β-arrestin2: an emerging player and potential therapeutic target in inflammatory immune diseases

Abstract

β-arrestin2, a pivotal protein within the arrestin family, is localized in the cytoplasm, plasma membrane and nucleus, and regulates G protein-coupled receptors (GPCRs) signaling. Recent evidence shows that β-arrestin2 plays a dual role in regulating GPCRs by mediating desensitization and internalization, and by acting as a scaffold for the internalization, kinase activation, and the modulation of various signaling pathways, including NF-κB, MAPK, and TGF-β pathways of non-GPCRs. Earlier studies have identified that β-arrestin2 is essential in regulating immune cell infiltration, inflammatory factor release, and inflammatory cell proliferation. Evidently, β-arrestin2 is integral to the pathological mechanisms of inflammatory immune diseases, such as inflammatory bowel disease, sepsis, asthma, rheumatoid arthritis, organ fibrosis, and tumors. Research on the modulation of β-arrestin2 offers a promising strategy for the development of pharmaceuticals targeting inflammatory immune diseases. This review meticulously describes the roles of β-arrestin2 in cells associated with inflammatory immune responses and explores its pathological relevance in various inflammatory immune diseases.

Introduction

β-arrestin2 is recognized as an essential component of the arrestin family, initially studied for its role in regulating the activity of β-adrenergic receptors (β-ARs). Ubiquitously expressed, β-arrestin2 is found in the cytoplasm, nucleus, and plasma membrane [1]. This pivotal protein can bind and regulate the activity of hundreds of G protein-coupled receptors (GPCRs), thus participating in numerous critical physiological functions [2]. The canonical role of β-arrestin2 involves the desensitization and internalization of GPCRs, accomplished by disrupting the combination of GPCRs to specific G proteins and promoting clathrin recruitment to form clathrin-coated vesicles [3]. Noncanonical functions of β-arrestin2 include acting as signal transducers, primarily as adaptor proteins for various intracellular signaling proteins or cascades. These actions depend on β-arrestin2 recruitment by activated GPCRs. Nevertheless, evidence suggests that β-arrestin2 can also regulate specific cellular functions independently of GPCRs, adding complexity to the study of its molecular mechanisms [4]. As a multifunctional scaffold, β-arrestin2 interacts with proteins including GPCRs, other cell membrane receptors, ion channels, transporters, and protein kinases. By binding to the intracellular domains of receptors, it modulates receptor signaling and regulates multiple signaling pathways [2]. Consequently, β-arrestin2 is widely involved in regulating physiological activities, such as cell invasion, migration [5], and anti-inflammation [6].

The inflammatory immune response (IIR) is a physiological or excessive systemic reaction triggered by IIR-associated cells in response to changes in internal and external environments [7]. A physiological IIR protects the body against pathological harm caused by such changes. However, an excessive IIR leads to cellular, molecular, organ, and systemic imbalances, forming the pathological basis for various inflammatory immune diseases [8]. These diseases, characterized by common inflammatory mechanisms and immune disorders, cause damage to multiple organs and systems. Despite this, the pathogenesis of some inflammatory immune diseases remains unclear [9]. Upon the occurrence of these diseases, IIR-associated cells respond rapidly. Various cytokines interact with corresponding receptors, affecting cellular functions through different or similar signaling pathways and manifesting disease features that can be non-specific or specific, congenital or acquired, acute or chronic [10].

Numerous studies have indicated that β-arrestin2 is extensively expressed in various IIR-associated cells, particularly immune cells. Additionally, these studies have observed that in IIR-associated cells and tissues, the expression of β-arrestin2 fluctuates in response to different specific IIR stimuli, suggesting that β-arrestin2 is a key player in the IIR and contributes to the progression of inflammatory immune diseases [7] (Fig. 1). Therefore, this review consolidates the functions of β-arrestin2 in IIR-associated cells and highlights the growing consensus on the roles of β-arrestin2 in inflammatory immune diseases. Future research posits that targeted regulation of β-arrestin2 expression represents a promising strategy for treating inflammatory immune diseases in clinical settings.

Fig. 1. β-arrestin2 participates in inflammatory immune diseases caused by regulating the IIR-associated cells.

β-arrestin2 plays a key role in modulating the physiological processes of various IIR-associated immune cells, including macrophage, neutrophil, T cell, B cell, and NK cell, as well as non-immune cells such as fibroblast, epithelial cell, fibroblast-like synoviocyte, hepatocyte, and endothelial cells. The regulation of β-arrestin2 significantly influences the progression of inflammatory immune diseases.

Distribution, structure, and function of β-arrestin2

In 1992, Attramadal et al. successfully cloned β-arrestin2 from a rat brain cDNA library. β-arrestin2 is a type of nonvisual structural protein, abundantly expressed in the heart and vascular system [11]. It plays a key role in desensitizing GPCRs and is primarily localized in the cytoplasm, plasma membrane, and nucleus.

Structurally, the primary components of β-arrestin2 are two antiparallel β-sheets, linked by a hinge and a short α-helix located at the back of the amino-terminal. Although the hinge does not engage in molecular interactions, it allows the β-sheets to move freely, contributing to the active conformation of β-arrestin2 [12]. The two β-sheets are symmetrically extended N-domain and C-domain, each consisting of 7-stranded beta sandwiches which have distinct binding sites for kinases [13]. The adjacent arrangement of the domains forms the central crest, which includes the finger loop—an essential receptor-binding element and a GPCR binding site—along with the middle loop and C-loop, which act as stabilizing components in maintaining the inactive basal state of β-arrestin2 [14]. Between the N-domain and C-domain, a polar core known as a phosphate sensor is present, and the phosphorylated C-tail of GPCRs interacts with basic residues in this region through a salt bridge. The maintenance of the inactivated state of β-arrestin2 depends on two types of interactions [15]: one is three-element interaction, mainly hydrophobic interactions between the C-tail, the N-terminal domain and the amino acids in the α-helix structure; the other is ionic interaction of the polar core, which consists of interactions between the N-terminal domain, the portal loop, and the charged amino acid residues distal to the C-tail. Together, these interactions stabilize β-arrestin2 by tightly linking the terminal structural domains and preventing the exposure of the N-terminal domain groove that binds to GPCRs. Once these stabilizing factors are disrupted, β-arrestin2 transitions from an inactive to a preactivated conformation [12]. In its preactivated state, β-arrestin2 is more likely to interact with the receptor [16, 17] (Fig. 2).

Fig. 2. The structure of β-arrestin2 (created by SWISS MODEL).

The structure of β-arrestin2 consists of two antiparallel β-sheets, the N-domain and C-domain, each with distinct binding sites for kinases. The central crest, stabilized by three loops (finger loop, middle loop, and C-loop) and a polar core, contributes to the overall structural stability of β-arrestin2.

The canonical function of β-arrestin2 is to terminate the conduction of GPCRs, exerting desensitizing and internalizing effects. GPCRs represent the largest group of cell surface receptors in the human genome, playing a vital role in regulating extensive physiological and pathophysiological processes with significant therapeutic implications [18]. Upon ligand binding to GPCRs, heterotrimeric G proteins are activated, leading to the transient stimulation of second messengers, including cyclic adenosine monophosphate (cAMP), MAPK, and intracellular calcium (Ca²⁺), which trigger intracellular signals [19]. β-arrestin2 was initially identified as an “arresting” protein in β-AR signaling, which desensitizes agonist-induced signaling. Upon agonist binding to GPCRs, the receptors undergo phosphorylation by GPCR kinases [20]. This phosphorylation facilitates the recruitment of β-arrestin2 to GPCRs, where it functions as a scaffolding protein. Subsequently, β2-adaptin and clathrin are recruited to the β-arrestin2 complex at the plasma membrane, initiating inward endocytosis. This internalization of GPCRs represents a common mechanism for receptor desensitization across most GPCRs [21]. Additionally, β-arrestin2 functions as an adaptor protein for E3 ubiquitin ligase, facilitating the ubiquitination (Ub) and degradation of GPCRs [22].

