Pierre Bouchelouche

Identification of Tyrosine Residues in the Intracellular Domain of the Growth Hormone Receptor Required for Transcriptional Signaling and Stat5 Activation

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, −3, and −5. In order to study the role of GH receptor tyrosine phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca channel activation since these pathways were normal in cells expressing a GH receptor in which all eight intracellular tyrosines were mutated to phenylalanines. Activation of Stat5 by GH was, however, abolished in cells expressing the GH receptor lacking intracellular tyrosines. This study demonstrates that specific tyrosines in the GH receptor are required for transcriptional signaling possibly by their role in the activation of transcription factor Stat5.

The cysteinyl leukotriene D4 receptor antagonist montelukast for the treatment of interstitial cystitis.

PURPOSE The presence of leukotriene D4 receptors in human detrusor myocytes and increased urinary leukotriene E4 in patients with interstitial cystitis and detrusor mastocytosis imply a role for cysteinyl containing leukotrienes as proinflammatory mediators in this disease. We examined the efficacy of the cysteinyl leukotriene 1 receptor antagonist montelukast for treating patients with interstitial cystitis and detrusor mastocytosis. MATERIALS AND METHODS Ten women in whom interstitial cystitis was diagnosed according to National Institute of Diabetes and Digestive and Kidney Diseases criteria and who also had detrusor mastocytosis with a minimum of 28 mast cells per mm.2 muscle tissue were included in this study. Patients received a single dose of montelukast daily for 3 months. The efficacy of treatment was determined by 24-hour urinary frequency, nocturia and pain using visual analog scales. RESULTS After 1 month of montelukast treatment there was a statistically significant decrease in 24-hour urinary frequency, nocturia and pain which persisted during the 3 months of treatment. After 3 months 24-hour urinary frequency had decreased from 17.4 to 12 voidings (p = 0.009), nocturia had decreased from 4.5 to 2.8 (p = 0.019) and pain had decreased from 46.8 to 19.6 mm. on a visual analog scale (p = 0.006). No side effects were observed during treatment. CONCLUSIONS Montelukast treatment resulted in significant improvement in urinary frequency and pain. Its efficacy for decreasing urinary frequency and pain imply a role of leukotriene receptor antagonists for managing interstitial cystitis but further placebo controlled clinical studies are needed.

Increased urinary leukotriene E4 and eosinophil protein X excretion in patients with interstitial cystitis

PURPOSE The role of cysteinyl containing leukotriene C4, D4 and E4, and eosinophil protein X in interstitial cystitis is unknown. Leukotriene E4, the end product of cysteinyl containing leukotrienes, and eosinophil protein X are markers of the activation of mast cells and eosinophils, respectively. Cysteinyl containing leukotrienes are potent and specific chemoattractants for eosinophils. We compared the urinary excretion of leukotriene E4 and eosinophil protein X in patients with interstitial cystitis and in healthy controls. MATERIALS AND METHODS Morning spot urine samples from nine patients with interstitial cystitis who fulfilled National Institute of Diabetes and Digestive and Kidney Diseases criteria were collected on the day of cystoscopy with biopsies. Aliquots of urine specimens were immediately centrifuged and the supernatants were stored at -80C until use. Urine samples from 9 healthy women served as controls. Urinary leukotriene E4 and eosinophil protein X were measured by enzyme immunoassay and radioimmunoassay, respectively. All determinations were performed in duplicate and normalized to urine creatinine. RESULTS Leukotriene E4 and eosinophil protein X were significantly increased in the morning urine of patients with interstitial cystitis compared with controls. The mean urinary excretion of leukotriene E4 plus or minus standard deviation was 148.8 +/- 62.5 and 62.2 +/- 17.5 ng./mmol. creatinine in patients and controls (p = 0.003), while the mean urinary excretion of eosinophil protein X was 109.7 +/- 70.4 and 43.7 +/- 22.0 microg./mmol. creatinine, respectively (p = 0.01). All urine cultures were negative. The mean mast cell count in detrusor biopsies in the interstitial cystitis group was 41 cells per mm.2 (range 5 to 84). Eosinophilic granulocytes were occasionally observed in the submucosa but not in the detrusor. CONCLUSIONS Our study shows that patients with interstitial cystitis and detrusor mastocytosis have increased urinary leukotriene E4 and eosinophil protein X. It is possible that cysteinyl containing leukotrienes and eosinophil protein X are involved in the pathogenesis of interstitial cystitis. Urinary leukotriene E4 and eosinophil protein X may be useful markers for assessing the grade of activation of mast cells and eosinophils in patients with interstitial cystitis and/or for confirming the diagnosis. However, it remains to be investigated whether the increase in urinary leukotriene E4 and eosinophil protein X correlates with interstitial cystitis symptoms.

