Biomedical subjects

Martina Schmidt

Publications and source records attributed to Martina Schmidt.

At least 19 recordsLinked to original sources

PubMed2006

Dynamic phospholipid signaling by G protein-coupled receptors.

G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP(2) by phospholipase C (PLC) into the second messengers IP(3) and DAG. Many receptors also stimulate phospholipase D (PLD), leading to the generation of the versatile lipid, phosphatidic acid. Particular PLC and PLD isoforms take differential positions in receptor signaling and are additionally regulated by small GTPases of the Ras, Rho and ARF families. It is now recognized that the PLC substrate, PIP(2), has signaling capacity by itself and can, by direct interaction, affect the activity and subcellular localization of PLD and several other proteins. As expected, the synthesis of PIP(2) by phosphoinositide 5-kinases is tightly regulated as well. In this review, we present an overview of how these signaling pathways are governed by GPCRs, explain the molecular basis for the spatially and temporally organized, highly dynamic quality of phospholipid signaling, and point to the functional connection of the pathways.

Humans↗

PubMed2006

Rho kinase: a target for treating urinary bladder dysfunction?

Urinary incontinence and other urinary storage symptoms are frequent in the general population but available treatments have limited efficacy and tolerability. Rho kinase (ROCK) has a central role in the regulation of smooth muscle contraction, including that of the urinary bladder. Recent experimental evidence indicates that this role could be deregulated and exacerbated in local and systemic pathological conditions that affect the bladder. In vitro studies with prototypical ROCK inhibitors such as Y27632 and in vivo data from animal models indicate that such drugs have potential as future treatments for bladder dysfunction.

Animals↗

PubMed2006

Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation.

The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.

Catalysis↗

PubMed2006

Defective RhoA/Rho-kinase signaling contributes to vascular hypocontractility and vasodilation in cirrhotic rats.

BACKGROUND & AIMS: Portal hypertension is associated with arterial hypotension and vascular hypocontractility, which persists despite elevated plasma levels of vasoconstrictors. We investigated the role of the RhoA/Rho-kinase pathway in vascular smooth muscle hypocontractility of rats with secondary biliary cirrhosis. METHODS: Aortic expressions of RhoA and Rho-kinase were analyzed in sham-operated and BDL rats by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblots. Activation of aortic RhoA was examined by pull down of guanosine triphosphate (GTP)-RhoA and membrane translocation of RhoA. Rho-kinase activity was assessed as phosphorylation of its substrate, moesin. Contractility of isolated aortic rings was determined myographically. The hemodynamic effect of the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) was determined in vivo by measuring changes in mean arterial pressure and systemic vascular resistance (SVR) (microspheres). RESULTS: Contraction of aortic rings from BDL rats was impaired in response to the alpha(1)-adrenergic receptor agonist methoxamine but not to high molar KCl. Aortic expression of RhoA was unchanged in cirrhotic rats, whereas Rho-kinase was down-regulated posttranscriptionally. Methoxamine-induced activation of RhoA as well as basal and methoxamine-induced phosphorylation of moesin were strongly reduced in aortas from cirrhotic rats. Aortic rings from cirrhotic rats precontracted with methoxamine showed an increased sensitivity to relaxation with Y-27632. The drop in SVR induced by Y-27632 was larger in cirrhotic rats than in sham-operated rats. CONCLUSIONS: An impaired vascular activation of RhoA and a down-regulation of Rho-kinase might contribute to vasodilation and vascular hypocontractility in BDL-induced cirrhosis.

Amides↗

PubMed2005

Proinflammatory stimulation and pioglitazone treatment regulate peroxisome proliferator-activated receptor gamma levels in peripheral blood mononuclear cells from healthy controls and multiple sclerosis patients.

The peroxisome proliferator-activated receptor gamma (PPAR-gamma) belongs to a receptor superfamily of ligand-activated transcription factors involved in the regulation of metabolism and inflammation. Oral administration of PPAR-gamma agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (MS), and PPAR-gamma agonist treatment of PBMCs from MS patients suppresses PHA-induced cell proliferation and cytokine secretion. These effects are pronounced when cells are preincubated with the PPAR-gamma agonists and reexposed at the time of stimulation, indicating a sensitizing effect. To characterize the mechanisms underlying this sensitizing effect, we analyzed PPAR-gamma expression in PMBCs of MS patients and healthy controls. Surprisingly, MS patients exhibited decreased PPAR-gamma levels compared with controls. PHA stimulation of PBMCs from healthy controls resulted in a significant loss of PPAR-gamma, which was prevented by in vitro preincubation of the cells or in vivo by long-term oral medication with the PPAR-gamma agonist pioglitazone. Differences in PPAR-gamma expression were accompanied by changes in PPAR-gamma DNA-binding activity, as preincubation with pioglitazone increased DNA binding of PPAR-gamma. Additionally, preincubation decreased NF-kappaB DNA-binding activity to control levels, whereas the inhibitory protein IkappaBalpha was increased. In MS patients, pioglitazone-induced increase in PPAR-gamma DNA-binding activity and decrease in NF-kappaB DNA-binding activity was only observed in the absence of an acute MS relapse. These results suggest that the sensitizing effect observed in the preincubation experiments is mediated by prevention of inflammation-induced suppression of PPAR-gamma expression with consecutive increase in PPAR-gamma DNA-binding activity.

