Elevated Endogenous Levels Research Articles

Targeted Disruption of Protein Kinase Cε Reduces Cell Invasion and Motility through Inactivation of RhoA and RhoC GTPases in Head and Neck Squamous Cell Carcinoma

Over 70% of patients with head and neck squamous cell carcinoma (HNSCC) present with locoregionally advanced stage III and IV disease. In spite of aggressive therapy, locoregional disease recurs in 60% and metastatic disease develops in 15% to 25% of patients causing a major decline in quality and length of life. Therefore, there is a need to identify and understand genes that are responsible for inducing an aggressive HNSCC phenotype. Evidence has shown that protein kinase C (PKC) epsilon is a transforming oncogene and may play a role in HNSCC progression. In this study, we determine the downstream signaling pathway mediated by PKC epsilon to promote an aggressive HNSCC phenotype. RNA interference knockdown of PKC epsilon in UMSCC11A and UMSCC36, two highly invasive and motile HNSCC cell lines with elevated endogenous PKC epsilon levels, resulted in cells that were significantly less invasive and motile than the small interfering RNA-scrambled control transfectants; 51 +/- 5% (P < 0.006) and 49 +/- 3% (P < 0.010) inhibition in invasion and 69 +/- 1% (P < 0.0005) and 66 +/- 3% (P < 0.0001) inhibition in motility, respectively. PKC epsilon-deficient UMSCC11A clones had reduced levels of active and serine-phosphorylated RhoA and RhoC. Moreover, constitutive active RhoA completely rescued the invasion and motility defect, whereas constitutive active RhoC completely rescued the invasion and partially rescued the motility defect of PKC epsilon-deficient UMSCC11A clones. These results indicate that RhoA and RhoC are downstream of PKC epsilon and critical for PKC epsilon-mediated cell invasion and motility. Our study shows, for the first time, that PKC epsilon is involved in a coordinated regulation of RhoA and RhoC activation, possibly through direct post-translational phosphorylation.

A FAAH-Regulated Class of N-Acyl Taurines That Activates TRP Ion Channels

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.

Effect of ABA upon anthocyanin synthesis in regenerated torenia shoots

To elucidate the mechanism of anthocyanin synthesis induction concomitant with chlorophyll degradation, we established a system in which anthocyanin synthesis and degradation of chlorophyll in regenerated torenia (Torenia fournieri) shoots was induced on medium containing 7% sucrose. Here, we studied the effect of several plant-growth regulators on anthocyanin synthesis and the degradation of chlorophyll in the torenia shoot regenerating system. Exogenous abscisic acid (ABA) could induce anthocyanin synthesis and chlorophyll senescence in regenerating torenia shoots on the medium containing a low concentration of sucrose (1.5%). We determined the changes in the amount of endogenous ABA in the regenerated shoots during the process of anthocyanin synthesis on the medium containing 7% sucrose. It was revealed that the 7% sucrose treatment elevated endogenous ABA levels before the induction of anthocyanin synthesis and chlorophyll degradation. However, while retransfer to the 1.5% sucrose medium resulted in a gradual decrease in the ABA level and a failure of induction of anthocyanin synthesis, normal shoot regeneration. These results suggest that changes in the amount of endogenous ABA may play an important role in the induction of anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots.

Involvement of rho kinase in the ouabain-induced contractions of the rat renal arteries

Agonist and depolarization-induced vascular smooth muscle contractions include the activation of rho/rho kinase pathway. However, there are no reports addressing the question whether this pathway is involved in ouabain-induced vascular smooth muscle contractions. Therefore, in this study, the possible participation of the rho/rho kinase pathway in ouabain-induced contractions was evaluated in rat renal arteries. Effects of rho kinase inhibitors (fasudil and Y-27632) on ouabain-induced contractions, and phosphorylation of myosin binding subunits (MYPT/MBS85) of myosin phosphatase were determined using isolated tissue and Western blot experiments, respectively. Fasudil and Y-27632 inhibited ouabain-induced contractions in a concentration-dependent manner. The phosphorylation of MYPT was not altered by ouabain. However, ouabain significantly increased MBS85 phosphorylation of myosin phosphatase. The phosphorylation of both subunits of myosin phosphatase was inhibited by Y-27632. These results indicate that activation of rho kinase and the subsequent phosphorylation of MBS85 are involved in ouabain-induced contraction of rat renal arteries. This mechanism may be important in essential hypertension with elevated endogenous ouabain levels.

