Inhibits cAMP Signaling Research Articles

Structural and pharmacological characterization of a medium-chain fatty acid receptor GPR84 in common carp (Cyprinus carpio)

The medium-chain fatty acid receptor GPR84, a member of the G protein-coupled receptor family, is mainly expressed in macrophages and microglia, and is involved in the regulation of inflammatory responses and retinal development in mammals and amphibians. However, structure, tissue distribution, and pharmacology of this receptor have rarely been reported in fish. In this study, we cloned the coding sequence (CDS) of common carp GPR84 (ccGPR84), examined its tissue distribution, and explored its cellular signaling function. The results showed that the CDS of ccGPR84 is 1191 bp and encodes a putative protein with 396 amino acids. Phylogenetic and chromosomal synteny analyses revealed that ccGPR84 was evolutionarily conserved with Cyprinids. Real-time quantitative PCR (qPCR) indicated that ccGPR84 was predominantly expressed in the intestine and spleen. Luciferase reporter assay demonstrated that nonanoic acid, capric acid (decanoic acid), undecanoic acid and lauric acid could inhibit cAMP signaling pathway and activate MAPK/ERK signaling pathway, while the potencies of these four fatty acids on the two signaling pathways were different. Lauric acid has the highest inhibitory potency on cAMP signaling pathway, followed by undecanoic acid, nonanoic acid, and capric acid. While for MAPK/ERK signaling pathway, nonanoic acid has the highest activation potency, followed by undecanoic acid, capric acid, and lauric acid. These findings lay the foundation for revealing the roles of different medium-chain fatty acids in the inflammatory response of common carp.

Molecular Cloning, Tissue Distribution, and Pharmacological Characterization of GPR84 in Grass Carp (Ctenopharyngodon Idella).

The G-protein-coupled receptor GPR84, activated by medium-chain fatty acids, primarily expressed in macrophages and microglia, is involved in inflammatory responses and retinal development in mammals and amphibians. However, our understanding of its structure, function, tissue expression, and signaling pathways in fish is limited. In this study, we cloned and characterized the coding sequence of GPR84 (ciGPR84) in grass carp. A phylogenetic analysis revealed its close relationship with bony fishes. High expression levels of GPR84 were observed in the liver and spleen. The transfection of HEK293T cells with ciGPR84 demonstrated its responsiveness to medium-chain fatty acids and diindolylmethane (DIM). Capric acid, undecanoic acid, and lauric acid activated ERK and inhibited cAMP signaling. Lauric acid showed the highest efficiency in activating the ERK pathway, while capric acid was the most effective in inhibiting cAMP signaling. Notably, DIM did not activate GPR84 in grass carp, unlike in mammals. These findings provide valuable insights for mitigating chronic inflammation in grass carp farming and warrant further exploration of the role of medium-chain fatty acids in inflammation regulation in this species.

Age-related changes in peripheral nociceptor function

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4–5 months) and aged (26–27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Dietary Bioactive Ingredients Modulating the cAMP Signaling in Diabetes Treatment.

As the prevalence of diabetes increases progressively, research to develop new therapeutic approaches and the search for more bioactive compounds are attracting more attention. Over the past decades, studies have suggested that cyclic adenosine monophosphate (cAMP), the important intracellular second messenger, is a key regulator of metabolism and glucose homeostasis in diverse physiopathological states in multiple organs including the pancreas, liver, gut, skeletal muscle, adipose tissues, brain, and kidney. The multiple characteristics of dietary compounds and their favorable influence on diabetes pathogenesis, as well as their intersections with the cAMP signaling pathway, indicate that these compounds have a beneficial effect on the regulation of glucose homeostasis. In this review, we outline the current understanding of the diverse functions of cAMP in different organs involved in glucose homeostasis and show that a diversity of bioactive ingredients from foods activate or inhibit cAMP signaling, resulting in the improvement of the diabetic pathophysiological process. It aims to highlight the diabetes-preventative or -therapeutic potential of dietary bioactive ingredients targeting cAMP signaling.