Adult↗

PubMed2005

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells.

BACKGROUND: Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. METHODS: Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. RESULTS: Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. CONCLUSION: Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Journal Article↗

PubMed2005

The guanine nucleotide exchange factor p63RhoGEF, a specific link between Gq/11-coupled receptor signaling and RhoA.

The monomeric GTPase RhoA, which is a key regulator of numerous cellular processes, is activated by a variety of G protein-coupled receptors, through either G12 or G(q) family proteins. Here we report that p63RhoGEF, a recently identified RhoA-specific guanine nucleotide exchange factor, enhances the Rho-dependent gene transcription induced by agonist-stimulated G(q/11)-coupled receptors (M3-cholinoceptor, histamine H1 receptor) or GTPase-deficient mutants of G alpha(q) and G alpha11. We further demonstrate that active G alpha(q) or G alpha11, but not G alpha12 or G alpha13, strongly enhances p63RhoGEF-induced RhoA activation by direct protein-protein interaction with p63RhoGEF at its C-terminal half. Moreover, the activation of p63RhoGEF by G alpha(q/11) occurs independently of and in competition to the activation of the canonical G alpha(q/11) effector phospholipase C beta. Therefore, our results elucidate a new signaling pathway by which G alpha(q/11)-coupled receptors specifically induce Rho signaling through a direct interaction of activated G alpha(q/11) subunits with p63RhoGEF.

Animals↗

PubMed2005

An audit of completeness of clinical histories: before and after introduction of a pro forma.

The completeness of a random sample of medical records, before and after introduction of a pro forma, was measured in an audit. Twenty key variables that were considered as an essential part of every clinical history were assessed. There was a significant improvement in nine of the 18 items for men, and 11 of the 20 items for women. The mean completeness score increased from 14.5 to 15.9 (out of 18) in men, and from 15 to 17.5 (out of 20) in women (P < 0.001). The improved completeness of medical records as a result of the pro forma should result in the improved management of clients, in addition to providing improved documentation in the case of a medicolegal case.

Documentation↗

PubMed2004

Regulation and cellular roles of phosphoinositide 5-kinases.

The membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), plays a critical role in various, apparently very different cellular processes. As precursor for second messengers generated by phospholipase C isoforms and class I phosphoinositide 3-kinases, PIP(2) is indispensable for cellular signaling by membrane receptors. In addition, PIP(2) directly affects the localization and activity of many cellular proteins via specific interaction with unique phosphoinositide-binding domains and thereby regulates actin cytoskeletal dynamics, vesicle trafficking, ion channel activity, gene expression and cell survival. The activity and subcellular localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms, which catalyze the formation of PIP(2), are actively regulated by membrane receptors, by phosphorylation and by small GTPases of the Rho and ARF families. Spatially and temporally organized regulation of PIP(2) synthesis by PIP5K enables dynamic and versatile PIP(2) signaling and represents an important link in the execution of cellular tasks by Rho and ARF GTPases.

Animals↗

PubMed2004

Epac- and Ca2+ -controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors.

We have recently reported that two typical Gs-coupled receptors, the beta2-adrenergic receptor and the receptor for prostaglandin E1, stimulate phospholipase C-epsilon (PLC-epsilon) and increase intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells and N1E-115 neuroblastoma cells, respectively, by a pathway involving Epac1, a cAMP-activated and Rap-specific guanine nucleotide exchange factor (GEF), and the GTPase Rap2B. Here we have demonstrated that these Gs-coupled receptors use this pathway to activate H-Ras and the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Specifically, agonist activation of the receptors resulted in activation of H-Ras and ERK1/2. The latter action was suppressed by dominant negative H-Ras, but not Rap1A. The receptor actions were independent of protein kinase A but fully mimicked by an Epac-specific cAMP analog as well as by a constitutively active Rap2B mutant. On the other hand, a cAMP-binding-deficient Epac1 mutant, the Rap GTPase-activating proteinII, and a dominant negative Rap2B mutant suppressed receptor- and Epac-mediated activation of H-Ras and ERK1/2. Finally, we have demonstrated that activation of H-Ras and ERK1/2 requires the lipase activity of PLC-epsilon and the subsequent [Ca2+]i increase, suggesting that H-Ras activation is mediated by a Ca2+ -activated GEF. In line with this hypothesis, receptor-mediated activation of H-Ras and ERK1/2 was strongly enhanced by expression of RasGRP1, a Ca2+ -regulated Ras-GEF. Collectively, our data indicated that Gs-coupled receptors can activate H-Ras and subsequently the mitogen-activated protein kinases ERK1/2 by a Ca2+ -activated Ras-GEF, possibly RasGRP1, mediated by cAMP-activated Epac proteins, which then lead via Rap2B and PLC-epsilon stimulation to [Ca2+]i increase.

