NE-activated ß2-AR/ß-arrestin 2/Src pathway mediates duodenal hyperpermeability induced by water-immersion restraint stress.

Stress causes a rapid spike in norepinephrine (NE) levels, leading to gastrointestinal dysfunction. NE reduces the expression of tight junctions (TJs) and aggravates intestinal mucosal damage, but the regulatory mechanism is still unclear. The present study aimed to investigate the molecular mechanisms underlying the regulation of stress-associated duodenal hyperpermeability by NE. Fluorescein isothiocyanate-dextran permeability, transepithelial resistance, immunofluorescence, Western blot, and high-performance liquid chromatography analysis were used in water-immersion restraint stress (WIRS) rats in this study. The results indicate that the duodenal permeability, degradation of TJs, mucosal NE, and ß2-adrenergic receptor (ß2-AR) increased in WIRS rats. The duodenal intracellular cyclic adenosine monophosphate levels were decreased, whereas the expression of ß-arrestin 2 negatively regulates G protein-coupled receptors signaling, was significantly increased. Src recruitment was mediated by ß-arrestin; thus, the levels of Src kinase activation were enhanced in WIRS rats. NE depletion, ß2-AR, or ß-arrestin 2 blockade significantly decreased mucosal permeability and increased TJs expression, suggesting improved mucosal barrier function. Moreover, NE induced an increased duodenal permeability of normal rats with activated ß-arrestin 2/Src signaling, which was significantly inhibited by ß2-AR blockade. The present findings demonstrate that the enhanced NE induced an increased duodenal permeability in WIRS rats through the activated ß2-AR/ß-arrestin 2/Src pathway. This study provides novel insight into the molecular mechanism underlying the regulation of NE on the duodenal mucosal barrier and a new target for treating duodenal ulcers induced by stress.

ß-Arrestin 2 and Epac2 Cooperatively Mediate DRD1-Stimulated Proliferation of Human Neural Stem Cells and Growth of Human Cerebral Organoids.

G-protein-coupled receptors (GPCRs) reportedly relay specific signals, such as dopamine and serotonin, to regulate neurogenic processes although the underlying signaling pathways are not fully elucidated. Based on our previous work, which demonstrated dopamine receptor D1 (DRD1) effectively induces the proliferation of human neural stem cells, here we continued to show the knockout of ß-arrestin 2 by CRISPR/Cas9 technology significantly weakened the DRD1-induced proliferation and neurosphere growth. Furthermore, inhibition of the downstream p38 MAPK by its specific inhibitors or small hairpin RNA mimicked the weakening effect of ß-arrestin 2 knockout. In addition, blocking of Epac2, a PKA independent signal pathway, by its specific inhibitors or small hairpin RNA also significantly reduced DRD1-induced effects. Simultaneous inhibition of ß-arrestin 2/p38 MAPK and Epac2 pathways nearly abolished the DRD1-stimulated neurogenesis, indicating the cooperative contribution of both pathways. Consistently, the expansion and folding of human cerebral organoids as stimulated by DRD1 were also mediated cooperatively by both ß-arrestin 2/p38 MAPK and Epac2 pathways. Taken together, our results reveal that GPCRs apply at least 2 different signal pathways to regulate neurogenic processes in a delicate and balanced manners.

ß-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice.

BACKGROUND: ß-Arrestin 2 (ß-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking ß-arr 2, suggesting a role for ß-arr 2 in the anabolic effects of PTH. The role of ß-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. OBJECTIVE: To assess the effects of cPTH administration on bone in mice lacking ß-arr 2 compared to wild-type (WT). METHODS: Groups of male and female WT or ß-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. RESULTS: ß-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. ß-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female ß-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. CONCLUSIONS: These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. ß-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Paeoniflorin-6'-O-benzene sulfonate protected the intestinal epithelial barrier by restoring the inhibitory effect of GRK2 and ß-arrestin 2 on ERK1/2-NF-κB.

Peoniflorin-6'-O-benzene sulfonate (CP-25) inhibited the activity of GRK2 to exert anti-inflammatory and immunomodulatory effects. This study aimed to investigate the effect of CP-25 the intestinal epithelial barrier and the mechanism. CaCO-2 cell monolayer and dextran sulfate salt (DSS)-induced colitis mouse model was used to evaluate intestinal epithelial barrier function in vitro and in vivo, respectively. Results showed that CP-25 prevented dysfunction of the intestinal epithelial barrier and inhibited NF-κB p65 activation in TNF-α-induced CaCO-2 cells. The colon structure destroyed in DSS-induced colitis mice was improved by CP-25. CP-25 has a role in inhibition membrane translocation of GRK2-ß-arrestin 2 complex, stabilization of the binding of GRK2 and ß-arrestin 2 to ERK1/2 in cytoplasm. Subsequently down-regulated the nuclear transcription and transactivation of NF-κB p65 via inhibiting its phosphorylation of Ser536, and Ser276, respectively and restored the epithelial barrier function. In conclusion, CP-25 inhibited ERK1/2-NF-κB activation and thereby protected the intestinal epithelial barrier, which was associated with restoring the inhibition of GRK2 and ß-arrestin 2 on ERK1/2.