Beyond its roles in the GPCR life cycle, the noncanonical function of β-arrestin2 involves acting as a scaffolding protein for numerous signaling pathways, both dependent on and independent of GPCRs [6]. β-arrestin2 serves as a scaffold regulating GPCRs activation of MAPKs, including ERK, JNK [23], and p38 kinases [24]. Recent studies have demonstrated that β-arrestin2 can initiate signaling cascades independently of G protein activation and provide scaffolding for various intracellular signaling networks through pathways such as Wnt, Notch, transforming growth factor-beta (TGF-β), and Hedgehog [25], as well as downstream kinases like Src protein tyrosine kinase, protein kinase B (PKB/Akt), and MAPK [26] (Fig. 3).

Fig. 3. Multiple functions of β-arrestin2.

β-arrestin2 mediates GPCR endocytosis and desensitization, receptor and membrane protein ubiquitylation, and signal transduction. β-arrestin2 regulates signaling pathways both in the presence and absence of G proteins and is involved in RTK (receptor tyrosine kinase), TβRIII (type III TGF-β receptor), Wnt and TLR-IL-1R (toll-like receptor-interleukin 1 receptor) pathways in various ways.

Effects of β-arrestin2 in IIR-associated cells

β-arrestin2 plays a pivotal role in various functions of IIR-associated cells, including immune and non-immune cells, by managing GPCR signals and mediating distinct downstream signals [17]. Beyond pathogen clearance, different types of IIR-associated cells are involved in tissue injury, inflammation, and repair processes [27]. In recent years, β-arrestin2 expression has been detected in numerous IIR-associated cells. Notably, β-arrestin2 regulates the functions of these cells by influencing their infiltration, proliferation, and release of inflammatory mediators [28]. A comprehensive understanding of β-arrestin2’s impact on IIR-associated cells aids in elucidating the pathogenesis of inflammatory immune diseases and identifying potential therapeutic targets.

IIR-associated immune cells

Defense by the innate immune cells, such as macrophages, neutrophils and natural killer (NK) cells, involves chemotaxis, cytokine release, pathogen clearance, and activation of the adaptive immune system through antigen presentation [29, 30]. Additionally, T lymphocytes and B lymphocytes, which are part of the adaptive immune system, aim to eliminate pathogens further. Increasing research indicates that β-arrestin2 is vital for modulating immune cell responses to cytokines and maintaining functional balance among immune cells [7] (Fig. 4).

Fig. 4. The roles of β-arrestin2 in IIR-associated immune cell types.

A schematic representation of signaling molecules and pathways related to the function of IIR-associated immune cells that interact with β-arrestin2.

Macrophages

Macrophages, as essential components of the human immune system, execute multiple functions, including bacterial phagocytosis, cytokine production, antigen presentation to naive T cells, and activation of the adaptive immune system. Phenotypically, macrophages are classified into type I (M1) and type II (M2) macrophages [31]. An imbalance between M1 and M2 macrophages can lead to pathological consequences and contribute to various inflammatory immune diseases. For instance, M1 macrophage activation in an inflammatory environment promotes the extensive production of pro-inflammatory cytokines and chemotactic factors, resulting in osteoclastogenesis, erosion, and progressive joint destruction. Conversely, M2 macrophage activation releases growth factors and cytokines such as IL-4 and IL-10, facilitating anti-inflammatory processes and clinical remission in rheumatoid arthritis (RA) [32]. Furthermore, macrophages are closely linked to several inflammatory immune diseases, including inflammatory bowel disease (IBD) [33], tumors, sepsis, and asthma [34].

β-arrestin2 influences the polarization of macrophages into M1 or M2 under various conditions. A previous study has shown that β-arrestin2 promotes RAW264.7 macrophage toward M2 polarization by inhibiting the LPS/NF-κB signaling pathway and reinforcing the IL-4/STAT6 signaling pathway [35]. Toll-like receptor 4 (TLR4) acts as a sensor, triggering an innate immune response in macrophages. Gαi and Gαs are key G proteins interacting with TLR4. Research has demonstrated that Gαi1/3 significantly contributes to M1 macrophage effector functions [36]. However, β-arrestin2 generally interacts with Gαi in peritoneal macrophages (PMs) to promote M1 polarization [37]. These findings suggest that β-arrestin2 may have opposing roles in regulating macrophage polarization depending on the different macrophage type and signaling pathway.

Extensive research has established that β-arrestin2 is critical in regulating the NF-κB pathway and pro-inflammatory factor secretion in macrophages. In collagen-induced arthritis (CIA) mice, reduced β-arrestin2 enhances the level and binding affinity of IκBα to NF-κB [37]. This reduction leads to decreased transcription of NF-κB-mediated cytokines, which modulate the activities of various immune cells including macrophages. Previous studies have shown that β-arrestin2 overexpression significantly reduces levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), p-p65, p-IκBα, and interleukin-1β (IL-1β) in an inflammation model induced by LPS in macrophages. These results indicate that β-arrestin2 can mitigate LPS-induced inflammatory responses mediated by NF-κB signaling and protect LPS-stimulated macrophages by inhibiting NLRP3 inflammasome activation [38]. Chen et al. [39] also found that the absence of β-arrestin2 alleviates autoimmune hepatitis (AIH) by suppressing monocyte migration and differentiation, reducing macrophage infiltration into the liver, and consequently alleviating hepatocyte apoptosis induced by inflammatory cytokines from monocyte-derived macrophages.

β-arrestin2 plays a significant role in translocated intimin receptor (Tir)-mediated immune evasion, presenting a potential novel target for bacterial infectious diseases. β-arrestin2 interacts with immunoreceptor tyrosine-based inhibition motifs (ITIMs) phosphorylated Tir and promotes the recruitment of SHP-1. This suppresses K63-linked ubiquitination and activation of tumor necrosis factor receptor-associated factor 6 (TRAF6). Reduced K63-linked ubiquitination of TRAF6 limits TAK1 binding protein 2 (TAB2) binding, inhibiting the TRAF6-transforming growth factor β-activated kinase 1 (TAK1) interaction and TAK1 activation. Simultaneously, SHP-1 interacts with TAK1, preventing TAK1/TAB1 interaction and decreasing TAK1 phosphorylation and ubiquitination. Consequently, β-arrestin2 mediates Tir’s inhibition of host innate immunity, blocking downstream signal transduction and cytokine cytokines [40]. However, infection with Mycobacterium tuberculosis (M. tuberculosis) increases β-arrestin2 expression in human macrophages. Silencing β-arrestin2 notably boosts bactericidal activity by enhancing pro-inflammatory cytokine expression, such as TNF-α. β-arrestin2 suppresses TLR2/ERK1/2 pathway activation and its transcriptional regulation activity during M. tuberculosis infection [41].

β-arrestin2 acts as a positive modulator accurately controlling the antiviral immune response of PMs by regulating the activation of cGMP-AMP synthase and interferon β production. Furthermore, β-arrestin2 deficiency reduces of PMs and disrupts the antiviral immune response in vivo [42]. In adenovirus-induced inflammatory responses, β-arrestin2 negatively modulates chemokine and cytokine production and the transcription of pro-inflammatory genes in PMs [43]. Conversely, β-arrestin2 exacerbates polymicrobial sepsis by acetylating high mobility group box-1 [44]. Therefore, β-arrestin2 regulates various macrophage functions, including cytokine secretion, macrophage polarization, and bacterial phagocytosis. Given the critical role of macrophages in regulating the occurrence and development of inflammatory immune diseases, elucidating the regulatory effects of β-arrestin2 on macrophages is of great significance.