Identification and Functional Characterization of Brainstem Cannabinoid CB 2 Receptors

The presence and function of CB2 receptors in central nervous system (CNS) neurons are controversial. We report the expression of CB2 receptor messenger RNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas.

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in alpha-ENaC and beta-ENaC expression, but no change in alpha1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.

Systemic Endothelial Dysfunction in Adults With Cyanotic Congenital Heart Disease

Secondary erythrocytosis results in increased shear stress in cyanotic congenital heart disease (CCHD), which may modify the balance between vasodilators and vasoconstrictors and affect systemic endothelial function. Because no data are available on systemic vasomotion, systemic endothelial function and nitric oxide (NO) availability were investigated in CCHD patients. Responses to arterial endothelium-dependent (acetylcholine [Ach]) and -independent (sodium nitroprusside [SNP]) vasodilation, NO synthase blockade (NG-monomethyl-L-arginine [L-NMMA]), endothelin-1 (ET-1), and ET-1 receptor blockade by BQ-123 in 11 CCHD patients (O2 saturation <90%; mean+/-SD, 79+/-1%; mean+/-SD age, 39+/-2 years) were compared with those in 10 age-matched healthy referents by using forearm venous occlusion plethysmography. Resting forearm blood flow (FBF) was lower in CCHD patients than in referents (2.4+/-0.2 versus 3.5+/-0.4 mL.min(-1).100 mL(-1) of forearm volume [FAV], P<0.05). Although the response to SNP was similar in both groups (CCHD, 2.0+/-0.3 to 8.3+/-1.0; referents, 3.6+/-0.7 to 11.9+/-1.2 mL.min(-1).100 mL(-1) of FAV; P>0.1), the response to Ach was markedly reduced in CCHD (maximal increase in FBF, 2.8+/-0.8 versus 37.5+/-4.4 mL.min(-1).100 mL(-1) of FAV; P<0.0001). l-NMMA was less effective in CCHD (decrease in FBF, 25+/-6% versus 40+/-4%; P<0.05). ET-1 caused less vasoconstriction in the CCHD group (-25+/-9% versus -51+/-7%, P<0.05), but the response to BQ-123 was similar in both groups (32+/-9% versus 27+/-9%). Systemic endothelial dysfunction is evident in CCHD patients as shown by strikingly reduced endothelial vasodilation to Ach. The response to exogenous ET-1 is reduced, possibly because of elevated endogenous ET-1 levels, but the effects of endogenous ET-1 on arterial tone are not enhanced, as indicated by the similar response to ET-1 blockade.

LH suppression following different low doses of the GnRH antagonist ganirelix in polycystic ovary syndrome

Elevated LH concentration is a common feature in polycystic ovary syndrome (PCOS). This study was designed to establish whether elevated LH levels in PCOS might be suppressed to normal range values by the administration of different low doses of GnRH antagonist, which subsequently might reverse the anovulatory status of these patients. Twenty-four PCOS patients with elevated endogenous LH concentrations were randomized into 3 different dose groups, receiving either 0.125 mg (Group A), 0.250 mg (Group B) or 0.500 mg (Group C) ganirelix sc daily for 7 subsequent days. During the first day of treatment, LH and FSH levels were assessed at 20 min intervals, during 8 h. Thereafter LH, FSH, androgens, estradiol (E2) and inhibins were assessed daily and frequent ultrasound scans were performed for 7 days to record follicle development. Repeated GnRH antagonist administration induced a significant suppression of LH (and to a lesser extent of FSH) serum levels, which was comparable between the different doses. Six hours after ganirelix administration, endogenous LH was suppressed by 49, 69 and 75%, and endogenous FSH was suppressed by 23, 19 and 25%, respectively. The decrease in serum LH and FSH levels was transient and lasted for 12 h, after which serum levels returned to baseline levels at 24 h after drug administration. Androgen levels were not significantly suppressed using this regimen. E2 levels decreased significantly (p < 0.001) and suppression was most pronounced in Group C. Spontaneous follicle development or ovulations were not recorded during the course of treatment. In conclusion, the present study demonstrates that the GnRH antagonist ganirelix is capable of normalising elevated LH levels in PCOS patients, in doses similar to the ones previously shown to prevent a premature LH rise during ovarian hyperstimulation for in vitro fertilization (IVF). In addition, the transient suppression of elevated endogenous LH levels per se does not re-establish normal follicle development in PCOS. However, follicle development may be insufficiently supported by the accompanied subtle suppression of endogenous FSH. Similarly, a transient decline in E2 levels does not effectively restore normal pituitary ovarian feedback. Moreover, these results support the contention of a limited role of LH in the pathogenesis of PCOS.