Biased Coupling to β-Arrestin of Two Common Variants of the CB2 Cannabinoid Receptor.

β-arrestins are partners of the G protein-coupled receptors (GPCRs), regulating their intracellular trafficking and signaling. Development of biased GPCR agonists, selectively targeting either G protein or β-arrestin pathways, are in the focus of interest due to their therapeutic potential in different pathological conditions. The CB2 cannabinoid receptor (CB2R) is a GPCR involved in various functions in the periphery and the central nervous system. Two common occurring variants of CB2R, harboring Q63R or L133I missense mutations, have been implicated in the development of a diverse set of disorders. To evaluate the effect of these mutations, we characterized the binding profile of these mutant CB2 receptors to G proteins and β-arrestin2. Although their ability to inhibit cAMP signaling was similar, the Q63R mutant had increased, whereas the L133I mutant receptor had decreased β-arrestin2 binding. In line with these observations, the variants also had altered intracellular trafficking. Our results show that two common variants of the CB2 receptor have biased signaling properties, which may contribute to the pathogenesis of the associated disorders and may offer CB2R as a target for further development of biased receptor activation strategies.

Regulated expression and function of the GABAB receptor in human pancreatic beta cell line and islets

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABAB receptor compared to human islet. In ECN90 cells, baclofen, a specific GABAB receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABAB receptor in human islets modulates insulin secretion.

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors.

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR’s protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

Pivotal role of cAMP-PKA-CREB signaling pathway in manganese-induced neurotoxicity in PC12 cells.

Manganese (Mn) plays a critical role in individual growth and development, yet excessive exposure can result in neurotoxicity, especially cognitive impairment. Neuronal apoptosis is considered as one of the mechanisms of Mn-induced neurotoxicity. Recent evidence suggests that cAMP-PKA-CREB signaling regulates apoptosis and is associated with cognitive function. However, whether this pathway participates in Mn-induced neurotoxicity is not completely understood. To fill this gap, in vitro cultures of PC12 cells were exposed to 0, 400, 500, and 600 μmol/L Mn for 24 hours, respectively. Another group of cells were pretreated with 10.0 μmol/L rolipram (a phosphodiesterase-4 [PDE4] inhibitor) for 1 hour followed by 500 μmol/L Mn exposure for 24 hours. Flow cytometry, immunofluorescence staining, enzyme-linked immunosorbent assay, and Western blot analysis were used to detect the apoptosis rate, protein levels of PDE4, cAMP signaling, and apoptosis-associated proteins, respectively. We found that Mn exposure significantly inhibited cAMP signaling and protein expression of Bcl-2, while increasing apoptosis rate, protein levels of PDE4, Bax, activated caspase-3, and activated caspase-8 in PC12 cells. Pretreatment of rolipram ameliorated Mn-induced deficits in cAMP signaling and apoptosis. These findings demonstrate that cAMP-PKA-CREB signaling pathway-induced apoptosis is involved in Mn-induced neurotoxicity.

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary.

Helicobacter pylori γ-Glutamyltranspeptidase Induces Tolerogenic Human Dendritic Cells by Activation of Glutamate Receptors.

Helicobacter pylori infection is characterized by chronic persistence of the bacterium. Different virulence factors, including H. pylori γ-glutamyltranspeptidase (gGT), have been reported to induce tolerogenicity by reprogramming dendritic cells (DCs). gGT is present in all bacterial isolates, indicating an important role for gGT in the course of infection. In the current study, we have analyzed the effect of H. pylori gGT on human DCs and the subsequent adaptive immune response. We show that glutamate produced due to H. pylori gGT enzymatic activity tolerizes DCs by inhibiting cAMP signaling and dampening IL-6 secretion in response to the infection. Together, our results provide a novel molecular mechanism by which H. pylori manipulates the host's immune response to persist within its host.

Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.

Salt-Inducible Kinase 1 Terminates cAMP Signaling by an Evolutionarily Conserved Negative-Feedback Loop in β-Cells.