Neutrophils

Neutrophils, the most abundant immune cells in the body, are a type of polymorphonuclear leukocyte. These cells are integral to various inflammatory immune diseases through different mechanisms. For instance, inhibiting the formation of neutrophil extracellular traps (NETs) could attenuate inflammatory signals in colitis mice induced by DSS [45]. And neutrophil infiltration in the intestine is a hallmark of active IBD [46]. Additionally, neutrophils are the first responders in innate immunity, possessing well-defined bactericidal functions against invading pathogens, including phagocytosis, immune mediator generation, granule exocytosis, and NETs formation [47].

When bacteria invade, neutrophils are mobilized to the infection site, playing a vital role in the initial defense by engaging in phagocytosis and bacterial killing. Neutrophils isolated from β-arrestin2 knockout (Arrb2^−/−) mice produced more IL-6 after LPS stimulation compared to wild-type (WT) cells. The deficiency of β-arrestin2 resulted in heightened levels of intercellular cell adhesion molecule-1 (ICAM-1) and L-selectin [48]. These observations suggest that β-arrestin2 negatively regulates the generation of neutrophil cytokines and adhesion receptors during the inflammatory response.

β-arrestin2 also significantly manages neutrophil chemotaxis [49]. In a model of zymosan-elicited peritoneal neutrophils from WT and Arrb2^−/− mice, chemokine receptor 2 (CXCR2)-mediated recruitment of neutrophils to inflammation sites was increased in Arrb2^−/− mice compared to WT mice. Su et al. [50] found that the β-arrestin2 deficiency led to reduced CXCR2 internalization compared to WT mice. These results indicate that β-arrestin2 exerts a negative regulatory influence on CXCR2-mediated neutrophil functions.

In addition to regulating neutrophil chemotaxis to specific chemokines, β-arrestin2 influences chemokine-induced granule exocytosis. Interleukin-8 (IL-8) triggers the formation of a β-arrestin2-hematopoietic cell kinase complex by stimulating the chemokine receptor CXCR1. This complex is then transported to granule-rich areas of granulocytes, leading to granule release. Conversely, this phenomenon is absent in β-arrestin2-deficient cells [51]. These investigations highlight that β-arrestin2 exerts a suppressive influence on granule release, cellular chemotaxis, and cytokine production in neutrophils during inflammatory responses.

T cells

An imbalance resulting from aberrant T cell activation, proliferation, migration, and apoptosis contributes to inflammatory immune diseases. Numerous studies have demonstrated that β-arrestin2 regulates T cell function and influences inflammatory immune diseases such as IBD [52] and asthma [53]. For instance, β-arrestin2 protects mice from DSS-induced colitis by inhibiting T cell activation [52].

CD4⁺ and CD8⁺ T cells are essential in adaptive immunity, and their dysregulation is implicated in inflammation and immune disorders. In purified CD4⁺ T cells from a mouse model of asthma, β-arrestin2 protein expression was significantly elevated compared to WT mice. Moreover, silencing β-arrestin2 in these CD4⁺ T cells downregulated ERK1/2 activation and interleukin-17 (IL-17) expression [54]. Similarly, CD4⁺ T cells isolated from allergic asthma mice, exhibited increased β-arrestin2 expression at both gene and protein levels. Deletion of β-arrestin2 in these cells partially reduced IL-4 and GATA3 expression through the β2-adrenoreceptor (β2AR) signaling pathway [53]. Studies have also demonstrated that β-arrestin2 influences the migration and proportion of CD8⁺ T cells. In a murine model of allergic contact hypersensitivity (CHS), a synthetic β-arrestin-biased CXCR3 agonist, VUF10661, enhances inflammation by increasing the recruitment of CD8⁺ T cell in a β-arrestin2-dependent manner [55, 56]. Under both homeostatic and colitis condition, Arrb2^−/− mice exhibited higher numbers of CD4⁺ and CD8⁺ T cells in the colonic lamina propria (cLP) compared to WT mice. Additionally, the cLP of Arrb2^−/− mice contained a significantly higher proportion of effector T cells (CD4⁺ and CD8⁺) compared to WT mice [52].

β-arrestin2 has also been found to mediate T cell apoptosis. The envelope protein gp120 is one of the most consistently studied HIV-1 viral proteins. The expression of β-arrestin2 was reduced in Jurkat cells (a type of human T lymphocyte cell line) when co-treated with gp120/anti-gp120 and morphine compared to treatment with gp120/anti-gp120 or morphine alone. Overexpression of β-arrestin2 in Jurkat cells inhibited gp120/morphine-mediated T cell apoptosis. Conversely, silencing β-arrestin2 increased T cell apoptosis under combined treatment [57]. These results suggest a novel role for β-arrestin2 signaling in T cell apoptosis.

β-arrestin2 is essential for T cell migration in vitro. In allergic asthma, macrophage-derived chemokine (MDC) acts as a potent chemoattractant for Th2 cells. While CD4⁺ T cells from the lungs of OVA-treated WT mice migrated effectively toward MDC, those lacking β-arrestin2 showed significantly impaired migration. MDC levels in lavage fluid were significantly elevated in OVA-treated WT mice, but not in similarly treated Arrb2^−/− mice. These data indicate that β-arrestin2 positively regulates MDC-mediated CD4⁺ T cell migration [58]. In another study, Fong et al. [59] examined the chemotactic responses of T cells from β-arrestin2-deficient mice to stromal cell-derived factor 1 (CXCL12) gradients. And the results have shown that T cells isolated from Arrb2^−/− mice were markedly impaired in their response to CXCL12. These observations suggest a positive regulatory role for β-arrestin2 in CXCL12-induced chemotaxis. Additionally, knockdown of β-arrestin2 inhibits chemokine-induced chemotaxis in primary Th2 cells by inhibiting p38 MAPK pathway activation [60]. And recent research found that β-arrestin2 promoted Jurkat cells migration by activating Akt signaling pathway.

Studies have shown that β-arrestin2 deficiency may exert a function in the proportion and activation of T cells [52, 61]. Sun et al. [61] found that deletion of β-arrestin2 decreased the Th17/Treg ratio and activated T cells, while increasing the number of naïve T cells in the spleens of fibrotic mice. In the current study, β-arrestin2 deficiency reduced the activated T cells in the liver and spleen of AIH mice [39]. However, in a DSS-induced colitis model, β-arrestin2 deficiency altered CD4⁺ and CD8⁺ T cell subsets, promoting T cell differentiation upon T cell receptor stimulation and resulting in higher quantities and percentages of activated T cells within the cLP [52]. Tao et al. [27] demonstrated that in immunized mice, β-arrestin2 deficiency led to increased circulating and spleen Th17 cells due to hyperactive IL-6-STAT3 signaling. Conversely, another study revealed that Arrb2^−/− mice exhibited reduced T cell recruitment into allergic lung tissue [62]. Accumulatively, these data implicated a functional role for β-arrestin2 as a mediator in the proportion and activation of T cells during multiple inflammatory conditions. The different functions of β-arrestin2 in regulating T cells activation might rely heavily on the inflammatory immune microenvironment in different inflammatory disease models.

These studies underscore the complex roles of β-arrestin2 in regulating T cell activation, differentiation, migration, and apoptosis. Based on these findings, we have reason to believe that β-arrestin2 may have important roles in regulating various functions of T cells. Further understanding between β-arrestin2 and T cell might facilitate the identification of potential therapeutic targets in inflammatory immune diseases driven by T cells.