Elevated endogenous GABA level correlates with decreased fMRI signals in the rat brain during acute inhibition of GABA transaminase

Vigabatrin and gabaculine, both highly specific inhibitors of GABA (gamma-aminobutyric acid) transaminase, cause significant elevation of endogenous GABA levels in brain. The time course of GABA concentration after acute GABA transaminase inhibition was measured quantitatively in the alpha-chloralose-anesthetized rat brain using in vivo selective homonuclear polarization transfer spectroscopy. The blood oxygenation level-dependent (BOLD) effect in functional magnetic resonance imaging (fMRI) has been considered to be coupled tightly to neuronal activation via the metabolic demand of associated glutamate transport. Correlated with the rise in endogenous GABA level after vigabatrin or gabaculine treatment, the intensity of BOLD-weighted fMRI signals in rat somatosensory cortex during forepaw stimulation was found to be reduced significantly. These results are consistent with previous findings that inhibition of GABA transaminase leads to augmented GABA release and potentiation of GABAergic inhibition.

Deficits in hippocampus-mediated pavlovian conditioning in endogenous hypercortisolism

Elevated endogenous levels of corticosteroids cause neural dysfunction and loss, especially within the hippocampus, as well as cognitive impairment in hippocampus-mediated tasks. Because Cushing's syndrome patients suffer from hypercortisolism, they represent a unique opportunity to study the impact of elevated glucocorticoids on cognitive functions. The aim of this study was to examine the performance of Cushing's syndrome patients on trace eyeblink conditioning, a cross-species, hippocampal-mediated test of learning and memory. Eleven Cushing's syndrome patients and 11 healthy control subjects participated in an eyeblink trace conditioning test (1000-msec trace) and a task of declarative memory for words. Salivary cortisol was collected in both the patients and the control subjects, and urinary free cortisol was collected in the patients only. The patients exhibited fewer conditional responses and remembered fewer words, compared with the control subjects. Cortisol levels correlated with immediate and delayed declarative memory only. Conditional response correlated with delayed recall after controlling for the magnitude of unconditional response. The integrity of the hippocampus seems to be compromised in Cushing's syndrome patients. Trace eyeblink conditioning might be useful both as a clinical tool to examine changes in hippocampus function in Cushing's disease patients and as a translational tool of research on the impact of chronic exposure of glucocorticoids.

In vivo activation of c-Jun N-terminal kinase signaling cascade prior to granule cell death induced by trimethyltin in the dentate gyrus of mice

The systemic administration of trimethyltin (TMT, 2.8 mg/kg, i.p.) induced granule cell death in the mouse dentate gyrus selectively 2 days later. The administration of TMT not only enhanced activator protein-1 DNA binding, along with an increase in expression of c-Jun and Fra-2, in the hippocampus 1 day later, but also facilitated phosphorylation of c-Jun N-terminal kinase (JNK) within the cytosol and nucleus. There was also a concomitant increase in the level of phosphorylated JNK kinase (MKK4/SEK1) in the cytosol 16–24 h after the administration. Moreover, TMT markedly elevated endogenous levels of both phosphorylated c-Jun and phosphorylated activating transcription factor-2 (ATF-2), in addition to activating JNK activity in the nuclear extracts obtained 16–24 h post-administration. Immunohistochemical analysis revealed that whereas Fra-2 and phosphorylated ATF-2 were expressed in the CA1 pyramidal cell layer predominantly, phosphorylated c-Jun was observed in both the CA1 pyramidal and dentate granule cell layers after TMT administration. Taken together, our data indicate that TMT activates the JNK pathway in the hippocampus prior to neuronal cell death. The prior activation of this pathway could be at least in part involved in the TMT-induced neural damage seen in the dentate granule cells of mice.

The effect of caffeine on peripheral vascular resistance in isolated perfused guinea pig hind limbs.

