CP-25 inhibits PGE2-induced angiogenesis by down-regulating EP4/AC/cAMP/PKA-mediated GRK2 translocation

Abstract G protein-coupled receptor kinase 2 (GRK2), a type of cytosolic enzyme, transiently translocates to the plasma membrane upon G protein-coupled receptors (GPCRs) activation, and it also binds to extracellular signal-regulated kinase (ERK) to inhibit the activation of ERK. GRK2 deficiency in endothelial cells (ECs) leads to increased pro-inflammatory signaling and promotes recruitment of leukocytes to activated ECs. However, the role of GRK2 in regulating angiogenesis remains unclear. Here, we show that GRK2 is a novel regulatory molecule on migration and tube formation of ECs, vessel sprouting ex vivo and angiogenesis in vivo. We identify that EP4/AC/cAMP/protein kinase A (PKA)-mediated GRK2 translocation to cells membrane decreases the binding of GRK2 and ERK1/2 to inhibit ERK1/2 activation, which promotes prostaglandin E2 (PGE2)-induced angiogenesis. GRK2 small interfering RNA (siRNA) inhibits the increase in PGE2-induced HUVECs migration and tube formation. In vivo, PGE2 increases ECs sprouting from normal murine aortic segments and angiogenesis in mice, but not from GRK2-deficient ones, on Matrigel. Further research found that Lys220 and Ser685 of GRK2 play an important role in angiogenesis by regulating GRK2 translocation. Paeoniflorin-6′-O-benzene sulfonate (CP-25), as a novel ester derivative of paeoniflorin (pae), has therapeutic potential for the treatment of adjuvant arthritis (AA) and collagen-induced arthritis (CIA), but the underlying mechanism of CP-25 on angiogenesis has not been elucidated. In our study, CP-25 inhibits the migration and tube formation of HUVECs, and angiogenesis in mice by down-regulating GRK2 translocation activation without affecting GRK2 total expression. Taken together, the present results revealed that CP-25 down-regulates EP4/AC/cAMP/PKA-mediated GRK2 translocation, restoring the inhibition of GRK2 for ERK1/2, thereby inhibiting PGE2-stimulated angiogenesis.

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The treatment effects of flaxseed-derived secoisolariciresinol diglycoside and its metabolite enterolactone on benign prostatic hyperplasia involve the G protein-coupled estrogen receptor 1

Applied Physiology Nutrition and Metabolism ◽

10.1139/apnm-2016-0332 ◽

2016 ◽

Vol 41 (12) ◽

pp. 1303-1310 ◽

Cited By ~ 13

Author(s):

Guan-Yu Ren ◽

Chun-Yang Chen ◽

Wei-Guo Chen ◽

Ya Huang ◽

Li-Qiang Qin ◽

...

Keyword(s):

Cell Cycle ◽

Estrogen Receptor ◽

Benign Prostatic Hyperplasia ◽

G Protein ◽

Therapeutic Potential ◽

Prostatic Hyperplasia ◽

Docking Simulations ◽

G Protein Coupled

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

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Chemogenetic Tools for Causal Cellular and Neuronal Biology

Physiological Reviews ◽

10.1152/physrev.00009.2017 ◽

2018 ◽

Vol 98 (1) ◽

pp. 391-418 ◽

Cited By ~ 23

Author(s):

Deniz Atasoy ◽

Scott M. Sternson

Keyword(s):

G Protein ◽

Ex Vivo ◽

Cell Types ◽

Cell Populations ◽

Specific Cell ◽

Cellular Processes ◽

Distinct Cell ◽

G Protein Coupled ◽

Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

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Vascular Abnormalities in Mice Deficient for the G Protein-Coupled Receptor GPR4 That Functions as a pH Sensor

Molecular and Cellular Biology ◽

10.1128/mcb.01909-06 ◽

2006 ◽

Vol 27 (4) ◽

pp. 1334-1347 ◽

Cited By ~ 79

Author(s):

Li V. Yang ◽

Caius G. Radu ◽

Meenakshi Roy ◽

Sunyoung Lee ◽

Jami McLaughlin ◽

...

Keyword(s):

G Protein ◽

Mesangial Cells ◽

Ex Vivo ◽

Extracellular Ph ◽

Ph Sensing ◽

G Protein Coupled Receptor ◽

Vascular Abnormalities ◽

G Protein Coupled

ABSTRACT GPR4 is a G protein-coupled receptor expressed in the vasculature, lung, kidney, and other tissues. In vitro ectopic overexpression studies implicated GPR4 in sensing extracellular pH changes leading to cyclic AMP (cAMP) production. To investigate its biological roles in vivo, we generated GPR4-deficient mice by homologous recombination. Whereas GPR4-null adult mice appeared phenotypically normal, neonates showed a higher frequency of perinatal mortality. The average litter size from GPR4−/− intercrosses was ∼30% smaller than that from GPR4+/+ intercrosses on N3 and N5 C57BL/6 genetic backgrounds. A fraction of knockout embryos and neonates had spontaneous hemorrhages, dilated and tortuous subcutaneous blood vessels, and defective vascular smooth muscle cell coverage. Mesangial cells in kidney glomeruli were also significantly reduced in GPR4-null neonates. Some neonates exhibited respiratory distress with airway lining cell metaplasia. To examine whether GPR4 is functionally involved in vascular pH sensing, an ex vivo aortic ring assay was used under defined pH conditions. Compared to wild-type aortas, microvessel outgrowth from GPR4-null aortas was less inhibited by acidic extracellular pH. Treatment with an analog of cAMP, a downstream effector of GPR4, abolished microvessel outgrowth bypassing the GPR4-knockout phenotype. These results suggest that GPR4 deficiency leads to partially penetrant vascular abnormalities during development and that this receptor functions in blood vessel pH sensing.