B cells

As key components of the immune system, B cells secrete antibodies that drive the development and progression of inflammatory immune diseases [63]. Zhou et al. [3] observed that Arrb2^−/− mice, compared to their WT counterparts, exhibited significantly more plasma cells and antibodies when exposed to LPS in isolated B lymphocytes in vitro and during a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-LPS challenge in vivo. The absence of β-arrestin2 upregulates TLR4 levels in B lymphocytes, thereby enhancing NF-κB activity and facilitating plasma cell formation in arthritis. Flow cytometry analysis revealed that Arrb2^−/− B lymphocytes generated more plasma cells and memory B cells than WT B lymphocytes upon LPS treatment. These outcomes suggest that β-arrestin2 deficiency may promote LPS-induced activation, differentiation, and antibody production in B lymphocytes. Despite this, research on the role of β-arrestin2 in B cell activity remains relatively limited.

NK cells

NK cells play a pivotal role in surveilling transformed and infected cells as part of the innate immune response [64]. They express activating receptors that identify and eradicate these aberrant cells, involving in the pathogenesis of various inflammatory diseases [65]. Moreover, NK cells modulate the survival and proliferation of T cells, thus influencing the regulation of inflammatory immune diseases such as colitis [66].

Yu et al. [67] demonstrated that the inhibitory receptor KIR2DL1 on NK cells is associated with β-arrestin2. This receptor is essential for recognizing major histocompatibility complex class I on target cells, protecting healthy cells. Upon ligand activation, β-arrestin2 facilitates the recruitment of tyrosine phosphatases SHP-1 and SHP-2 to KIR2DL1, thus enhancing signaling. Studies on Arrb2^−/− mice revealed that the absence of β-arrestin2 increased NK cell cytotoxicity, reducing susceptibility to murine cytomegalovirus infection. These effects were not observed in mice lacking NK cells compared to WT mice. Collectively, these results suggest that β-arrestin2 inhibits NK cell activation [67].

IIR-associated non-immune cells

IIR-associated non-immune cells, such as epithelial cells, endothelial cells, hepatocytes, fibroblasts, and fibroblast-like synoviocytes (FLS), play a key role in maintaining organ homeostasis and pathogen protection in inflammatory immune diseases [18]. Beyond regulating immune cell responses, β-arrestin2 also influences the function of these non-immune cells, such as modulating epithelial cell apoptosis, promoting lipid deposition in hepatocytes, and adjusting fibroblast proliferation (Fig. 5).

Fig. 5. The roles of β-arrestin2 in IIR-associated non-immune cell types.

Schematic representation of signaling molecules and pathways related to IIR-associated non-immune cell function that have been demonstrated to interact with β-arrestin2.

Epithelial cells

Epithelial cells, located in the superficial layers of the skin and cavities, maintain organ homeostasis and protect against pathogens [68]. β-arrestin2 has been shown to regulate the function of intestinal epithelial cells (IECs) and salivary gland epithelial cells.

Inflammatory cytokines secreted by IECs during inflammation modulate the accumulation and function of various immune cells [69]. And increased apoptosis of IECs may contribute to the development of intestinal cancer and IBD [70]. In inflammatory conditions, β-arrestin2 recruitment by G protein-coupled receptor kinase 2 (GRK2) to the membrane is essential for NF-κB phosphorylation and ERK1/2 activation [71]. Jiang et al. [72] found that IECs with TNF-α stimulation increased the translocation of the GRK2-β-arrestin2 complex across the membrane while reducing the cytoplasmic binding of GRK2 and β-arrestin2 to ERK1/2, resulting in elevated ERK1/2 and NF-κB activation. In Caco-2 cells, β-arrestin2 bound to GPR120 (free fatty acid receptor 4, FFAR4), attenuating NF-κB activation, an effect negated by silencing β-arrestin2 [73]. These findings underscore the importance of the NF-κB pathway in β-arrestin2-mediated anti-inflammatory responses in IECs. Furthermore, β-arrestin2 is implicated in IEC apoptosis. Fu et al. [74] demonstrated that LPS-treated IECs exhibited increased β-arrestin2 expression. Overexpression of β-arrestin2 enhanced apoptosis of IECs treated by LPS, while silencing β-arrestin2 reduced apoptosis of IECs. Further experiments revealed that β-arrestin2 facilitated endoplasmic reticulum stress (ERS)-induced apoptosis by binding to binding immunoglobulin protein (BiP), promoting its polyubiquitination and degradation, thereby releasing the pro-apoptotic molecule BIK from BiP.

Similarly, β-arrestin2 promotes apoptosis of salivary gland epithelial cells under interferon-α (IFN-α) stimulation. Huang et al. [75] showed that IFN-α significantly increased β-arrestin2 expression in human salivary gland epithelial cells. Furthermore, inhibition of β-arrestin2 protein can significantly reduce the apoptosis of salivary gland epithelial cells induced by IFN-α, suggesting that β-arrestin2 may facilitate inflammation-induced apoptosis in salivary gland epithelial cells.

Endothelial cells

Endothelial cells are distributed throughout the liver, brain, blood vessels, lungs, spleen, and other tissues and organs. Necrotic endothelial cells could activate neighboring normal endothelial cells, promoting them to release inflammatory cytokines and upregulate adhesion molecules, thus promoting the progression of inflammation [76]. Mice with endothelial cell-specific deletion of β-arrestin2 exhibit impaired retinal angiogenesis [77].

β-arrestin2 extensively colocalizes with endothelial cell nitric oxide (NO) synthase (eNOS) and VE-cadherin, which are exclusive to liver endothelial cells. Liu et al. [78] demonstrated that β-arrestin2 is an indispensable component of the G protein- coupled receptor kinase-interacting protein 1 (GIT1)/eNOS/NO pathway and is associated with the pathogenesis of sinusoidal portal hypertension. Overexpressing β-arrestin2 in injured or β-arrestin2-deficient sinusoidal endothelial cells (SECs) restored eNOS function by promoting eNOS complex formation and NO production. While overexpression of GIT1 in WT SECs significantly enhanced eNOS activation, introducing GIT1 into β-arrestin2-deficient SECs failed to restore eNOS activity, indicating that β-arrestin2 is essential for GIT1-mediated enhancement of eNOS activity. Further experiments revealed that the formation of the GIT1/eNOS complex mediated by β-arrestin2 depends on the activation of ERK1/2 and Src.

Hepatocytes

The liver is the body’s most important organ for metabolism and detoxification. Hepatocytes, the primary functional cells of the liver, comprise 70%–80% of the liver cell population. Persistent hepatocyte death in chronic liver disease induces inflammation, activates hepatic stellate cells (HSCs), and transforms them into myofibroblasts, eventually leading to liver cirrhosis and liver cancer [79].

β-arrestin2 regulates hepatocyte apoptosis in various liver disease models. Sun et al. [80] found that β-arrestin2 expression was significantly elevated in the livers of alcoholic liver disease mice. Ethanol stimulation significantly increased the apoptosis of hepatocyte compared to the control group, while β-arrestin2 deficiency significantly decreased the apoptosis of hepatocyte. Similarly, silencing β-arrestin2 in AML-12 cells significantly suppressed the ethanol-induced expression of apoptosis-related genes such as Bax and cleaved-caspase 3. Further studies revealed that β-arrestin2 promotes hepatocyte apoptosis by inhibiting the Akt signaling pathway. Studies showed that the expression of the full-length or N-terminus of β-arrestin2 reduced Akt phosphorylation and its co-immunoprecipitation with Akt, thereby increasing apoptosis in hepatocytes [81]. Thus, β-arrestin2 promotes hepatocyte apoptosis through multiple signaling pathways.

Additionally, Sun et al. [82] discovered that β-arrestin2 plays a role in lipid deposition in hepatocytes. They found that β-arrestin2 interacts with adenylate-activated protein kinase (AMPK) α1 to inhibit its phosphorylation, leading to the suppression of AMPK signaling and promoting lipid deposition in AML-12 cells.

Fibroblasts

Fibroblasts, the predominant cells in connective tissue, differentiate from mesenchymal cells. Inappropriate fibroblast activation can contribute to disease persistence by inducing pro-inflammatory and immunosuppressive properties in inflammatory immune diseases, such as organ fibrosis, IBD, RA, and tumors [83, 84]. Studies have revealed that β-arrestin2 plays a key role in regulating fibroblast biology, including myofibroblast transformation, collagen synthesis, and organ fibrosis [85].