The role of caffeine in cardiovascular disease is controversial. Most of its pharmacologic actions are attributed to its role as an adenosine antagonist. Adenosine is one of the most important endogenous vasodilatative substances and is released under ischemic conditions, for example, in the skeletal muscle of patients with peripheral arterial occlusive disease. We aimed to investigate the influence of caffeine on peripheral vascular resistance and on the beneficial vasodilatory effect of adenosine in isolated perfused guinea pig hind limbs. (1) Caffeine was administered at 0.5, 5, and 50 micromol/L under normoxic conditions. (2) The vasculature of the perfused guinea pig hind limb was precontracted with noradrenaline (3 micromol/L), followed by adenosine (10 micromol/L) under normoxic conditions. When vascular resistance (VR) had reached a steady state, caffeine was administered additionally at dosages of 0.5, 5, and 50 micromol/L. (3) This protocol was repeated using iloprost 0.1 micromol/L instead of adenosine as vasodilatory substance. (4) Under hypoxia, caffeine was again administered at the above dosages. (5) Under hypoxia, experiments with adenosine A2-receptor antagonists (alloxazine 10 micromol/L and ZM 241385 100 nmol/L) were done. Under normoxic conditions, 0.5 and 5 micromol/L caffeine had nearly no effect on vascular resistance compared with baseline conditions. A slight, but statistically not significant decrease in VR was achieved with 50 micromol/L caffeine. In the presence of noradrenaline, the vasodilatory effect of adenosine was reduced by 7.6 +/- 1.6% after the addition of 0.5 micromol/L caffeine, and by 37.3 +/- 3.8% at a dosage of 5 micromol/L caffeine. A dosage of 50 micromol/L caffeine completely abolished the vasodilatative effect of adenosine. In the presence of iloprost, only a slight but statistically insignificant inhibitory influence (0.9%) of caffeine at a dosage of 50 micromol/L could be seen. Hypoxia significantly reduced VR. Caffeine at 0.5 micromol/L diminished this effect by about 53.2 +/- 4.6% and abolished it at 5 and 50 micromol/L. The hypoxia-induced adenosine-mediated vasodilatation seems to be an adenosine A2A-receptor-mediated effect. The observed effect of hypoxia-induced vasodilatation in peripheral arteries may be the result of the vasodilatory effect of elevated endogenous adenosine during hypoxia. For patients with peripheral arterial disease, drinking of caffeine-containing beverages may reduce the beneficial vasodilatory effect of elevated endogenous adenosine levels.

Effects of bovine alpha-lactalbumin on gastric defense mechanisms in naive rats.

We recently investigated the effects of the major proteins in cow's milk on gastric mucosal injuries in rat ulcer models. We found that alpha-lactalbumin (alpha-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of alpha-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of alpha-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, alpha-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of alpha-LA). Thus, we demonstrated that alpha-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.

Increased phasic activity of dopaminergic neurones in the rat ventral tegmental area following pharmacologically elevated levels of endogenous kynurenic acid.

Kynurenic acid (KYNA) is an antagonist of ionotropic glutamate receptors, preferentially blocking the glycine-site of the N-methyl-D-aspartate (NMDA) receptor. In the present electrophysiological study, the firing pattern of dopamine (DA) neurones of rat ventral tegmental area (VTA) was investigated following pharmacologically elevated endogenous levels of KYNA by means of an inhibitor of kynurenine 3-hydroxylase (PNU 156561A). Pre-treatment with PNU 156561A (40 mg kg-1, i.v., 5-9 h) caused a threefold increase in endogenous KYNA in whole brain levels and also evoked a significant increase in firing rate and bursting activity of VTA DA neurones. Administration of D-cycloserine (2-128 mg kg-1, i.v.), a partial agonist at the glycine-site of the NMDA-receptor, was found to reverse the increase in firing rate and bursting activity as induced by elevated concentrations of KYNA. The electrophysiological effects of elevated KYNA levels were in all essential mimicked by administration of the NMDA-receptor antagonist MK 801 (0.05-1.6 mg kg-1, i.v.). Thus, the effects of elevated endogenous brain KYNA observed in the present study are likely to be carried out by NMDA receptor antagonism. In conclusion, this study shows that an increase in endogenous KYNA levels produces significant actions on the tonic afferent control of the firing pattern of VTA DA neurones. Given the psychotomimetic effects of NMDA-receptor antagonists, e.g. phencyclidine and ketamine, the state of hyperactivity of mesocorticolimbic DA system induced by elevated levels of KYNA may represent a pathophysiological condition analogous to that seen in schizophrenic patients.