Pancreatic β-cells are critical in the regulation of glucose homeostasis by controlled secretion of insulin in mammals. Activation of protein kinase A by cAMP is shown to be responsible for enhancing this pathway, which is countered by phosphodiesterase (PDE) that converts cAMP to AMP and turns off the signal. Salt-inducible kinases (SIKs) were also known to inhibit cAMP signaling, mostly by promoting inhibitory phosphorylation on CREB-regulated transcription coactivators. Here, we showed that SIK1 regulates insulin secretion in β-cells by modulating PDE4D and cAMP concentrations. Haploinsufficiency of SIK1 led to the improved glucose tolerance due to the increased glucose-stimulated insulin secretion. Depletion of SIK1 promoted higher cAMP concentration and increased insulin secretion from primary islets, suggesting that SIK1 controls insulin secretion through the regulation of cAMP signaling. By using a consensus phosphorylation site of SIK1, we identified PDE4D as a new substrate for this kinase family. In vitro kinase assay as well as mass spectrometry analysis revealed that the predicted Ser(136) and the adjacent Ser(141) of PDE4D are critical in SIK1-mediated phosphorylation. We found that overexpression of either SIK1 or PDE4D in β-cells reduced insulin secretion, while inhibition of PDE4 activity by rolipram or knockdown of PDE4D restored it, showing indeed that SIK1-dependent phosphorylation of PDE4D is critical in reducing cAMP concentration and insulin secretion from β-cells. Taken together, we propose that SIK1 serves as a part of a self-regulatory circuit to modulate insulin secretion from pancreatic β-cells by controlling cAMP concentration through modulation of PDE4D activity.

Chronic stimulation of alpha-2A-adrenoceptors with guanfacine protects rodent prefrontal cortex dendritic spines and cognition from the effects of chronic stress

The prefrontal cortex (PFC) provides top-down regulation of behavior, cognition, and emotion, including spatial working memory. However, these PFC abilities are greatly impaired by exposure to acute or chronic stress. Chronic stress exposure in rats induces atrophy of PFC dendrites and spines that correlates with working memory impairment. As similar PFC grey matter loss appears to occur in mental illness, the mechanisms underlying these changes need to be better understood. Acute stress exposure impairs PFC cognition by activating feedforward cAMP-calcium- K+ channel signaling, which weakens synaptic inputs and reduces PFC neuronal firing. Spine loss with chronic stress has been shown to involve calcium-protein kinase C signaling, but it is not known if inhibiting cAMP signaling would similarly prevent the atrophy induced by repeated stress. The current study examined whether inhibiting cAMP signaling through alpha-2A-adrenoceptor stimulation with chronic guanfacine treatment would protect PFC spines and working memory performance during chronic stress exposure. Guanfacine was selected due to 1) its established effects on cAMP signaling at post-synaptic alpha-2A receptors on spines in PFC, and 2) its increasing clinical use for the treatment of pediatric stress disorders. Daily guanfacine treatment compared to vehicle control was found to prevent dendritic spine loss in layer II/III pyramidal neurons of prelimbic PFC in rats exposed to chronic restraint stress. Guanfacine also protected working memory performance; cognitive performance correlated with dendritic spine density. These findings suggest that chronic guanfacine use may have clinical utility by protecting PFC gray matter from the detrimental effects of stress.

Review: evolution of GnIH and related peptides structure and function in the chordates.