Interleukin-4 and 13 Induce the Expression and Release of Monocyte Chemoattractant Protein 1, Interleukin-6 and Stem Cell Factor From Human Detrusor Smooth Muscle Cells: Synergy With Interleukin-1β and Tumor Necrosis Factor-α

Purpose: Interstitial cystitis is characterized by an increased number of activated MCs in the detrusor muscle. However, to our knowledge the factors that influence the anatomical relationship between MCs and HDSMCs are unknown. MCP-1, IL-6 and SCF have a critical role in the regulation of MC development, signaling and function. We investigated whether HDSMCs are capable of expressing and releasing MCP-1, IL-6 and SCF in response to IL-4, IL-13, IL-1β and tumor necrosis factor-α.Materials and Methods: HDSMCs were isolated and cultured using an explant technique. Protein expression, and the secretion of MCP-1, IL-6 and SCF were assayed by semiquantitative reverse transcriptase-polymerase chain reaction and specific enzyme-linked immunosorbent assay.Results: Unstimulated cells released low amounts of MCP-1, IL-6 and SCF. In cells stimulated by IL-4 MCP-1 mRNA was up-regulated by a mean factor ± SD of 3.5 ± 1.3, IL-6 mRNA was up-regulated by 3.8 ± 1.3, the soluble form of SCF was up-regulated by 3.2 ± 0.6 an...

Effect of the SK/IK channel modulator 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle

What’s known on the subject? and What does the study add?

Cysteinyl Leukotriene D4 Increases Human Detrusor Muscle Responsiveness to Histamine

PURPOSE Leukotriene D(4) and histamine are proinflammatory mediators that are released concomitantly by activated mast cells. There is the possibility of mutual potentiation of their actions in inflammatory diseases such as interstitial cystitis. We investigated whether human detrusor smooth muscle cells showed increased responsiveness to histamine in the presence of leukotriene D(4). MATERIALS AND METHODS Cold cup detrusor biopsies were obtained from patients undergoing cystoscopy for benign noninvasive bladder diseases. Human detrusor smooth muscle cells in culture were obtained using an explantation technique and subcultivated for a maximum of 3 passages. The cytosolic free Ca(2+) concentration and contractile force were measured by spectrofluorometry and myograph techniques, respectively. RESULTS Low doses of leukotriene D(4) (10 nM or less), which usually do not produce a significant effect on the free Ca(2+) concentration or on muscle contraction when administered 30 to 60 seconds beforehand, significantly enhanced the transient increase in the free Ca(2+) concentration and isometric force induced by 50 to 200 nM histamine. Increased histamine responses were associated with an upward shift in the fura-2 fluorescence ratio, suggesting that histamine hyperresponsiveness was due to the appearance of additional histamine receptors on the sarcolemma or to more efficient signaling per receptor. Leukotriene D(4) concentrations greater than 10 nM had no potentiating effects. CONCLUSIONS To our knowledge this is the first demonstration in the human detrusor that leukotriene D(4) potentiates the effect of histamine. These inflammatory mediators, which are often released concomitantly from mast cells, may interact mutually to potentiate the spasmogenic effect of histamine. These results suggest that the combination of leukotriene D(4) and histamine H1 receptor antagonists may be more effective for the treatment of interstitial cystitis than when given alone.

