence, of arachidonic acid. The effect of DuP-697 was found to be irreversible, however, washing away the agent reversed the action of NS-398. Similar phenomena have been reported by us in bovine aortic endothelial cells and in human dermal fibroblasts. Attenuation of the inhibition by aspirin and indomethacin, without altering the enzyme's basal activity or the inhibition induced by ibuprofen or naproxen may suggest the possibility that the prostaglandin H synthase-2 specific inhibitors DuP-697 and NS-398 affect prostaglandin H synthase-1 by interaction with a site different from the enzyme's catalytic site.

European Journal of Pharmacology, 1998

Corticotropin releasing factor (CRF) is a hypothalamic hormone that also displays autocrine/parac... more Corticotropin releasing factor (CRF) is a hypothalamic hormone that also displays autocrine/paracrine roles at peripheral sites. High concentrations of CRF have been identified in endothelial cells and other inflammatory tissues. We investigated the effects of CRF and antagonists in the regulation of prostaglandin synthesis in bovine aortic endothelial cells, and also characterized the binding of CRF in these cells. Interleukin-1alpha increased prostacyclin (prostaglandin I2) synthesis in endothelial cells and this response to interleukin-1alpha was abolished by simultaneous exposure to CRF. The effect of CRF on interleukin-1alpha-induced prostaglandin synthesis was antagonised by the CRF receptor antagonist alpha-helical CRF-(9-41). In addition, this as well as another CRF receptor antagonist, namely [D-Phe12]CRF-(12-41), when applied alone at low concentrations inhibited the interleukin-1alpha-induced prostaglandin synthesis similarly to CRF, suggesting partial agonistic action. Binding of [125I]-labeled CRF in endothelial cells was saturable and fitted a two sites model. Kd for the higher-affinity class of receptors was 0.2 +/- 0.02 nM, and Bmax 0.79 +/- 0.095 fmol/mg protein. The lower-affinity class of receptors had a Kd of 1.77 +/- 0.14 microM and Bmax 0.97 +/- 0.12 fmol/mg protein. These findings suggest a direct role for CRF in the local regulation of inflammation.

European Journal of Pharmacology, 1999

Bovine aortic endothelial cells produce prostacyclin as their major arachidonic acid metabolite. ... more Bovine aortic endothelial cells produce prostacyclin as their major arachidonic acid metabolite. cAMP, in turn, is the second messenger for prostacyclin. In the present study, we investigated the effects of cAMP-elevating agents on prostacyclin production by bovine aortic endothelial cells. Treatment of resting bovine aortic endothelial cells with cAMP-elevating agents inhibited prostacyclin production and cyclooxygenase activity, without affecting arachidonic acid release. No change was detected in cyclooxygenase-1 protein expression. The specific inhibitor of protein kinase A, Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt), and the phosphatase inhibitor, okadaic acid, both suppressed cAMP-induced inhibition, suggesting that this inhibition is mediated by a phosphorylation-dephosphorylation cascade, which is possibly protein kinase A-dependent. In lipopolysaccharide-treated cyclooxygenase-2 expressing bovine aortic endothelial cells, where cyclooxygenase-1 activity was selectively inhibited, dibutyryl cAMP failed to inhibit cyclooxygenase-2 activity. Cyclooxygenase-2 protein was induced upon treatment with dibutyryl cAMP and further induction of cyclooxygenase-2 protein was effected by IBMX (3-isobutyl-1-methyl-xanthine) and dibutyryl cAMP in bacterial lipopolysaccharide-stimulated cells. These results suggest that increased cellular cAMP selectively inhibits cyclooxygenase-1 activity without altering cyclooxygenase-1 protein expression, and at the same time, up-regulates cyclooxygenase-2 protein. This complex regulation of cyclooxygenase activity and protein expression by cAMP may represent a prostacyclin-induced autoregulatory mechanism in bovine aortic endothelial cells.

European Journal of Pharmacology, 2006

Prostaglandins are known to transduce their signals via 7 transmembrane prostanoid receptors, whi... more Prostaglandins are known to transduce their signals via 7 transmembrane prostanoid receptors, which typically signal through coupling to G proteins and downstream second messenger molecules and protein kinase activation. Recently we have shown that cyclic nucleotides affect prostaglandins binding to bovine aortic endothelial cells independent of protein kinases. Here we show that incubation of bovine aortic endothelial cells with permeable analogs of cAMP or cGMP leads to a rapid and reversible reduction in PGE 2 binding to the cells. Since cyclic nucleotides are known modulators of cyclic nucleotide gated channels, we examined the effect of a specific cyclic nucleotide gated channel blocker l-cis-diltiazem on prostaglandin E 2 (PGE 2 ) binding to bovine aortic endothelial cells. L-cis-diltiazem is shown to displace PGE 2 binding to bovine aortic endothelial cells in a dose dependent manner. In addition the effect of PGE 2 and l-cis-diltiazem on thapsigargin induced calcium elevation in the cells was compared. Both agents reduced in bovine aortic endothelial cells the thapsigargin induced calcium elevation by about half. PGE 2 also retarded the time course of the response to thapsigargin. Simultaneous treatment of the cells with both PGE 2 and l-cis-diltiazem did not yield an inhibitory effect beyond that observed with l-cis-diltiazem alone. Together our data point at the cyclic nucleotide gated channels as a feasible candidate for association with the PGE 2 binding site in bovine aortic endothelial cells.