Li et al. [86] found that β-arrestin2 levels in adult human cardiac fibroblasts (CFs) isolated from failing left ventricles were approximately twofold higher compared to those from normal ventricles. Knockdown of β-arrestin2 in failing CFs decreased α-SMA expression and collagen synthesis, suggesting that β-arrestin2 is essential for CF transformation into myofibroblasts and collagen synthesis. Further experiments showed that β-arrestin2 knockdown reduced TGF-β-induced collagen synthesis in failing CFs by suppressing ERK1/2 and Smad2/3 phosphorylation. In CFs, β-arrestin2 not only influences collagen synthesis but also promotes fibroblast proliferation. Chen et al. [87] demonstrated that β-arrestin2 also mediated ERK1/2 signaling, enhancing the proliferation of neonatal rat cardiac fibroblasts (NRCFs).

Beyond CFs, β-arrestin2 also plays a significant role in kidney and lung fibroblasts. Wang et al. [88] overexpressed β-arrestin2 in NRK-49F (rat kidney fibroblast cell line) and found increased levels of collagen I and fibronectin induced by SII ([1-sar, 4, 8-ile]-angiotensin II), an effect linked to ERK1/2 signaling. And in primary lung fibroblasts, β-arrestin2 deficiency resulted in a less invasive phenotype and altered expression of genes related to matrix synthesis, basement membrane degradation, and cell adhesion [89]. In summary, β-arrestin2 promotes collagen synthesis, myofibroblast transformation, and fibroblast proliferation, primarily through the ERK1/2 signaling pathway.

Fibroblast-like synoviocytes (FLS)

In inflamed joints, FLS act as primary effector cells, secreting numerous pro-inflammatory and matrix-degrading molecules. These molecules induce chemotaxis and activation of resident parenchymal cells and infiltrating immune cells, contributing to joint damage [90].

Li et al. [91] found that β-arrestin2 protein and mRNA levels were elevated in FLS isolated from CIA mice. Overexpressing β-arrestin2 in FLS significantly decreased hyaluronan (HA)-induced production of inflammatory factors like TNF-α and IL-6 compared to the control group, suggesting that β-arrestin2 exerts anti-inflammatory effects in experimental arthritis. When FLS were stimulated with TLR2/4 endogenous ligands (HA and TNF-α), β-arrestin2 expression was enhanced, which associated with the p38 MAPK pathway. Wu et al. [92] isolated and cultured human FLS, stimulating them with IL-1β, and found that both β-arrestin2 expression and FLS proliferation were increased, showing a positive correlation.

Table 1 summarizes the regulatory effects of β-arrestin2 on migration, apoptosis, proliferation, and cytokine secretion in various types of IIR-associated cells, suggesting that β-arrestin2 may play a role in the development of IIR-associated cell-mediated inflammatory immune diseases.

Table 1.

The alteration of β-arrestin2 expression and implication in IIR-associated cells.

Cell type	Model	β-arrestin2 expression	Cell function	Ref.
RAW264.7	LPS stimulation	↓	Promoting macrophage M1 polarization; activating NF-κB pathway and NLRP3 inflammasome	[35, 38]
THP-1	MCP-1 stimulation	↑	Promoting migration and differentiation of monocytes	[39]
	Mycobacterium tuberculosis infection	↑	Suppressing the bactericidal activity of macrophages	[41]
Primary macrophage	PMs isolated from CIA mice	↑	Facilitating macrophage M1 polarization	[37]
	PBMCs isolated from RA patients	↓	Promoting the expression of TNF-α and IL-6	[105]
CD4+ T cell	Isolated from asthma mice	↑	Mediating ERK1/2 activation; stimulating IL-17, IL-4, and GATA3 expression	[53, 54]
	Isolated from colitis mice	↓	Promoting T cell differentiation and activation	[52]
	Isolated from asthma mice	↑	Promoting MDC-mediated CD4+ T cell migration	[58]
Th17 cell	Isolated from CIA mice and induced from CD4+ T cell	↓	Increasing Th17 cell generation through IL-6-STAT3 signaling	[27]
Jurkat cell	gp120/morphine stimulation	↓	Promoting T cell apoptosis	[57]
B cell	Isolated from CAIA mice and stimulated by LPS	↑	Increasing plasma cell differentiation and antibody production by inhibiting TLR4 endocytosis and aggravating NF-κB signaling	[3]
Neutrophil	Zymosan stimulation	↑	Reducing CXCR2 signaling	[50]
Epithelial cell	TNF-α-induced Caco-2 cells	↑	Decreasing the binding with ERK1/2 and promoting the activation of ERK1/2 and NF-κB	[72]
	LPS-induced IEC-6 cells	↑	Promoting ERS-induced IECs apoptosis by inhibiting BiP	[74]
	IFN-α-induced HSGECs	↑	Encouraging epithelial apoptosis through GRP78-ATF6-CHOP signaling	[75]
Endothelial cell	Primary SECs	↓	Decreasing eNOS complex formation and NO production by interrupting GIT1/eNOS/NO signaling	[78]
Hepatocyte	Ethanol-induced AML-12 cells	↑	Promoting hepatocyte apoptosis via Akt suppression; inducing hepatic lipid accumulation via AMPK pathway	[80, 82]
Fibroblast	TGF-β-stimulated primary CFs	↑	Increasing ERK1/2 and Smad2/3 phosphorylation and enhancing collagen synthesis	[86]
	AVP-stimulated primary NRCFs	↑	Promoting NRCFs proliferation through GRK2/β-arrestin2/ERK1/2 signaling	[87]
	SII-stimulated NRK-49F cells	↑	Increasing the expression of collagen I and fibronectin by ERK1/2 signaling	[88]
	TGF-β stimulated primary lung fibroblasts isolated from IPF mice and patients	↑	Increasing the expression of genes involved in matrix production, basement membrane degradation, and cell adhesion	[89]
FLS	TNF-α and HA induced primary FLS isolated from CIA mice	↑	Enhancing anti-inflammation effects by p38 MAPK signaling	[91]
	IL-1β induced primary FLS isolated from human FLS	↑	Promoting FLS proliferation by down-regulating cAMP-PKA signaling	[92]

LPS lipopolysaccharides, MCP-1 monocyte chemoattractant protein-1, PMs peritoneal macrophages, CIA collagen-induced arthritis, RA rheumatoid arthritis, TNF-α tumor necrosis factor-α, MDC macrophage-derived chemokine, STAT3 signal transducer and activator of transcription 3, CAIA collagen antibody-induced arthritis, TLR4 toll-like receptor 4, SECs sinusoidal endothelial cells, HSGECs human salivary gland epithelial cells, ERS endoplasmic reticulum stress, eNOS endothelial nitric oxide synthase, CFs cardiac fibroblasts, AVP arginine vasopressin, NRCFs neonatal rat cardiac fibroblasts, IPF Idiopathic pulmonary fibrosis, SII [1-sar, 4, 8-ile]-angiotensin II, HA hyaluronan, FLS fibroblast-like synoviocytes.

β-arrestin2 in the progression of inflammatory immune diseases

In response to damaging stimuli such as pathogens, senescent cells, and toxic substances, inflammation acts as a protective mechanism by activating the immune system to eliminate harmful agents and initiate healing [93]. Inflammatory immune diseases, characterized by immune dysregulation and persistent inflammation, can inflict multi-organ and multi-system damage [94]. The excessive IIR is the pathological basis of the occurrence and development of multiple inflammatory immune disease. In fact, moderating IIR plays an important role in protecting the body from pathological damage to the internal and external environment [95].