002 Multiple exposures to blue light does not improve psoriasis in patients with elevated endogenous levels of protoporphyrin IX

Endogenous levels of protoporphyrin IX (PpIX) are known to be elevated in psoriatic plaques. Activation of PpIX by visible light after topical application of aminolevulinic acid has been shown to improve psoriasis. This study was designed to determine whether multiple exposures to blue light alone could improve psoriasis in patients exhibiting elevated endogenous PpIX levels. Patients and methods: Seventeen patients with at least two psoriatic plaques of 4 × 4 cm exhibiting elevated endogenous PpIX levels (as detected by in vivo fluorescence spectroscopy) were included in the study. Patients were required to discontinue all topical therapies for at least 2 weeks and systemic therapy for at least 8 weeks before treatment. One of the two plaques on each patient was exposed to 10 J/cm2 Of blue light from a fluorescence panel three times per week for 4 consecutive weeks. The other plaque was used as a non‐exposed control. Blinded clinical evaluations were performed at baseline and every week during the 4‐week treatment period as well as at 1 and 3 weeks after the last exposure. Psoriasis severity was assessed by evaluating on a scale of 0–4 the presence of erythema, induration, and desquamation. PpIX levels were measured before and after light exposure by in vivo fluorescence spectroscopy at week 1 and 4. Results: All patients completed the study without presenting treatment related side‐effects. In vivo fluorescence spectroscopy demonstrated an almost complete photobleaching of PpIX in exposed plaques immediately after light exposure (P = 0.005). There was no significant difference between the mean psoriasis severity score of the exposed or control plaques before and after 12 exposures to blue light. Conclusion: Under the current conditions multiple exposures to blue light did not improve psoriasis.

Calcium-Mediated Telomerase Activity in Ovarian Epithelial Cells

Though the potential of telomerase as an anti-cancer target is evident, information about regulation of telomerase remains fragmentary. In the present study, we examined the role of calcium, an essential cellular signaling molecule, in the regulation of telomerase. We found that calcium induced de novo telomerase activity in telomerase-negative ovarian surface epithelial (OSE) cell lines but not in primary cultures of OSE. In addition, we showed that calcium elevated endogenous telomerase levels in a telomerase-positive ovarian cancer cell line. The use of calcium channel blockers or calcium chelators inhibited this calcium-mediated induction of telomerase activity. Furthermore, cadmium and chromium appeared to cause a moderate induction of telomerase activity while several other metal salts did not. Our data provide the first example of calcium-induced telomerase activity in human cell lines, provide a novel avenue for possible intervention of telomerase, and permit development of therapeutic agents for adjunctive chemotherapy.

Expansion of CD14+CD16+ Blood Monocytes in Patients with Chronic Renal Failure Undergoing Dialysis: Possible Involvement of Macrophage Colony-Stimulating Factor

A novel subpopulation of blood monocytes coexpressing CD16 antigen and low levels of CD14 antigen (CD14+CD16+ monocytes) has recently been identified, and expansion of these CD14+CD16+ monocytes has been reported under some pathological conditions. In this study, we examined the immunophenotype of blood monocytes in patients with chronic renal failure (CRF) who were undergoing hemodialysis (HD, n = 52) or continuous ambulatory peritoneal dialysis (CAPD, n = 36) using two-color immunofluorescence flow cytometry. The percentage and absolute number of CD14+CD16+ monocytes were significantly higher (p < 0.001) in both HD and CAPD patients compared with those in healthy control subjects. We also determined the plasma concentrations of hematopoietic growth factors and cytokines using an enzyme-linked immunosorbent immunoassay. The plasma levels of macrophage colony-stimulating factor (M-CSF) were markedly increased in both HD and CAPD patients relative to the normal controls. The plasma M-CSF levels correlated significantly with the number of CD14+CD16+ monocytes in the whole group of subjects. These findings suggest that elevated endogenous M-CSF levels may participate in the expansion of CD14+CD16+ monocytes in CRF patients undergoing dialysis.

The Wilms' Tumor Gene Product (WT1) Modulates the Response to 1,25-Dihydroxyvitamin D3 by Induction of the Vitamin D Receptor