Cellular Signalling, 1996

PH]PGE2 and [3H]PGF2,~ were shown to bind with similar binding capacity and dissociation constant... more PH]PGE2 and [3H]PGF2,~ were shown to bind with similar binding capacity and dissociation constants to bovine aorta endothelial cells. The similarity in the binding parameters suggests that both agonists may bind to the same binding site. Displacement of [3H]PGE2 performed with PGE2, PGF2,~ or U-46619, a thromboxane agonist, shows that all three prostanoids displaced the bound PH]PGE2 with comparable potency (ICs0 = 10 -7 M). These results indicated that the three different prostanoids, which serve as specific agonists to different prostanoid receptors, also compete for the same binding site in bovine endothelial cells with similar affinity. Comparison of the displacement of PH]PGE2 or [3H]PGF2~ by a number of prostaglandin agonists and antagonists further supports the notion that the natural prostanoids bind with similar affinities to the same binding site. Thus, sulprostone, an EPI/EP3 agonist, displaced bound PH]PGE2 and [3H]PGF2~ with ICs0 of about 10 7 M. On the other hand, thromboxane antagonists (BAY u-3405 and GR-32191B), EPI specific antagonist (SC-19220) EPI/DP antagonist (AH-6809) and iloprost, a stable prostacyclin agonist, failed to displace bound pH]PGE2 or [3H]PGF2~ at a concentration range of 10 -9 --10 -6 m. Gradual increase of sodium fluoride (NaF), a general activator of G binding proteins, or incubation of permeabilized cells with GTPyS resulted in a decrease in PH]PGE2 binding, suggesting that the binding site represents a low-affinity common prostanoid receptor which, similar to other prostanoid receptors, is probably coupled with G binding proteins.

Cellular Signalling, 1999

PGE and prostacyclin each enhance cAMP synthesis in the osteoblast-like cell line UMR-106. The am... more PGE and prostacyclin each enhance cAMP synthesis in the osteoblast-like cell line UMR-106. The amount of cAMP induced by PGE was 5᎐7-fold greater than the amount induced by cicaprost or 2 iloprost, stable prostacyclin analogues. Both PGE and the two prostacyclin analogues enhanced cAMP 2 synthesis with similar time dependence. The EC values of PGE and cicaprost were 3 = 10 y6 and 5 = 10 y8 50 2 Ž . M, respectively. Short-term incubation of the cells with 12-o-tetradecanoylphorbol 13-acetate TPA markedly reduced the PGE -induced cAMP synthesis. In contrast, cells that were incubated with the same concentra-2 tions of TPA in the presence of cicaprost or iloprost showed a 1.6-fold increase in cAMP formation. The marked disparity between the cAMP response to cicaprost and PGE in the presence of TPA suggests that the 2 two prostanoids induce cAMP synthesis in the UMR-106 cells by interaction with different receptors. These observations support the idea that the osteoblastic UMR-106 cells may express specific prostacyclin receptors and suggest that prostacyclin may have a unique role in osteoblasts. CELL SIGNAL 11;3:165᎐169, 1999. ᮊ 1999

Cellular Signalling, 1989

Both NaCl and NaF promoted PGE2 binding to epididymal adipocyte membranes by apparent increase in... more Both NaCl and NaF promoted PGE2 binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the 'salt effect' of sodium on PGE2 binding, the effects of Mg2+ and guanyl nucleotides on PGE2 binding in the presence of NaCl or NaF were compared. Mg2+ decreased PGE2 binding; high NaF concentration abolished this inhibition, while increased NaCl concentrations did not affect the Mg2+ inhibition. In the presence of Mg2+ the effects of NaCl and NaF were additive. The enhancement of PGE2 binding by fluoride, unlike sodium, was dependent on the presence of Mg2+. Incubation of the membranes with GDP beta S, Gpp(NH)p, GTP or GTP gamma S increased PGE2 binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE2 binding at low NaF concentrations and inhibition of PGE2 binding at high NaF concentrations. No changes in the stimulatory action of NaCl on PGE2 binding were observed in the simultaneous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE2 binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE2 binding. The implications of these findings are discussed.

Cellular Signalling, 1992

Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated f... more Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.