Animals↗

PubMed2004

p63RhoGEF and GEFT are Rho-specific guanine nucleotide exchange factors encoded by the same gene.

Activation of Rho GTPases, which play pivotal roles in diverse cellular functions, is catalysed by specific guanine nucleotide exchange factors (GEFs). We and others (Souchet et al. (2002)) independently cloned a human cDNA encoding a 580 aa protein (p63RhoGEF), which contains a tandem of Dbl homology and pleckstrin homology domains typical for RhoGEFs. In accordance with Souchet et al., recombinant p63RhoGEF interacted with and catalysed GDP/GTP exchange at RhoA, but not Rac1 or Cdc42. Recently, an N-terminally truncated form of p63RhoGEF, termed GEFT, was described as a Rac/Cdc42-specific GEF (Guo et al. 2003). As judged by RT-PCR with specific primers, we were able to detect mRNA variants encoding p63RhoGEF and GEFT within several tissues and cell lines. Apparently, they co-exist within one cell and are derived from the same gene. When expressed in human embryonic kidney cells, both p63RhoGEF and GEFT caused activation of RhoA, but not Rac1 or Cdc42, and induced serum response factor-mediated gene transcription, which was fully blunted by the Rho-inactivating C3 transferase. In line with these data, expression of either p63RhoGEF or GEFT in J82 human bladder carcinoma cells induced the formation of actin stress fibres. We therefore conclude that p63RhoGEF and GEFT are apparently isoforms derived from the same gene and that GEFT, similar to p63RhoGEF, activates RhoA in several cell types.

Cells, Cultured↗

PubMed2004

The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15(Ink4b) in murine myeloid cells.

CDKN2B (INK4B), which encodes the cyclin-dependent kinase inhibitor p15(INK4b), is up-regulated by many cytokines found in hematopoietic environments in vivo. In human acute myeloid leukemias (AMLs), it is inactivated with high frequency. To gain insight into the regulatory pathways leading to the normal activation of p15(Ink4b) expression, we examined interferon beta (IFNbeta)-induced transcription. Using reporter gene assays in murine myeloid cells M1, we determined that a 328-bp fragment, located 117 to 443 bp upstream of the translation initiation site, was sufficient to activate transcription. Both the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) and PU.1 were able to increase transcription from this region. It was determined that both ICSBP and PU.1 must bind to DNA to form a stable PU.1/ICSBP binding complex. Interestingly, introduction of the ICSBP into ICSBP-null Tot2 cells led to a significant increase in p15(Ink4b) RNA expression. This regulation of the Ink4b promoter is apparently myeloid specific because both ICSBP and PU.1 are myeloid commitment factors. Importantly, this provides a mechanism to explain in part the tumor suppressor activity of ICSBP, since ICSBP-deficient mice develop a chronic myelogenous leukemia (CML)-like disease and a high percentage of human AML and CML lack ICSBP transcripts.

Acute Disease↗

PubMed2004

Inhibition of phospholipase C-epsilon by Gi-coupled receptors.

We recently reported that several Gs-coupled receptors stimulate phospholipase C (PLC)-epsilon via increased formation of cyclic AMP and subsequent activation of the small GTPase Rap2B by the cyclic AMP-activated exchange factor Epac1. Here we show by studies in HEK-293 and N1E-115 neuroblastoma cells that this stimulation induced by Gs-coupled receptors or the direct adenylyl cyclase activator, forskolin, is potently inhibited by Gi-coupled receptors, known to inhibit cyclic AMP formation. PLC inhibition by the overexpressed M2 muscarinic receptor and the endogenously expressed sphingosine-1-phosphate and delta-opioid receptors was fully pertussis toxin-sensitive and accompanied by a reduction in Rap2B activation induced by Gs-coupled receptors. In contrast, Rap2B activation and PLC stimulation induced by membrane-permeable cyclic AMP analogues, including an Epac-specific activator, or PLC stimulation caused by constitutively active Rap2B were not affected by the Gi-coupled receptors. In summary, our data indicate that Gi-coupled receptors can inhibit PLC-epsilon, most likely by suppressing formation of cyclic AMP required for Epac-mediated Rap2B activation.

Cells, Cultured↗

PubMed2004

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3.

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.

CSK Tyrosine-Protein Kinase↗

PubMed2003

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42.