Disrupted circadian expression of ß-arrestin 2 affects reward-related µ-opioid receptor function in alcohol dependence.

There is increasing evidence for a daily rhythm of µ-opioid receptor (MOR) efficacy and the development of alcohol dependence. Previous studies show that ß-arrestin 2 (bArr2) has an impact on alcohol intake, at least partially mediated via modulation of MOR signaling, which in turn mediates the alcohol rewarding effects. Considering the interplay of circadian rhythms on MOR and alcohol dependence, we aimed to investigate bArr2 in alcohol dependence at different time points of the day/light cycle on the level of bArr2 mRNA (in situ hybridization), MOR availability (receptor autoradiography), and MOR signaling (Damgo-stimulated G-protein coupling) in the nucleus accumbens of alcohol-dependent and non-dependent Wistar rats. Using a microarray data set we found that bArr2, but not bArr1, shows a diurnal transcription pattern in the accumbens of naïve rats with higher expression levels during the active cycle. In 3-week abstinent rats, bArr2 is up-regulated in the accumbens at the beginning of the active cycle (ZT15), whereas no differences were found at the beginning of the inactive cycle (ZT3) compared with controls. This effect was accompanied by a specific down-regulation of MOR binding in the active cycle. Additionally, we detect a higher receptor coupling during the inactive cycle compared with the active cycle in alcohol-dependent animals. Together, we report daily rhythmicity for bArr2 expression linked to an inverse pattern of MOR, suggesting an involvement for bArr2 on circadian regulation of G-protein coupled receptors in alcohol dependence. The presented data may have implications for the development of novel bArr2-related treatment targets for alcoholism.

G-protein biased signaling agonists of Dopamine D3 receptor promote distinct activation patterns of ERK1/2.

Dopamine D3 receptors (D3R) have a causal role in neurological and psychiatric disorders. We have developed a novel class of G-protein biased (GPB) signaling D3R agonists with minimal ß-arrestin2 (ßarr2) recruitment and demonstrated efficacy in rodent model of Parkinson's disease. This contrasts with unbiased (UB) D3R agonists like Pramipexole which recruit both ß-arrestin and G-proteins for signaling. In this study, we investigated the effects of GPB and UB agonists on D3R mediated activation of mono and dual phosphorylation of ERK1/2. We used the neuronal-like SH-SY5Y cells stably expressing D3R and ßarr2 knockdown (ßarr2KD) to delineate the roles of Gi/o and ßarr2 on phosphorylation patterns of ERK1/2 induced by D3R agonists. Results indicate GPB and UB agonists promote similar early and late phase mono activation patterns of ERK1/2. On the contrary, GPB and UB agonists promote either early or early and late phase dual activation of ERK1/2, respectively. The early phase dual activation of ERK1/2 is predominantly promoted by Gi/o while the late phase dual activation by ßarr2 recruitment. PKC plays a significant role in both the early and late phase dual phosphorylation of ERK1/2. ßarr2KD significantly increased short- and long-term dual phosphorylation levels of ERK1/2 induced by GPB agonists which was inhibited by a combination of Gi/o and PKC inhibitors. Interestingly, ßarr2KD significantly reduced the short and long-term dual phosphorylation of ERK1/2 by UB agonists. Overall, this study highlights that biased signaling agonists of D3R have differential effects on ERK1/2 which may be advantageous to develop better drugs.

Remifentanil preconditioning promotes liver regeneration via upregulation of ß-arrestin 2/ERK/cyclin D1 pathway.