Discovery of gonadotropin-inhibitory hormone (GnIH) in the Japanese quail in 2000 was the first to demonstrate the existence of a hypothalamic neuropeptide inhibiting gonadotropin release. We now know that GnIH regulates reproduction by inhibiting gonadotropin synthesis and release via action on the gonadotropin-releasing hormone (GnRH) system and the gonadotrope in various vertebrates. GnIH peptides identified in birds and mammals have a common LPXRF-amide (X = L or Q) motif at the C-terminus and inhibit pituitary gonadotropin secretion. However, the function and structure of GnIH peptides are diverse in fish. Goldfish GnIHs possessing a C-terminal LPXRF-amide motif have both stimulatory and inhibitory effects on gonadotropin synthesis or release. The C-terminal sequence of grass puffer and medaka GnIHs are MPQRF-amide. To investigate the evolutionary origin of GnIH and its ancestral structure and function, we searched for GnIH in agnathans, the most ancient lineage of vertebrates. We identified GnIH precursor gene and mature GnIH peptides with C-terminal QPQRF-amide or RPQRF-amide from the brain of sea lamprey. Lamprey GnIH fibers were in close proximity to GnRH-III neurons. Further, one of lamprey GnIHs stimulated the expression of lamprey GnRH-III peptide in the hypothalamus and gonadotropic hormone β mRNA expression in the pituitary. We further identified the ancestral form of GnIH, which had a C-terminal RPQRF-amide, and its receptors in amphioxus, the most basal chordate species. The amphioxus GnIH inhibited cAMP signaling in vitro. In sum, the original forms of GnIH may date back to the time of the emergence of early chordates. GnIH peptides may have had various C-terminal structures slightly different from LPXRF-amide in basal chordates, which had stimulatory and/or inhibitory functions on reproduction. The C-terminal LPXRF-amide structure and its inhibitory function on reproduction may be selected in later-evolved vertebrates, such as birds and mammals.

The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 β-Cells

Prolonged exposure to melatonin improves glycemic control in animals. Although glucose metabolism is controlled by circadian clock genes, little is known about the role of melatonin signaling and its duration in the regulation of clock gene expression in pancreatic β-cells. Activation of MT1 and MT2 melatonin receptors inhibits cAMP signaling, which mediates clock gene expression. Therefore, this study investigated exposure duration-dependent alterations in cAMP element-binding protein (CREB) phosphorylation and clock gene expression that occur during and after exposure to ramelteon, a selective melatonin agonist used to treat insomnia. In rat INS-1 cells, a pancreatic β-cell line endogenously expressing melatonin receptors, ramelteon persistently decreased CREB phosphorylation during the treatment period (2–14 h), whereas the subsequent washout induced an enhancement of forskolin-stimulated CREB phosphorylation in a duration- and concentration-dependent manner. This augmentation was blocked by forskolin or the melatonin receptor antagonist luzindole. Similarly, gene expression analyses of 7 clock genes revealed the duration dependency of the effects of ramelteon on Rev-erbα and Bmal1 expression through melatonin receptor-mediated cAMP signaling; longer exposure times (14 h) resulted in greater increases in the expression and signaling of Rev-erbα, which is related to β-cell functions. Interestingly, this led to amplified oscillatory Rev-erbα and Bmal1 expression after agonist washout and forskolin stimulation. These results provide new insights into the duration-dependent effects of ramelteon on clock gene expression in INS-1 cells and may improve the understanding of its effect in vivo. The applicability of these results to pancreatic islets awaits further investigation.

Central and Direct Regulation of Testicular Activity by Gonadotropin-Inhibitory Hormone and Its Receptor

Gonadotropin-inhibitory hormone (GnIH) was first identified in Japanese quail to be an inhibitor of gonadotropin synthesis and release. GnIH peptides have since been identified in all vertebrates, and all share an LPXRFamide (X = L or Q) motif at their C-termini. The receptor for GnIH is the G protein-coupled receptor 147 (GPR147), which inhibits cAMP signaling. Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and the dorsomedial hypothalamic area (DMH) in most mammals. GnIH neurons in the PVN or DMH project to the median eminence to control anterior pituitary function via GPR147 expressed in gonadotropes. Further, GnIH inhibits gonadotropin-releasing hormone (GnRH)-induced gonadotropin subunit gene transcription by inhibiting the adenylate cyclase/cAMP/PKA-dependent ERK pathway in an immortalized mouse gonadotrope cell line (LβT2 cells). GnIH neurons also project to GnRH neurons that express GPR147 in the preoptic area (POA) in birds and mammals. Accordingly, GnIH can inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as by directly inhibiting pituitary gonadotrope activity. GnIH and GPR147 can thus centrally suppress testosterone secretion and spermatogenesis by acting in the hypothalamic–pituitary–gonadal axis. GnIH and GPR147 are also expressed in the testis of birds and mammals, possibly acting in an autocrine/paracrine manner to suppress testosterone secretion and spermatogenesis. GnIH expression is also regulated by melatonin, stress, and social environment in birds and mammals. Accordingly, the GnIH–GPR147 system may play a role in transducing physical and social environmental information to regulate optimal testicular activity in birds and mammals. This review discusses central and direct inhibitory effects of GnIH and GPR147 on testosterone secretion and spermatogenesis in birds and mammals.