The Wilms' tumor gene (wt1) encodes a transcription factor involved in urogenital development, in particular in renal differentiation, and in hematopoietic differentiation. Differentiation of a number of solid tumor and leukemic cells lines can be mediated by 1,25-dihydroxyvitamin D(3). This is predominantly mediated by the nuclear receptor for 1,25-dihydroxyvitamin D(3), the vitamin D receptor (VDR). In initial experiments addressing a possible link between WT1 and VDR, we observed a correlated expression of WT1 and VDR mRNA in samples from renal tissues. HT29 colon carcinoma cells, stably transfected to express WT1, exhibited elevated endogenous VDR levels compared with control cells transfected with a control construct. Elevated VDR expression was found in wt1-transfected human embryonic kidney 293 cells, as well. In transient cotransfection experiments, we observed an activation of a vdr promoter reporter by WT1 through a WT1 recognition element, indicating transcriptional regulation of the vdr gene expression by WT1. The responsive sequence element was specifically bound by wild-type, but not by mutated WT1, in electrophoretic mobility shift assays. HT29 colon carcinoma cells, which respond to 1,25-dihydroxyvitamin D(3) with slow induction of growth arrest, were investigated for the influence of WT1 on 1,25-dihydroxyvitamin D(3)-mediated growth suppression. Although HT29 cells transfected with a control construct responded moderately to 1,25-dihydroxyvitamin D(3), the response of HT29 cells expressing WT1 was strikingly enhanced. Stimulation with dihydroxyvitamin D(3) caused an up to 3-fold reduction in the growth rate of different HT29 clones expressing WT1 as compared with control cells lacking WT1 expression. Thus, induction of VDR by WT1 leads to an enhanced response to 1,25-dihydroxyvitamin D(3). We conclude that the vitamin D receptor gene is a target for transcriptional activation by WT1, suggesting a possible physiological role of this regulatory pathway.

Cross-talk between steroid-receptor-mediated and cell-membrane-receptor-mediated signalling pathways results in the in vivo modulation of c-Met and ornithine decarboxylase gene expression in mouse kidney.

The cross-talk in vivo between two signalling pathways activated by testosterone via intracellular androgen receptor, and induced by damage to renal tubules evoked by anti-folate [N(10)-propargyl-5,8-dideazafolic acid (CB 3717)] or folate is reported. We show that CB 3717/folate induces the expression of the hepatocyte growth factor (HGF)/c-Met signalling system in injured kidneys in which a significant, but transient, elevation of the HGF mRNA level occurs. It is followed by a severalfold increase in the c-Met transmembrane receptor message that persists for up to 24 h. The c-Met expression is also positively controlled by testosterone, which induces a significant increase in its mRNA level that is abolished by an anti-androgen, casodex. However, when testosterone and anti-folate/folate are administered sequentially, a substantial (3.5-4.0-fold) decrease in the increase of c-Met expression caused by CB 3717/folate alone occurs. Similarly, testosterone-induced ornithine decarboxylase (ODC) mRNA level and activity are decreased 2.8-7.7-fold when the androgen is applied together with CB 3717. Antagonism between these pathways is also visible under physiological conditions in the kidneys of male mice in which, owing to elevated endogenous testosterone levels, neither the ODC activity nor the mRNA level is induced by anti-folate/folate, whereas the c-Met message response to these drugs is significantly decreased. Our results document a substantial negative regulation of c-Met and ODC gene expression as a result of the cross-talk between testosterone-activated and HGF-activated pathways and suggest a sex-differentiated response to injury of mouse kidneys.

Cross-talk between steroid-receptor-mediated and cell-membrane-receptor-mediated signalling pathways results in the in vivo modulation of c-Met and ornithine decarboxylase gene expression in mouse kidney

The cross-talk in vivo between two signalling pathways activated by testosterone via intracellular androgen receptor, and induced by damage to renal tubules evoked by anti-folate [N10-propargyl-5,8-dideazafolic acid (CB 3717)] or folate is reported. We show that CB 3717/folate induces the expression of the hepatocyte growth factor (HGF)/c-Met signalling system in injured kidneys in which a significant, but transient, elevation of the HGF mRNA level occurs. It is followed by a severalfold increase in the c-Met transmembrane receptor message that persists for up to 24h. The c-Met expression is also positively controlled by testosterone, which induces a significant increase in its mRNA level that is abolished by an anti-androgen, casodex. However, when testosterone and anti-folate/folate are administered sequentially, a substantial (3.5Ő4.0-fold) decrease in the increase of c-Met expression caused by CB 3717/folate alone occurs. Similarly, testosterone-induced ornithine decarboxylase (ODC) mRNA level and activity are decreased 2.8Ő7.7-fold when the androgen is applied together with CB 3717. Antagonism between these pathways is also visible under physiological conditions in the kidneys of male mice in which, owing to elevated endogenous testosterone levels, neither the ODC activity nor the mRNA level is induced by anti-folate/folate, whereas the c-Met message response to these drugs is significantly decreased. Our results document a substantial negative regulation of c-Met and ODC gene expression as a result of the cross-talk between testosterone-activated and HGF-activated pathways and suggest a sex-differentiated response to injury of mouse kidneys.