Remifentanil is a potent, short-acting opioid analgesic drug that can protect tissues from ischemia and reperfusion injury though anti-inflammatory effects. However, the utility of remifentanil in liver regeneration after hepatectomy is not known. Using a 70% hepatectomy mouse model (PHx), we found that preconditioning animals with 4 µg/kg remifentanil enhanced liver regeneration through supporting hepatocyte proliferation but not through anti-inflammatory effects. These effects were also phenocopied in vitro where 40 mM remifentanil promoted the proliferation of primary mouse hepatocyte cultures. We further identified that remifentanil treatment increased the expression of ß-arrestin 2 in vivo and in vitro. Demonstrating specificity, remifentanil preconditioning failed to promote liver regeneration in liver-specific ß-arrestin 2 knockout (CKO) mice subjected to PHx. While remifentanil increased the expression of activated (phosphorylated)-ERK and cyclin D1 in PHx livers, their levels were not significantly changed in remifentanil-treated CKO mice nor in WT mice pretreated with the ERK inhibitor U0126. Our findings suggest that remifentanil promotes liver regeneration via upregulation of a ß-arrestin 2/ERK/cyclin D1 axis, with implications for improving regeneration process after hepatectomy.

Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on AKT Phosphorylation and Cell Proliferation in Pituitary Tumors.

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for prolactin-secreting pituitary tumors but are poorly effective in nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a ß-arrestin 2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test ß-arrestin 2 involvement. We found that the DRD2 agonist BIM53097 induced a reduction of the p-AKT/total-AKT ratio in MMQ (-32.8 ± 17.6%, p < 0.001 vs. basal) and in a subset (n = 15/41, 36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase in p-AKT. The ability of BIM53097 to reduce p-AKT correlated with its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097, and nearly all subgroup 2 NF-PitNETs were resistant. ß-Arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, ß-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, ß-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that ß-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of ß-arrestin 2 as a biomarker predicting NF-PitNETs' responsiveness to treatment with dopamine agonists.

Engineering Quick- and Long-acting Naloxone Delivery Systems for Treating Opioid Overdose.

PURPOSE: Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick- and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. METHODS: The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 µm and 50 µm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with ß-arrestin recruitment assays. RESULTS: The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 µm to 23 µm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 µm to 23 µm helped eliminate the lag phase and obtain the steady-state drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a µ-opioid receptor, to recruit ß-arrestin 2. CONCLUSIONS: Naloxone released from the three selected formulations could inhibit DAMGO-induced ß-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration.

ß-Arrestin 2 mediates arginine vasopressin-induced IL-6 induction via the ERK1/2-NF-κB signal pathway in murine hearts.

Evidence to date suggests that ß-arrestins act beyond their role as adapter proteins. Arginine vasopressin (AVP) may be a factor in inflammation and fibrosis in the pathogenesis of heart failure. In the present study we investigated the effect of AVP on inflammatory cytokine IL-6 production in murine hearts and the impact of ß-arrestin 2-dependent signaling on AVP-induced IL-6 production. We found that administration of AVP (0.5 U/kg, iv) markedly increased the levels of IL-6 mRNA in rat hearts with the maximum level occurred at 6 h. In ß-arrestin 2 KO mouse hearts, deletion of ß-arrestin 2 decreased AVP-induced IL-6 mRNA expression. We then performed in vitro experiments in adult rat cardiac fibroblasts (ARCFs). We found that AVP (10-9-10-6 M) dose-dependently increased the expression of IL-6 mRNA and protein, activation of NF-κB signaling and ERK1/2 phosphorylation, whereas knockdown of ß-arrestin 2 blocked AVP-induced IL-6 increase, NF-κB activation and ERK1/2 phosphorylation. Pharmacological blockade of ERK1/2 using PD98059 diminished AVP-induced NF-κB activation and IL-6 production. The selective V1A receptor antagonist SR49059 effectively blocked AVP-induced NF-κB phosphorylation and activation as well as IL-6 expression in ARCFs. In AVP-treated mice, pre-injection of SR49059 (2 mg/kg, iv) abolished AVP-induced NF-κB activation and IL-6 production in hearts. The above results suggest that AVP induces IL-6 induction in murine hearts via the V1A receptor-mediated ß-arrestin2/ERK1/2/NF-κB pathway, thus reveal a novel mechanism of myocardial inflammation in heart failure involving the V1A/ß-arrestin 2/ERK1/2/NF-κB signaling pathway.

ß-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane.

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane ß-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of ß-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that ß-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that ß-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and ß-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

ß-arrestin 2 negatively regulates NOD2 signalling pathway through association with TRAF6 in microglia after cerebral ischaemia/reperfusion injury.