Role of protein kinase C during interferon-γ and phorbol ester-stimulated immunocytochemical expression of ICAM-1 in human renal carcinoma cells

Incubation of the human renal carcinoma cell line CaKi‐1 with interferon‐γ (IFN‐γ) or the phorbol ester, phorbol‐12‐myristate 13‐acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule‐1 (ICAM‐1) in a dose‐dependent manner. Since PMA is capable of activating the Ca2+/phospholipid‐dependent protein kinase C (PKC), we investigated the role of this kinase during IFN‐γ signal transduction. Calcium ionophore A23187 significantly enhanced IFN‐γ‐ and PMA‐induced ICAM‐1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN‐γ. Finally, 24 h of PMA pretreatment with subsequent IFN‐γ stimulation enhanced ICAM‐1 staining above values from cultures where IFN‐γ was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN‐γ on PKC activation, as determined by acetylated myelin basic protein 4–14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM‐1 antigen by IFN‐γ in the whole cell population. Hence, other Ca2+‐dependent signalling pathway(s) mediated by IFN‐γ receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN‐γ stimulation in our model.

Arachidonic acid and calcium metabolism in rnelittin stimulated neutrophils

Melittin, the predominant fraction of bee venom proteins, was studied in an experimental model of human neutrophil granulocytes to reveal its influence on eicosanoid release, metabolism and receptor function in relation to intracellular calcium metabolism. Melittin (2 μmol/l) was as potent as the calcium ionophore A23187 (10 μmol/l) for activation of 5-lipoxygenase, releasing arachidonate only from phosphatidyl-choline and phosphatidyl-ethanolamine of cellular membranes, as judged from the decreases in radioactivity by 15.4% and 30.5%, respectively. The mechanism responsible for the release of arachidonate from cellular membranes is closely coupled to cellular calcium metabolism, and melittin was found to promote calcium entry through receptor gated calcium channels, probably due to an activation of phospholipase A2. Furthermore, a down-regulation of leukotriene B4 receptors was seen. The maximal number of binding sites per cell was reduced from a median of 1520 to 950 with melittin (1 μmol/l). The study has revealed some factors important for the inflammatory mechanisms mediated by melittin.

Interferon-gamma increases inositol phosphate formation and cellular calcium ion concentration independent of ICAM-1 antigen enhancement in renal tubular cells

In the present study, we investigated the effect of interferon‐gamma (IFN‐γ) on cellular inositol phosphate formation and cellular calcium ion concentration [Ca2+]i in human renal proximal tubular (HRPT) cells. We also examined the possible role of the inositol phosphate‐Ca2+ signalling pathway during IFN‐γ‐induced intercellular adhesion molecule‐1 (ICAM‐1) antigen expression. IFN‐γ caused an increase in the formation of inositol 1‐monophosphate (Ins 1‐P), inositol 1,4‐bisphosphate (Ins 1,4‐P2), inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3) and inositol 1,3,4,5‐tetrakisphosphate (Ins 1,3,4,5‐P4). A rapid time‐dependent rise in [Ca2+]i was observed upon IFN‐γ stimulation, with maximal levels reached after 1 min. A lower rise in [Ca2+]i was observed when cells were stimulated in Ca2+‐free medium. This correlated with the generation of Ins 1,4,5‐P3 by IFN‐γ, a well‐known secondary messenger capable of releasing Ca2+ from intracellular stores. The induction of ICAM‐1 antigen expression was enhanced by IFN‐γ, 4‐bromocalcium ionophore A23187 (Bromo‐A23187), and their combinations. However, the calcium antagonist diltiazem and calcium chelator EGTA had no effect on IFN‐γ antigen induction. In conclusion, our data suggest that IFN‐γ stimulation of HRPT cells results in the cleavage of phosphatidylinositol bisphosphate by phospholipase C, generating inositol phosphates, of which Ins 1,4,5‐P3 probably releases Ca2+ from intracellular stores. A further increase in [Ca2+]i upon IFN‐γ stimulation results from influx of extracellular Ca2+. IFN‐γ signal transduction in HRPT cells may not be limited to the inositol phosphate‐Ca2+ pathway since IFN‐γ‐induced ICAM‐1 antigen expression was unaffected by calcium antagonist/chelator.

Effects of pertussis and cholera toxin on the interferon-γ stimulated immunocytochemical staining of ICAM-1 and inositol phosphate formation in a human renal carcinoma cell line

SummaryWe have recently shown that interferon-γ (IFN-γ) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1. In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-γ signalling. Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-γ induced ICAM-1 staining. Preincubation with PT or CT for 24 h also inhibited IFN-γ induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation. Our findings suggest that IFN-γ induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway. This may reflect a role for G-proteins in the coupling of IFN-γ receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.