Inflammatory immune diseases have emerged as a major cause of morbidity and mortality globally in recent decades. Despite numerous proposed factors, the primary etiology of these diseases remains elusive [96]. Identifying key regulatory molecules in these diseases is essential for elucidating their pathogenesis and developing effective treatments. Extensive research has demonstrated the involvement of β-arrestin2 in inflammatory immune diseases. Table 2 provides detailed information on β-arrestin2 expression in various inflammatory immune diseases and under specific treatments, highlighting their underlying mechanisms.

Table 2.

The expression and roles of β-arrestin2 in inflammatory immune diseases.

Disease	Model	β-arrestin2 expression	Mechanism/effect	Ref.
IBD	Piroxicam-induced colitis mice; TNBS-induced colitis mice	↑	Impeding NK1R recycling and causing retention of the NK1R in endosomes	[101]
	DSS-induced UC mice; TNBS-induced CD mice	↑	Encouraging epithelial apoptosis through ER stress/PUMA pathway	[102]
	NEC in humans and mice	↑	Facilitating the release of the pro-apoptotic molecule BIK from BiP	[74]
RA	CIA mice	↓	Promoting NLRP3 inflammasome and NF-κB activation	[38]
	CAIA mice	↓	Promoting B lymphocyte differentiation by TLR4-NF-κB pathway	[3]
Sepsis	CLP-induced sepsis mice	↓	Elevating pulmonary microvascular permeability	[108]
	CLP-induced late sepsis mice	↑	Exacerbating the mice mortality and immunosuppression	[111]
Asthma	AASP-induced airway inflammation mice	↑	Increasing airway inflammation and mucus hyperplasia by PAR2/β-arrestin2 signaling axis	[115]
	IL-13-induced asthma mice	↑	Increasing mucous metaplasia and lung eosinophilia	[119]
Organ fibrosis	CCl4-induced liver fibrosis mice	↑	Regulating collagen deposition, ROS and NOX4 expression; activating ERK and JNK pathways	[61, 122]
	Bleomycin-induced lung fibrosis mice	↑	Increasing the expression of genes involved in matrix production, basement membrane degradation, and cell adhesion	[89]
Tumor	DEN-induced liver tumor mice; HCC sample of patient	↓	Increasing HCC cell migration and invasion ability	[129]
	Lewis lung cancer mice	↑	Modulating tumor growth and metastasis through the activation of CXCR2 and NF-κB	[132]
	Breast cancer sample of patient	↑	Promoting breast cancer cell migration and invasion	[136]
	Ovarian cancer sample of patient	↑	Impairing the overall survival of ovarian cancer patients	[137]
	CRC sample of patient	↑	Promoting CRC cell apoptosis via the NF‑κB pathway	[138]
	RCC sample of patient	↑	Promoting RCC cell proliferation and migration	[139]
HSE	HSV-1-induced HSE mice	↓	Decreasing HSE mice survival and promoting brain pro-inflammatory responses	[142]
PD	MPTP-induced PD mice; LPS-induced PD mice	↓	Promoting the activation of microglia and the secretion of pro-inflammatory factors	[143]

IBD inflammatory bowel disease, TNBS trinitrobenzenesulfonic acid, NEC necrotizing enterocolitis, AASP Alternaria alkaline serine protease, CLP cecal ligation and puncture, DEN diethylnitrosamine, HCC hepatocellular carcinoma, CRC colorectal cancer, RCC renal cell carcinoma, HSE herpes simplex encephalitis, HSV-1 herpes simplex virus type-1, PD Parkinson’s disease, MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Inflammatory bowel disease (IBD)

IBD, encompassing ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic inflammatory disorder marked by recurrent episodes that damage the gastrointestinal tract [97, 98]. Both CD and UC are characterized by dysregulated host immune responses against components of the intestinal microbiota [99, 100]. Recently, the global prevalence of IBD has been increasing, but its molecular pathology remains unclear.

The role of β-arrestin2 in IBD is contentious. In an intestinal inflammation model, an elevated presence of the neurokinin 1 receptor produces continuous pro-inflammatory signals, disrupting neuronal function. In Piroxicam-induced and trinitrobenzenesulfonic acid (TNBS)-induced colitis models, increased β-arrestin2 expression has been observed in enteric neurons, contributing to the accumulation of neurokinin 1 receptor and exacerbating clinical symptoms [101]. Moreover, a key pathological change in IBD is intestinal epithelial apoptosis. Zeng et al. [102] demonstrated that β-arrestin2 levels were elevated in both UC patients and DSS-induced colitis models. β-arrestin2 binds and phosphorylates eukaryotic initiation factor 2 (eIF2a), activating ERS signaling and upregulating the p53-upregulated modulator of apoptosis (PUMA). In colitis, ERS/PUMA mediates IEC apoptosis via mitochondria-dependent pathways. In the DSS-induced UC model, the mortality rate of WT mice was 80%, compared to 10% in the Arrb2^−/− group, and the TUNEL assay showed significantly reduced IEC apoptosis in the Arrb2^−/− group. These observations indicate that in colitis, β-arrestin2 promotes epithelial cell apoptosis via the ERS/PUMA pathway, damaging the intestinal barrier and exacerbating IBD [102]. Furthermore, in the LPS-induced colitis model, the survival rate of the WT group was 30%, compared to 75% in the Arrb2^−/− group. The results demonstrated that intestinal epithelial apoptosis ratio and expression of CHOP (a transcription factor in ERS) is significantly higher in the WT model group compared to Arrb2^−/− model group [74]. These results suggest that β-arrestin2 may promote the progression of IBD by stimulating ERS-induced apoptosis of IECs.

In contrast, other studies have demonstrated that β-arrestin2 exerts a beneficial role in intestinal inflammation. Substantial evidence indicates that macrophages undergo polarization to distinct phenotypes in response to the intestinal microenvironment, which is closely linked to the progression of IBD. The enteric nervous system releases oxytocin, triggering β-arrestin2 activation in macrophages. This activation suppresses NF-κB and enhances the STAT6 pathway, facilitating M2 polarization in macrophages and alleviating DSS-induced colitis. Depletion of β-arrestin2 diminished oxytocin’s capacity to augment STAT6 phosphorylation levels stimulated by IL-4. Similarly, after β-arrestin2 siRNA lentivirus interference, LPS retained its ability to stimulate the production of inflammatory factors like TNF-α mRNA in macrophages; however, oxytocin pretreatment had no effect on LPS-stimulated responses [35]. Additionally, in Arrb2^−/− mice, activated T cells exhibited increased migration to the cLP, exacerbating DSS-induced colitis [52].

These studies suggest that β-arrestin2 exhibits opposite regulatory roles in the subsection of IBD. β-arrestin2 may exhibit distinct roles in different cell types. In inflammatory immune cells, β-arrestin2 inhibits T cell infiltration and facilitates macrophage M2 polarization, exerting a protective role in IBD [52]. However, β-arrestin2 may promote IECs apoptosis by activating ERS through different signaling pathways, thereby may aggravate IBD. Therefore, β-arrestin2 may exert opposite effects in IBD by depending on the modulation of different types of cells. And the different functions of β-arrestin2 in IBD might rely heavily on the cell specificity.

Rheumatoid arthritis (RA)

RA is an inflammatory immune disease affecting over five million people worldwide. The primary clinical manifestations of RA include synovial inflammation and persistent destructive joint damage [103]. Currently, β-arrestin2 is considered a protective factor in RA, acting to attenuate the inflammatory response and slow disease progression.

The activation of the NLRP3 inflammasome significantly contributes to the pathogenesis of RA. The NF-κB signaling pathway stimulates the production of the NLRP3 inflammasome and cytokines like IL-1β and IL-18 [104]. Cao et al. [38] revealed that CIA mice with Arrb2-Ad (β-arrestin2 overexpressing adenovirus) injection showed lower levels of p-p65, p-IκBα, NLRP3, caspase-1 p20, and IL-18 compared to the control and RA model group. This indicates that β-arrestin2 diminishes the inflammatory response by inhibiting the NF-κB/NLRP3 inflammasome activation in CIA mice.