We previously reported that nucleotide-binding oligomerization domain-containing protein (NOD) 2 was involved in the inflammatory responses to cerebral ischaemia/reperfusion (I/R) insult. However, the mechanism by which NOD2 participates in brain ischaemic injury and the regulation of NOD2 in the process are still obscure. Increased ß-arrestin 2 (ARRB2) expression was observed in microglia following cerebral I/R in wild-type mice besides the up-regulation of NOD2 and TRAF6. Stimulation of NOD2 by muramyl dipeptide (MDP) in BV2 cells induced the activation of NF-κB by the phosphorylation of p65 subunit and the degradation of IκBα. Meanwhile, the protein level of Cyclooxygenase-2 (COX-2), the protein expression and activity of MMP-9 were significantly increased in BV2 cells after administration of MDP. Furthermore, overexpression of ARRB2 significantly suppressed the inflammation induced by MDP, silence of ARRB2 significantly enhanced the inflammation induced by MDP in BV2 cells. In addition, we observed endogenous interaction of TRAF6 and ARRB2 after stimulation of MDP or cerebral I/R insult, indicating ARRB2 negatively regulates NOD2-triggered inflammatory signalling pathway by associating with TRAF6 in microglia after cerebral I/R injury. Finally, the in vivo study clearly confirmed that ARRB2 negatively regulated NOD2-induced inflammatory response, as ARRB2 deficiency exacerbated stroke outcomes and aggravated the NF-κB signalling pathway induced by NOD2 stimulation after cerebral I/R injury. These findings revealed ARRB2 negatively regulated NOD2 signalling pathway through the association with TRAF6 in cerebral I/R injury.

Biased signaling agonist of dopamine D3 receptor induces receptor internalization independent of ß-arrestin recruitment.

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration - which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while ß-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608-promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.

ß-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting Akt Protein.

Recent studies reveal that multifunctional protein ß-arrestin 2 (Arrb2) modulates cell apoptosis. Survival and various aspects of liver injury were investigated in WT and Arrb2 KO mice after bile duct ligation (BDL). We found that deficiency of Arrb2 enhances survival and attenuates hepatic injury and fibrosis. Following BDL, Arrb2-deficient mice as compared with WT controls displayed a significant reduction of hepatocyte apoptosis as demonstrated by the TUNEL assay. Following BDL, the levels of phospho-Akt and phospho-glycogen synthase kinase 3ß (GSK3ß) in the livers were significantly increased in Arrb2 KO compared with WT mice, although p-p38 increased in WT but not in Arrb2-deficient mice. Inhibition of GSK3ß following BDL decreases hepatic apoptosis and decreased p-p38 in WT mice but not in Arrb2 KO mice. Activation of Fas receptor with Jo2 reduces phospho-Akt and increases apoptosis in WT cells and WT mice but not in Arrb2-deficient cells and Arrb2-deficient mice. Consistent with direct interaction of Arrb2 with and regulating Akt phosphorylation, the expression of a full-length or N terminus but not the C terminus of Arrb2 reduces Akt phosphorylation and coimmunoprecipates with Akt. These results reveal that the protective effect of deficiency of Arrb2 is due to loss of negative regulation of Akt due to BDL and decreased downstream GSK3ß and p38 MAPK signaling pathways.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development.

The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in ß-arrestin knockout mice suggest that interaction of certain GPCRs, including µ-, δ-, κ-opioid and hCB1Rs, with ß-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to ß-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no ß-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to ß-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced ß-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to similar treatment with CP-55,940. PNR-4-20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non-biased full agoinst JWH-018 or Δ9-THC. Finally, studies of a structurally similar analog PNR- 4-02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to ß-arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically.

GRK2/ß-arrestin mediates arginine vasopressin-induced cardiac fibroblast proliferation.

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl-tetrazolium assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α-smooth muscle actin (α-SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK1/2 were determined by western blotting. AVP, in a time- and concentration-dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein-coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2-Ct or knock-down of GRK2 suppressed AVP-induced BrdU incorporation and the expression of MMP2 and α-SMA in NRCFs. Moreover, shRNA-mediated silencing of ß-arrestin1 or ß-arrestin 2 abolished AVP-induced BrdU incorporation and MMP2 expression. AVP-induced NRCF proliferation depended on the phosphorylation of ERK1/2 , and inhibition of GRK2 or silencing of ß-arrestins blocked AVP-induced ERK1/2 phosphorylation. The effects of AVP on NRCF proliferation and α-SMA expression were blocked by SR45059, a vasopressin receptor type1A (V1A R) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V1A R-mediated GRK2/ß-arrestin/ERK1/2 signalling.

Sevoflurane prevents stroke-induced depressive and anxiety behaviors by promoting cannabinoid receptor subtype I-dependent interaction between ß-arrestin 2 and extracellular signal-regulated kinases 1/2 in the rat hippocampus.

