3D structural model of the G-protein-coupled cannabinoid CB2 receptor

The potential for therapeutic specificity in regulating diseases and for reduced side effects has made cannabinoid (CB) receptors one of the most important G-protein-coupled receptor (GPCR) targets for drug discovery. The cannabinoid (CB) receptor subtype CB2 is of particular interest due to its involvement in signal transduction in the immune system and its increased characterization by mutational and other studies. However, our understanding of their mode of action has been limited by the absence of an experimental receptor structure. In this study, we have developed a 3D model of the CB2 receptor based on the recent crystal structure of a related GPCR, bovine rhodopsin. The model was developed using multiple sequence alignment of homologous receptor sub-types in humans and mammals, and compared with other GPCRs. Alignments were analyzed with mutation scores, pairwise hydrophobicity profiles and Kyte-Doolittle plots. The 3D model of the transmembrane segment was generated by mapping the CB2 sequence onto the homologous residues of the rhodopsin structure. The extra- and intracellular loop regions of the CB2 were generated by searching for homologous C(alpha) backbone sequences in published structures in the Brookhaven Protein Databank (PDB). Residue side chains were positioned through a combination of rotamer library searches, simulated annealing and minimization. Intermediate models of the 7TM helix bundles were analyzed in terms of helix tilt angles, hydrogen-bond networks, conserved residues and motifs, possible disulfide bonds. The amphipathic cytoplasmic helix domain was also correlated with biological and site-directed mutagenesis data. Finally, the model receptor-binding cavity was characterized using solvent-accessible surface approach.     

CB2 cannabinoid receptors promote neural progenitor cell proliferation via mTORC1 signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.     

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via NMDA receptor phosphorylation

Development of drug addiction involves complex molecular changes in the CNS. The mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse. We previously showed that dopamine D(1) and D(3) receptors play different roles in regulating cocaine-induced MAPK activation. Although there are functional and physical interactions between dopamine and glutamate receptors, little is known regarding the involvement of D(1) and D(3) receptors in modulating glutamate-induced MAPK activation and underlying mechanisms. In this study, we show that D(1) and D(3) receptors play opposite roles in regulating N-methyl-d-aspartate (NMDA) -induced activation of extracellular signal-regulated kinase (ERK) in the caudate putamen (CPu). D(3) receptors also inhibit NMDA-induced activation of the c-Jun N-terminal kinase and p38 kinase in the CPu. NMDA-induced activation of the NMDA-receptor R1 subunit (NR1), Ca(2+)/calmodulin-dependent protein kinase II and the cAMP-response element binding protein (CREB), and cocaine-induced CREB activation in the CPu are also oppositely regulated by dopamine D(1) and D(3) receptors. Finally, the blockade of NMDA-receptor reduces cocaine-induced ERK activation, and inhibits phosphorylation of NR1, Ca(2+)/calmodulin-dependent protein kinase II, and CREB, while inhibiting ERK activation attenuates cocaine-induced CREB phosphorylation in the CPu. These results suggest that dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via phosphorylation of NR1.     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.     

p53 regulates ERK1/2/CREB cascade via a novel SASH1/MAP2K2 crosstalk to induce hyperpigmentation

We previously reported that three point mutations in SASH1 and mutated SASH1 promote melanocyte migration in dyschromatosis universalis hereditaria (DUH) and a novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype. However, the underlying mechanism of molecular regulation to cause this hyperpigmentation disorder still remains unclear. In this study, we aimed to investigate the molecular mechanism undergirding hyperpigmentation in the dyschromatosis disorder. Our results revealed that SASH1 binds with MAP2K2 and is induced by p53‐POMC‐MC1R signal cascade to enhance the phosphorylation level of ERK1/2 and CREB. Moreover, increase in phosphorylated ERK1/2 and CREB levels and melanogenesis‐specific molecules is induced by mutated SASH1 alleles. Together, our results suggest that a novel SASH1/MAP2K2 crosstalk connects ERK1/2/CREB cascade with p53‐POMC‐MC1R cascade to cause hyperpigmentation phenotype of DUH.     

Visualization of 2-arachidonoylglycerol accumulation and cannabinoid CB1 receptor activity in rat brain cryosections by functional autoradiography

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.     

IGFBP7 regulates sepsis-induced acute kidney injury through ERK1/2 signaling

IGFBP7 as an early biomarker has been used to identify patients at risk of developing acute kidney injury (AKI). Nevertheless, its role in AKI remains obscure. The aim of our study is to determine the role and mechanism of IGFBP7 in lipopolysaccharide (LPS)-induced HK-2 cells in vitro and on sepsis-induced AKI by cecal ligation and puncture (CLP) in vivo. Here, we identified that IGFBP7 expression was increased in patients with AKI and HK-2 cells with LPS (1, 2, and 5 μg/mL) induction. HK-2 cells with LPS induction showed cell cycle arrest at G1-G0 phases and cell apoptosis and activated ERK1/2 parallel with the changes in the proteins belonging to the ERK1/2 pathway, including Cyclin D1, P21, Bax, and Bcl-2, which were inhibited by the IGFBP7 knockdown. Moreover, IGFBP7 overexpression significantly induced cell cycle arrest at G1-G0 phases and cell apoptosis of HK-2 cells, which were inhibited by PD98509, an ERK1/2 signaling inhibitor. IGFBP7 knockdown effectively alleviated the severity of the renal injury, evidenced by decreases in the urinary levels of creatinine, blood urea nitrogen, and albumin, cell apoptosis, and activation of ERK1/2 signaling in CLP mice. Taken together, our findings indicate that IGFBP7 regulates sepsis-induced AKI through ERK1/2 signaling.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Striatal CB1 and D2 receptors regulate expression of each other,CRIP1A and delta opioid systems

Although biochemical and physiological evidence suggests a strong interaction between striatal CB₁ cannabinoid (CB₁R) and D₂ dopamine (D₂R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB₁R or D₂R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB₁R and D₂R knockdown reduced striatal dopaminergic‐stimulated [³⁵S]GTPγS binding, and D₂R knockdown reduced pallidal WIN55212‐2‐stimulated [³⁵S]GTPγS binding. Decreased D₂R and CB₁R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB₁Rs or D₂Rs, and over‐expression of CRIP1a. Down‐regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D‐Pen2, D‐Pen5]‐enkephalin‐stimulated [³⁵S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB₁R or D₂R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.     

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A_2A, cannabinoid CB₁ and metabotropic glutamate 5 (mGlu₅) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB₁R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A_2ARs. Such a permissive effect of A_2ARs towards CB₁Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A_2ARs. The selective mGlu₅R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A_2AR blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A_2ARs regulates the synaptic effects of CB₁Rs and that A_2ARs may control CB₁ effects also indirectly, namely through mGlu₅Rs.     

Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24 h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.