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.

Animals↗

PubMed2003

Anti-inflammatory and antiproliferative actions of PPAR-gamma agonists on T lymphocytes derived from MS patients.

Peroxisomal proliferator-activated receptors (PPARs) belong to a nuclear receptor superfamily of ligand-activated transcription factors. The PPAR-gamma isoform is expressed in human T lymphocytes, and oral administration of PPAR-gamma agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, suggesting a potential role for PPAR-gamma agonists in the treatment of autoimmune diseases. To assess a potential therapeutic role of PPAR-gamma agonists in multiple sclerosis, we compared the immunomodulatory effects of the thiazolidinedione (TZD) drugs pioglitazone (PIO) and ciglitazone and the non-TZD PPAR-gamma agonist GW347845 on human T leukemia cells (Jurkat cells) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) derived from 21 multiple sclerosis patients and 12 healthy donors. PIO, ciglitazone, and GW347845 suppressed PHA-induced T cell proliferation by 40-50% and secretion of interferon-gamma and tumor necrosis factor alpha, by 30-50%. Inhibition of proliferation was increased to approximately 80% and that of proinflammatory cytokine secretion, to 80-90% when PBMCs were first preincubated with PPAR-gamma agonists and re-exposed at the time of PHA stimulation, indicating a sensitizing effect of PPAR-gamma agonists. Inhibition of proliferation was also observed in the tetanus toxoid-specific T cell line KHS.TT2, albeit to a lesser extent. The antiproliferative effects of PIO and GW347845 were accompanied by a decrease of cell viability. Electron microscopy and Western blot analysis revealed DNA condensation and down-regulation of bcl-2, suggesting the induction of apoptosis in activated T lymphocytes. In summary, the data support the potential use of PPAR-gamma agonists as immunomodulatory, therapeutic agents for autoimmune diseases.

Adjuvants, Immunologic↗

PubMed2003

Differential agonist-induced regulation of human M2 and M3 muscarinic receptors.

We have compared the regulation of M(2) and M(3) muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration-dependently reduced M(2) receptor density with a maximum reduction of about 60%. Treatment with 1mM carbachol for 24hr was accompanied by desensitisation of carbachol-induced Ca(2+) elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein alpha-subunits. Consistently, heterologous desensitisation of Ca(2+) elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time- and concentration-dependently up-regulated M(3) receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M(3) receptor was a reduction in the expression of the alpha-subunit of G(q/11). The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca(2+) elevation. However, the reduction of G(q/11) was accompanied by a heterologous desensitisation of Ca(2+) elevations by ATP and sphingosine-1-phosphate. Levels of M(2) and M(3) receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M(2) and M(3) receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.

Carbachol↗

PubMed2003

Consistent inactivation of p19(Arf) but not p15(Ink4b) in murine myeloid cells transformed in vivo by deregulated c-Myc.

Cyclin-dependent kinase inhibitors p16(INK4a) and p15(INK4b), encoded by the CDKN2A and B loci, play an important role in negative regulation of the cell cycle. Furthermore, p19(ARF) also encoded by the CDKN2A locus, has been shown to regulate positively the p53 pathway leading to growth arrest and apoptosis. All three genes have been inactivated in human tumors. In myeloid cells, p15(INK4b) mRNA is upregulated during cytokine-induced differentiation and/or growth arrest, and hypermethylation of the p15(INK4b) gene promoter region is a common event in acute myeloid leukemia. In the present study, we examined murine monocyte/macrophage tumors with deregulated c-myc for evidence of Ink4 gene inactivation. p15(Ink4b) mRNA and protein were detected in the majority of leukemias, and p16(Ink4a) mRNA and protein were highly expressed in two of them. pRb was in a hypophosphorylated state in most of the neoplasms indicating that the Cdk inhibitors that were expressed in the cells were functional. The observed expression of p15(Ink4b) is inconsistent with their proliferation state, although it might be expected to be expressed owing to the maturity of the cells. These data suggest, therefore, that deregulated c-Myc bypasses the pRb restriction point and cell cycle arrest in these tumors. An examination of p19(Arf) exons revealed deletions of the gene in up to 94% of the tumors. Since this gene shares exon 2 with p16(Ink4a), it is often difficult to determine which gene is the relevant tumor suppressor. However, the loss of only the p19(Arf)-specific exon 1 beta was observed in a tumor that had normal p16(Ink4a) protein expression. In addition, the p19(Arf)-specific exon was deleted in another tumor that expressed a functional chimeric protein, p15Ex1-p16Ex2-3; it was demonstrated here that this fusion protein is capable of inducing G1 arrest. These data overall supports the hypothesis that the critical inactivation event in these hematopoietic neoplasms is elimination of p19(Arf), and not Ink4 function.

Animals↗