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Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo

10.1101/2021.11.19.469234 ◽

2021 ◽

Author(s):

Amro Elgheznawy ◽

Patricia Oeftering ◽

Maximilian Englert ◽

Friederike Kaiser ◽

Charly Kusch ◽

...

Keyword(s):

G Protein ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Platelet Response ◽

G Protein Coupled Receptor ◽

Icp Ms ◽

And Function ◽

G Protein Coupled

Zinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

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The G-protein-coupled receptor kinase 5 inhibits NFκB transcriptional activity by inducing nuclear accumulation of IκBα

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0804446105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17818-17823 ◽

Cited By ~ 77

Author(s):

Daniela Sorriento ◽

Michele Ciccarelli ◽

Gaetano Santulli ◽

Alfonso Campanile ◽

Giovanna Giuseppina Altobelli ◽

...

Keyword(s):

G Protein ◽

Transcriptional Activity ◽

Underlying Mechanism ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

Physical Interaction ◽

G Protein Coupled Receptor ◽

G Protein Coupled

G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5–IκBα interaction on NFκB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IκBα, leading to the inhibition of NFκB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IκBα, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IκBα as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFκB, we evaluated the effects of GRK5-RH on NFκB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFκB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFκB activity.

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PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway

Blood ◽

10.1182/blood-2011-04-350587 ◽

2011 ◽

Vol 118 (19) ◽

pp. 5355-5364 ◽

Cited By ~ 70

Author(s):

Yushan Zhang ◽

Yehia Daaka

Keyword(s):

Glycogen Synthase ◽

Ex Vivo ◽

Cell Types ◽

Tube Formation ◽

Microvascular Endothelial Cells ◽

Selective Agonists ◽

Interfering Rna ◽

Subtype Selective

Abstract Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3β that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.

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Protective Effect of Genistein against Compound 48/80 Induced Anaphylactoid Shock via Inhibiting MAS Related G Protein-Coupled Receptor X2 (MRGPRX2)

Molecules ◽

10.3390/molecules25051028 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1028 ◽

Cited By ~ 2

Author(s):

Mukesh Kumar ◽

Kailash Singh ◽

Karthi Duraisamy ◽

Ahmed A. Allam ◽

Jamaan Ajarem ◽

...

Keyword(s):

G Protein ◽

Inhibitory Activity ◽

Cell Activation ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Mast Cell Activation ◽

Anaphylactoid Shock ◽

G Protein Coupled

Anaphylactoid shock is a fatal hypersensitivity response caused by non-IgE mediated mast cell activation. These reactions are mediated by a family of G protein-coupled receptors (GPCRs) known as Mas related GPCRX2 (MRGPRX2). Several US FDA approved drugs which are used in day to day life have been reported to cause anaphylactoid shock. Surprisingly, no therapeutic drugs are available which can directly target MRGPRX2 for treatment of anaphylactoid shock. Genistein is a non-steroidal polyphenol known for its diverse physiological and pharmacological activities. In recent studies, Genistein has been reported for its anti-inflammatory activity on mast cells. However, the effects and mechanistic pathways of Genistein on anaphylactoid reaction remain unknown. In the present study, we designed a battery of in-vitro, in-silico and in-vivo experiments to evaluate the anti-anaphylactoid activity of Genistein in order to understand the possible molecular mechanisms of its action. The in-vitro results demonstrated the inhibitory activity of Genistein on MRGPRX2 activation. Further, a mouse model of anaphylactoid shock was used to evaluate the inhibitory activity of Genistein on blood vessel leakage and hind paw edema. Taken together, our findings have demonstrated a therapeutic potential of Genistein as a lead compound in the treatment of anaphylactoid shock via MRGPRX2.

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Studies of involvement of G-protein coupled receptor-3 in cannabidiol effects on inflammatory responses of mouse primary astrocytes and microglia

PLoS ONE ◽

10.1371/journal.pone.0251677 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0251677

Author(s):

Jun Wu ◽

Nu Chen ◽

Yongqing Liu ◽

Grzegorz Godlewski ◽

Henry J. Kaplan ◽

...

Keyword(s):

G Protein ◽

Glial Cells ◽

Inflammatory Responses ◽

Therapeutic Potential ◽

G Protein Coupled Receptor ◽

Primary Astrocytes ◽

Anti Inflammatory ◽

G Protein Coupled

Cannabidiol (CBD) exhibits anti-inflammatory and neuroprotective properties and is suggested to be effective in the pre-clinical and clinical treatment of illnesses of the central nervous system (CNS). Two major types of CNS glial cells, astrocytes and microglia, play critical roles in the development and pathogenesis of CNS diseases. However, the mechanisms by which CBD plays an anti-inflammatory and neuroprotective role for these glial cells have not been fully elucidated. In this study, we examined the effects of CBD on the inflammatory response of mouse primary astrocytes and microglia. We also investigated whether the effect of CBD on cytokine release is mediated by the G protein coupled receptor 3 (GPR3), which was recently identified as a novel receptor for CBD. Our results showed that CBD inhibited inflammatory responses of astrocytes and microglia stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand in vitro and in vivo. In addition, CBD reduced the phosphorylation of STAT3 and NF-κB signaling pathways in LPS-stimulated astrocytes. However, the inhibitory effect of CBD on pro-inflammatory cytokine production was independent of GPR3 expression in both types of glial cells. Thus, although CBD is effective in ameliorating the activation of astrocytes and microglia, its mechanism of action still requires further study. Our data support the concept that CBD may have therapeutic potential for neurological disorders that involve neuroinflammation.

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