Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non-osteogenic medium with or without purmorphamine (2 mu M) for periods of up to 14 days. Purmorphamine up-regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204208, 2012. (C) 2011 Wiley Periodicals, Inc.

Immunohistochemical Analysis of Human Homologue of Drosophila Patched (PTCH) in Dental Follicles of Impacted Third Molars

This study investigated the immunodetection of PTCH in epithelial components of dental follicles associated with impacted third molars without radiographic signs of pathosis. One hundred and five specimens of dental follicles associated with impacted third molars with incomplete rhizogenesis (between Nolla's stage 6 and 9) were surgically removed from 56 patients. Epithelial cell proliferation was determined by using immunohistochemical labeling. Statistical analysis was performed using Fisher exact test and a level of significance of 5%. Of the 105 dental follicles collected, 3 were PTCH-positive. The specimens with squamous metaplasia and epithelial hyperplasia had higher rates of positivity for PTCH, as well as those with active remnants of odontogenic epithelium. This study suggests that the odontogenic cells of the dental follicle might be proliferating during the rhizogenesis, while the squamous metaplasia and hyperplasia of the epithelial lining and proliferative odontogenic epithelial rests show the differentiation potential of dental follicles.

The IgA1 immune complex-mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN. Kidney International (2012) 82, 1284-1296; doi:10.1038/ki.2012.192; published online 5 September 2012

Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum

We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7- parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7- parasites.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Insulin alone or with captopril: effects on signaling pathways (AKT and AMPK) and oxidative balance after ischemia-reperfusion in isolated hearts

Insulin and the inhibition of the reninangiotensin system have independent benefits for ischemiareperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemiareperfusion. Isolated hearts were perfused (Langendorff technique) with KrebsHenseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was similar to 31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (similar to 2.2 times) in all groups vs. group KH and was similar to 35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was similar to 28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

MyD88 signaling is directly involved in the development of murine placental Malaria

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster development

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Conditional and transgenic mouse models as tools to study the role of TGFbeta, BMP signaling in skeletal development

Die TGFbeta/BMP Signaltransduktionskaskade ist wichtig für viele Entwicklungsprozesse fast aller embryonaler sowie extraembryonaler Gewebe und sie ist ebenso essentiell bei der Aufrechterhaltung der Homöostase im adulten Organismus. In vielen Mausmodellen und Zellkulturversuchen wurde gezeigt, dass Liganden dieses Signalweges in verschiedene Stadien der Knorpel- und Knochenentwicklung involviert sind. BMPs sind beispielsweise maßgeblich an der frühen Kondensation und Bildung des Knorpels und später an Proliferation und Hypertrophie der Chondrozyten beteiligt. BMPs können ektopisch Knochenbildung auslösen und das Expressionsmuster der Liganden und spezifischen Rezeptoren in der Wachstumsfuge lässt auf eine wichtige Rolle der BMPs in der Wachstumsfuge schließen. Der gezielte knock out der BMP-Rezeptoren Bmpr1a und Bmpr1b in proliferierenden Chondrozyten führt zur Ausbildung einer generellen Chondrodysplasie. Smad1, Smad5 und Smad8 sind die Mediatoren der BMP-Signalkaskade. Im Rahmen der vorliegenden Arbeit sollte die Rolle und Funktion der Smad1- und Smad5-Proteine in der Wachstumsfuge untersucht werden. Hierzu wurden konditionale Smad1-knock out-Mäuse mit einer transgenen Mauslinie gekreuzt, die die Cre-Rekombinase spezifisch in proliferierenden Chondrozyten exprimiert. Diese Mäuse wurden mit und ohne heterozygotem Smad5-Hintergrund charakterisiert. Bei einem knock out von Smad1 allein konnte ein leichte Verkürzung der Wachstumsfuge beobachtet werden, wobei prähypertrophe und hypertrophe Zone gleichermaßen betroffen waren. Dieser Phänotyp war verstärkt in Mäusen mit zusätzlichem heterozygotem Smad5-Hintergrund. Eine Verringerung der Proliferationsrate konnte zusammen mit einer verminderten Ihh-Expression nachgewiesen werden. Zusätzlich konnte anhand von Röntgenaufnahmen eine Dysorganisation der nasalen Region und ein fehlendes nasales Septum beobachtet werden. Produktion und Mineralisation der extrazellulären Matrix waren nicht beeinträchtigt. Um die Rolle der BMP- und TGFbeta-Signalkaskaden während der endochondralen Ossifikation zu vergleichen, wurden transgene Mäuse generiert, in denen die TGFbeta-Signalkaskade spezifisch in proliferierenden Chondrozyten gestört war. Zwei Mauslinien, die ähnliche Phänotypen zeigten, wurden untersucht. Esl1 ist ein TGFbeta-bindendes Protein, von dem man annimmt, dass es die TGFbeta-Signalkaskade inhibieren kann. Esl1-knock out-Mäuse sind kleiner als Wildtypmäuse und die Überexpression von Esl1 in proliferierenden Chondrozyten führt zu einer Verlängerung der Wachstumsfuge und einer verstärkten Proliferationsrate. Knorpelmarker, wie Col2a1 und Sox9 sind in diesen Mäusen herunterreguliert, während Col10a1 und Ihh als Marker für die hypertrophe und prähypertrophe Zone herunterreguliert waren. Dies führt zu der Annahme, dass mehr Zellen in die terminale Differenzierung eintreten. Bei transgenen Mäusen, in denen ein dominant-negativer (dn) TGFbeta-Rezeptor in proliferierenden Chondrozyten überexprimiert wurde, konnte eine verlängerte prähypertrophe Zone, eine erhöhte Ihh-Expression, sowie eine verstärkte Proliferationsrate beobachtet werden. Zusätzlich konnte in homozygoten Tieren ein craniofacialer Phänotyp beschrieben werden, der zu Problemen bei der Nahrungsaufnahme und damit zu einer starken Wachstumsbeeinträchtigung führte. Die BMP- und TGFbeta-Signalkaskaden haben möglicherweise antagonistische Effekte in der Wachstumsfuge. Während der Ausfall von BMP in proliferierenden Chondrozyten aufgrund einer gesunkenen Proliferationsrate zu einer Verkürzung der Wachstumsfuge führte, kann man in Mäusen mit einer Störung der TGFbeta-Signalkaskade eine verstärkte Proliferation in einer daher verlängerten Wachstumsfuge beobachten. Ein weiteres Ziel dieser Arbeit war die Generation einer transgenen Mauslinie, die die Cre-Rekombinase spezifisch in hypertrophen Chondrozyten exprimiert. Promoterstudien mit transgenen Mäusen weisen darauf hin, dass ein putatives AP1-Element, etwa 4 kb vor dem ersten Exon des Col10a1 gelegen, wichtig für die spezifische Expression in hypertrophen Chondrozyten ist. Ein Konstrukt, dass vier Kopien dieses Elements und den basalen Promoter enthält, wurde benutzt, um die Cre-Rekombinase spezifisch zu exprimieren. Diese Mauslinie befindet sich in der Testphase und erste Daten deuten auf eine spezifische Expression der Cre-Rekombinase in hypertrophen Chondrozyten hin.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling

Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/beta-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase beta-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3beta. Finally, beta-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects beta-catenin activity in the heart and its implications for disease pathogenesis.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.