Further studies revealed that β-arrestin2 deficiency could exacerbate collagen antibody-induced arthritis (CAIA) by increasing plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling [3]. Another study involving the stimulation of peripheral blood mononuclear cells (PBMCs) from RA patients under the treatment of tanshinone IIA (Tan IIA) showed a notable increase in β-arrestin2 protein levels, alongside reduced p65 protein expression. However, when β-arrestin2 was silenced in PBMCs, Tan IIA did not inhibit the secretion of inflammatory factors including TNF-α and IL-6 [105]. These results indicated that Tan IIA exerts a protective function in RA patients through upregulating β‑arrestin2 expression, thus inhibiting the inflammatory response in RA patients. To sum up, these studies indicate that β-arrestin2 plays a key role in the development and pathogenesis of RA, suggesting that targeting β-arrestin2 may be an effective strategy for future RA treatments.

Sepsis

Sepsis is a prevalent and severe complication that poses a major threat to health, causing substantial morbidity and mortality through multi-organ damage. The current definition of sepsis characterizes it as a condition where life-threatening organ dysfunction results from an uncontrolled host immune response to infection [106]. A critical aspect of organ damage in sepsis is microvascular barrier dysfunction, leading to the leakage of protein-rich edema fluid into various organs, particularly affecting the lungs [107]. Zhan et al. [108] found that β-arrestin2 expression decreased in the cecal ligation and puncture (CLP) group compared to the control group, suggesting that β-arrestin2 plays a significant role in CLP-induced sepsis. Furthermore, penehyclidine hydrochloride treatment upregulated β-arrestin2 expression, reducing pulmonary microvascular permeability. In a previous study, male rats underwent CLP and were randomly allocated to either a CO₂ pneumoperitoneum or laparotomy procedure. Compared to the laparotomy group, β-arrestin2 expression in the liver, lung, and kidney was maintained in the CO₂ pneumoperitoneum group, demonstrating a protective effect in an abdominal sepsis model [109]. However, the mechanisms by which β-arrestin2 affects the inflammatory process and preserves immune function in sepsis are not fully identified [110].

Studies have demonstrated that β-arrestin2 enhances survival and mitigates cardiac dysfunction in septic mice. Yan et al. [111] established an abdominal sepsis model of CLP using C57BL/6J WT mice, Arrb2^−/− mice and β-arrestin2 overexpression mice. The 24 h survival rates were 40%, 13.3%, and 80%, respectively, suggesting that β-arrestin2 significantly improves survival in CLP mice. Hemodynamic analysis revealed that cardiac output was lower in Arrb2^−/− mice and higher in β-arrestin2 overexpression mice compared to the WT group. Western blot analysis showed that β-arrestin2 expression and p38 MAPK phosphorylation levels in myocardial tissues of WT mice increased significantly after CLP compared to control mice, and overexpression of β-arrestin2 markedly reduced the elevated p38 MAPK phosphorylation levels in CLP-induced cardiac tissue. This finding suggests that β-arrestin2 ameliorates CLP-induced cardiac dysfunction and enhances survival by inhibiting p38 MAPK activation. Conversely, Zhou et al. [112] found that transfection of a microRNA-155 mimic reduced β-arrestin2 expression, thereby restoring immunocompetence and protecting against cardiac dysfunction in late sepsis. Moreover, β-arrestin2 overexpression worsened mortality and immune suppression in late-stage sepsis in mice. These studies indicate that β-arrestin2 may exert opposing effects at different stages of sepsis, necessitating further exploration of its role in sepsis-related cardiac function. Thus, targeting β-arrestin2 could provide a new direction for developing clinical treatments for cardiac insufficiency in sepsis patients.

Asthma

Asthma is an inflammatory disease primarily characterized by persistent airway inflammation and reversible airway obstruction, significantly contributing to a decline in quality of life. Current treatments for asthma focus on managing symptoms such as bronchoconstriction and airway inflammation [113].

Proteinase-activated receptor-2 (PAR2) has long been linked to allergic airway inflammation and asthma, with β-arrestin2 identified as a key downstream signaling molecule in asthma development [114]. Activation of airway PAR2 induces mucus production, leukocyte recruitment, and epithelial thickening, all dependent on β-arrestin2. Furthermore, β-arrestin2 is essential for PAR2-mediated Alternaria alkaline serine protease (AASP)-induced airway inflammation [115]. Recently, several potent PAR2 antagonists have been shown to mediate PAR2 physiological responses and downstream β-arrestin2/MAPK signaling pathways in vitro, while preventing airway hyperresponsiveness, inflammation, and mucus overproduction in vivo [116, 117].

Research has demonstrated that β-arrestin2 plays a significant role in the development of allergic inflammatory airway diseases, particularly allergic asthma. This condition is marked by substantial Th cell and eosinophil infiltration into the lungs, coupled with chemokine receptor activation [118]. Among the Th cell subsets involved in asthma pathogenesis, Th2 cells are particularly crucial. In a study, primary Th2 cells isolated from Arrb2^−/− and WT asthma mice were stimulated by CCL22. And the CCL22-induced chemotaxis of Th2 cells from Arrb2^−/− mice was significantly impaired compared to the control group, suggesting that β-arrestin2 plays a crucial role in Th2 cell chemotaxis [60]. Moreover, allergic asthmatic mice exhibit elevated β-arrestin2 levels, which promote IL-4 production and Th2 cell differentiation via β2AR stimulation, thereby advancing the disease [53]. Activation of β2AR on airway smooth muscle cells results in airway relaxation, and thus, β2AR agonists are commonly used as bronchodilators to manage asthma. Nguyen et al. found that knockout of Arrb2^−/− attenuates the IL-13-induced asthma phenotype, including mucous metaplasia and lung eosinophilia, similar to β2AR^−/− mice. Additional research has shown that targeted deletion of β-arrestin2 in airway epithelial cells reduces the asthma phenotype response induced by IL-13 [119]. These studies indicate that the pro-inflammatory pathway downstream of β2AR involves β-arrestin2, making it a potential target for future asthma therapies.

Organ fibrosis

Hepatic fibrosis frequently serves as the terminal stage of various chronic liver diseases, contributing significantly to morbidity and mortality [120]. The persistence of harmful stimuli leads to excessive deposition of extracellular matrix, resulting in hepatic fibrosis. In human hepatic fibrosis and porcine serum-induced or CCl₄-induced hepatic fibrosis models, β-arrestin2 levels increase in hepatic tissues as the disease progresses, correlating positively with collagen levels [61, 121]. Sun et al. [61] found that Arrb2^−/− mice exhibited reduced expression of TGF-β1, p-Smad2, p-Smad3, p-Akt, and collagen, while showing increased expression of TβRIII compared to WT mice. These results suggest that β-arrestin2 deficiency downregulates TGF-β1 signaling, and may participate in the pathological process of liver fibrosis. In a CCl₄-induced hepatic fibrosis model, pathological analysis revealed that inflammatory cell infiltration, steatosis, and collagen synthesis were significantly reduced in the hepatic tissues of Arrb2^−/− mice compared to WT mice, with markedly lower levels of ROS and NOX4. In vitro, silencing the β-arrestin2 in LX-2 cells inhibited the activation of JNK and ERK pathways, reducing ROS, NOX4, and collagen production. Thus, knockdown β-arrestin2 may alleviate hepatic fibrosis progression by inhibiting JNK and ERK pathway activation, thereby reducing NOX4 expression and ROS levels [122].