One of the most frequent psychological consequences of stroke is depression. Previous animal studies have demonstrated that post-conditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury. Thus, we hypothesized that repeated exposure to sevoflurane after transient ischemia can prevent the development of depressive-like behavior. To test this hypothesis, we induced transient cerebral ischemia via transient occlusion of bilateral common carotid arteries and examined the effects of subsequent repeated exposure to sevoflurane on sucrose preference, locomotor activity, and rearing activity in rats. To explore the putative neurobiological mechanisms, we further investigated the roles of hippocampal CB1 receptor in the behavioral effects of sevoflurane. We found that repeated sevoflurane exposures reversed ischemia-induced depressive-like behaviors. Furthermore, CB1 receptor inhibition in the dorsal hippocampus (DH) abolished the effects of sevoflurane exposures on ischemia-induced depressive-like behaviors. In addition, repeated sevoflurane exposures increased CB1 receptor expression and endocannabinoids levels in the DH of ischemic rats. Moreover, repeated sevoflurane exposures enhanced the expression of ß-arrestin 2, increased the activation of extracellular signal-regulated kinases (ERK)1/2, and promoted the interaction of ß-arrestin 2 and ERK1/2 in the DH, and such effects were reversed by CB1 receptor antagonism in the DH. Finally, ß-arrestin 2 expression and ERK1/2 activation in the DH were critical for the preventative effects of sevoflurane exposures on ischemia-induced depressive-like behaviors. Taken together, our results suggested that sevoflurane exposure after brain ischemia may prevent the development of depression, and such preventative effects of sevoflurane are likely ascribed to the activation of CB1 receptor-mediated ß-arrestin 2-ERK1/2 signaling pathways. We propose that the following mechanisms are critical for the preventative effects of sevoflurane against post-stroke depressive and anxiety behaviors: repeated sevoflurane exposure after transient brain ischemia enhances N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) levels and normalize cannabinoid receptor type 1 (CB1) receptor expression in the dorsal hippocampus, which results in enhanced interaction of ß-arrestin 2 and extracellular signal-regulated kinases (ERK1/2) and increased ERK1/2 activation, leading to decreased depressive and anxiety behaviors. We think these findings should provide a new strategy for treatment of post-stroke depression.

ß-Arrestin 2 negatively regulates Toll-like receptor 4 (TLR4)-triggered inflammatory signaling via targeting p38 MAPK and interleukin 10.

The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that ß-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. In vitro studies with cells including peritoneal macrophages and HEK293/TLR4 cells have demonstrated that ß-arrestin 2 forms complexes with p38 and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of ß-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking ß-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which ß-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.

Myeloid beta-arrestin 2 depletion attenuates metabolic dysfunction-associated steatohepatitis via the metabolic reprogramming of macrophages.

Macrophage-mediated inflammation has been implicated in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH); however, the immunometabolic program underlying the regulation of macrophage activation remains unclear. Beta-arrestin 2, a multifunctional adaptor protein, is highly expressed in bone marrow tissues and macrophages and is involved in metabolism disorders. Here, we observed that ß-arrestin 2 expression was significantly increased in the liver macrophages and circulating monocytes of patients with MASH compared with healthy controls and positively correlated with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Global or myeloid Arrb2 deficiency prevented the development of MASH in mice. Further study showed that ß-arrestin 2 acted as an adaptor protein and promoted ubiquitination of immune responsive gene 1 (IRG1) to prevent increased itaconate production in macrophages, which resulted in enhanced succinate dehydrogenase activity, thereby promoting the release of mitochondrial reactive oxygen species and M1 polarization. Myeloid ß-arrestin 2 depletion may be a potential approach for MASH.

Loss of ß-arrestin 2 exacerbates experimental autoimmune encephalomyelitis with reduced number of Foxp3+ CD4+ regulatory T cells.

ß-Arrestins are well-known regulators and mediators of G protein-coupled receptor signalling, and accumulating evidence reveals that they are functionally involved in inflammation and autoimmune diseases. Of the two ß-arrestins, ß-arrestin 1 is documented to play regulatory roles in an animal model of multiple sclerosis (MS), whereas the role of ß-arrestin 2 is less clear. Here, we show that ß-arrestin 2-deficient mice displayed the exacerbated and sustained symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. At the cellular level, deficiency of ß-arrestin 2 led to a decreased number of Foxp3(+) CD4(+) regulatory T (Treg) cells in peripheral lymphoid organs of EAE mice. Consistently, our in vitro observations also revealed that loss of ß-arrestin 2 impaired the conversion of Foxp3(-) CD4(+) T cells into Foxp3(+) CD4(+) inducible Treg cells. Taken together, our data suggest that ß-arrestin 2 plays a regulatory role in MS, that is opposite to that of ß-arrestin 1, in autoimmune diseases such as MS, which is at least partially through regulation of iTreg cell differentiation.