C5aR and C3aR antagonists each inhibit diet‐induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling

Mammalian survival depends on metabolizing nutrients, storing energy, and combating infection. Complement activation in blood triggers energy-depleting immune responses to fight infections. Here we identify surprising energy-conserving roles for complement proteins C5a and C3a and their receptors, C5aR and C3aR, roles that are contraindicated in complement biology. Rats fed a high-carbohydrate high-fat diet developed obesity, visceral adiposity, adipose inflammation, glucose/insulin intolerance, and cardiovascular dysfunction that correlated with increased plasma C3a, adipose C5aR, and C3aR. These in vivo changes were dramatically attenuated by receptor-selective antagonists of either C5aR (5 mg/kg/d p.o.) or C3aR (30 mg/kg/d p.o.), which both reduced proinflammatory adipokines and altered expression of inflammatory genes in adipose tissue. In vitro C5a and C3a (100 nM) exhibited novel insulin-like effects on 3T3-L1 adipocytes, promoting energy conservation by increasing glucose and fatty acid uptake while inhibiting cAMP signaling and lipolysis, and induced PGE(2) release from macrophages, effects all blocked by each respective antagonist (10 μM). These studies reveal important new links between complement signaling and metabolism, highlight new complement functions on adipocytes and in adipose tissue, demonstrate how aberrant immune responses may exacerbate obesity and metabolic dysfunction, and show that targeting C3aR or C5aR with antagonists is a new strategy for treating metabolic dysfunction.

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and β-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

ERK and PDE4 cooperate to induce RAF isoform switching in melanoma

Melanocytes use BRAF to activate the MAP kinase (MAPK) pathway because CRAF is inhibited by the cyclic AMP (cAMP) pathway in these cells. By contrast, melanomas harboring Ras mutations use CRAF to activate the MAPK pathway. We describe the molecular mechanism of Raf isoform switching and cAMP pathway disruption, which take place during melanocyte transformation. We show that overactivation of the MAPK pathway, induced by the oncogenic Ras in melanoma, induces constitutive phosphorylation of BRAF on Ser151 by ERK, which inhibits NRAS-BRAF interaction . We also demonstrate that melanoma cells have elevated cAMP phosphodiesterase activity owing to overexpression of the cAMP-specific phosphodiesterase-4 enzymes; this activity inhibits cAMP signaling and allows CRAF reactivation in these cells. Reactivating the cAMP pathway inhibits proliferation and induces apoptosis of Ras-mutated melanoma cells, suggesting a new therapeutic approach for treating melanomas harboring Ras mutations.

The use of α-2A adrenergic agonists for the treatment of attention-deficit/hyperactivity disorder

Neuropsychiatric disorders involve dysfunction of the prefrontal cortex (PFC), a highly evolved brain region that mediates executive functioning. The dorsolateral PFC is specialized for regulating attention and behavior, while the ventromedial PFC is specialized for regulating emotion. These abilities arise from PFC pyramidal cell networks that excite each other to maintain goals and rules ‘in mind’. Imaging studies have shown reduced PFC gray matter, weaker PFC connections and altered PFC function in patients with attention-deficit/hyperactivity disorder. Thus, medications that strengthen PFC network connections may be particularly useful for the treatment of attention-deficit/hyperactivity disorder and related disorders. Recent data show that compounds such as guanfacine can enhance PFC function by stimulating postsynaptic α-2A receptors on the dendritic spines of PFC pyramidal cells where networks interconnect. Stimulation of these receptors inhibits cAMP signaling, thus closing potassium channels and strengthening physiological connections. These actions may benefit patients with weak PFC function.