Pulmonary fibrosis is a chronic and progressive lung condition characterized by thickened fibrotic walls in the alveoli, ultimately resulting in respiratory insufficiency [123]. Lovgren et al. [89] conducted a study using Arrb2^−/− mice in the widely recognized bleomycin mouse model of lung fibrosis. The research revealed that WT mice exhibited a mortality rate of ~50%, whereas the Arrb2^−/− mice showed remarkable resistance to mortality. Knockdown β-arrestin2 in idiopathic pulmonary fibroblasts suppressed fibroblast invasion. Additionally, the absence of β-arrestin2 in primary lung fibroblasts altered the expression of genes involved in basement membrane degradation, matrix generation, and cell adhesion. Consequently, β-arrestin2 induces abnormalities in fibroblast proliferation pathways, potentially facilitating the progression of pulmonary fibrosis. These outcomes suggest that β-arrestin2 is a key mediator of fibroblast invasion and the development of certain types of organ fibrosis.

Tumor

Under the influence of various tumor-inducing factors, cells in local tissues undergo abnormal proliferation, resulting in neoplasm development [124]. Extensive research has demonstrated that alterations in GPCR signaling regulate tumorigenesis and tumor progression. As a pivotal regulator of GPCRs, β-arrestin2 is intricately linked to the pathogenesis of multiple tumor types [125, 126]. β-arrestin2 modulates cancer cell proliferation, migration, invasion, and cell cycle by engaging in diverse signaling pathways or interacting with various receptors and ligands, thus either promoting or inhibiting tumor development [13, 127].

β-arrestin2 expression progressively decreases during the progression of hepatocellular carcinoma (HCC) [128, 129]. Research indicates a decline in β-arrestin2 levels during liver tumorigenesis in a diethylnitrosamine (DEN)-induced mouse model. Additionally, in HCC patients, β-arrestin2 expression is significantly lower in cancerous tissues compared to normal tissues, correlating with unfavorable patient outcomes and aggressive pathological features. In vitro studies, have shown that overexpressing β-arrestin2 significantly reduces cell migration and invasion by increasing E-cadherin levels, suppressing vimentin expression and Akt activation in HCC cells [129]. Li et al. [128] also revealed that the absence of β-arrestin2 reduces the ratio of activated T cells and worsens DEN-induced HCC, accompanied by increased β2AR expression. Furthermore, β-arrestin2 overexpression enhances the interaction between β-arrestin2 and β2AR in activated HSCs, reducing Akt phosphorylation and inhibiting HCC cell proliferation and migration. These results suggest that β-arrestin2 modulates HCC cell proliferation and migration via the Akt pathway.

Lung cancer accounts for approximately two million deaths globally and stands as the leading cause of cancer mortality worldwide [130]. Chronic inflammation is recognized as a risk factor for lung cancer, promoting its development and progression [131]. Raghuwanshi et al. [132] found that Lewis lung cancer cells grow more rapidly and form more metastatic nodules in Arrb2^−/− mice compared to WT mice. Furthermore, Arrb2^−/− mice exhibited elevated expression of vascular endothelial growth factor in lung tumor tissues (LTTs), along with significantly reduced infiltration of NK cells and neutrophils. In vitro, experiments showed that stimulating neutrophils from WT and Arrb2^−/− mice with the chemokine CXCL1 resulted in significantly reduced phosphorylated p65 protein expression in Arrb2^−/− neutrophils, indicating that β-arrestin2 may inhibit tumor growth and metastasis by suppressing the NF-κB pathway. In another study, Kim et al. [133] demonstrated that β-arrestin2 negatively regulates lung cancer progression. Gene set enrichment analysis comparing three primary non-small cell lung cancer patient LTTs with significantly reduced ARRB2 expression to three LTTs with slightly decreased ARRB2 levels revealed significant enrichment in toll-like receptor (TLR) signaling and autophagy-related genes in the former group. This suggests a potential inhibitory role of β-arrestin2 in TLR-mediated autophagy pathways in lung cancer. Additionally, molecular investigations showed that β-arrestin2 suppresses the TRAF6-TAB2 signaling for NF-κB activation and the TRAF6-BECN1 signaling pathway for autophagy induced by TLR3 and TLR4. Lung cancer cells lacking β-arrestin2 demonstrated significantly increased proliferation, migration, invasion, and colony formation upon TLR3 and TLR4 stimulation. Thus, by inhibiting TLR3- and TLR4-induced autophagy, β-arrestin2 plays a vital role in negatively regulating lung cancer progression.

Breast cancer is a prevalent malignancy in women, results from the uncontrolled proliferation of breast epithelial cells. Numerous studies have shown that β-arrestin2 regulates the cell cycle, invasion, and proliferation of breast cancer cells. The Iverson Lab [134] recently demonstrated that β-arrestin2 directly interacts with the MELK, a serine/threonine kinase essential for cell cycle regulation and proliferation. Notably, co-expression of β-arrestin2 and MELK significantly decreased the number of breast cancer cells in the S-phase, indicating that β-arrestin2 interferes with cell proliferation via this mechanism. Invadopodia formation, closely associated with the invasive and metastatic potential of tumors [135] has been shown to involve a β-arrestin2 and ERK1/2-dependent mechanism in breast cancer cells [5]. Additionally, breast cancer cell proliferation is stimulated by Ral GTPases, whose activity can be regulated by β-arrestin2. Li et al. [136] reported that mRNA levels of β-arrestin2 and Ral GTPases are elevated in advanced stages of breast cancer. These findings highlight a novel role for β-arrestin2 in mediating the cell cycle, invasion, and proliferation of breast cancer cells.

β-arrestin2 also plays vital roles in various other tumors, such as ovarian cancer, colorectal cancer (CRC), and renal cell carcinomas (RCC). Czogalla et al. [137] used immunohistochemistry to analyze β-arrestin2 expression in samples from 156 ovarian cancer patients, finding that its cytoplasmic expression level correlates with clinical and pathological characteristics, as well as prognosis. Supporting this observation, β-arrestin2 overexpression enhanced the viability of ovarian cancer cells in vitro. Furthermore, recent research found that β-arrestin2 levels are increased in CRC tissues compared with normal colon tissues. In vitro studies with CRC cell lines showed that β-arrestin2 knockdown decreased 5-fluorouracil-induced apoptosis, while β-arrestin2 overexpression promoted cancer cell apoptosis [138]. Masannat et al. [139] recently reported that ARRB2 transcript levels are elevated in RCC and that that high levels of ARRB2 correlate with worse patient survival. Deletion of β-arrestin2 inhibited RCC cell proliferation and migration both in vitro and in vivo. Additional studies revealed that β-arrestin2 regulates RCC cell cycle progression through c-Src activation and cyclin A expression.

These studies indicate that β-arrestin2 plays a vital role in regulating the formation and development of various tumors. Given the highly heterogeneous nature of tumors, which exhibit different molecular characteristics depending on their stage and site of origin, variations in the tumor microenvironment caused by different in vivo and in vitro models may explain the opposing effects of β-arrestin2 on tumor formation in different contexts. These outcomes strongly suggest that targeting β-arrestin2 and the signaling pathways it regulates—such as those involved in cell proliferation, invasion, and survival, could lead to the development of novel anti-tumor therapies.

Other diseases

In addition to the inflammatory immune diseases previously mentioned, β-arrestin2 also plays roles in herpes simplex disease, Parkinson’s disease (PD), and other inflammatory conditions. It may exert regulatory effects by inhibiting the secretion of pro-inflammatory cytokines by microglia.

Herpes simplex encephalitis (HSE) results in severe neurological impairments, including behavioral disturbances, paralysis, and epilepsy [140]. Despite antiviral treatments aimed at inhibiting viral replication, the mortality rate of HSE remains around 30%, with many survivors experiencing profound neurological complications [141]. In a previous study, C57BL/6 WT mice and β-arrestin2 overexpression transgenic mice were injected with human herpesvirus 1 (HSV-1) to establish an HSE mouse model. Transgenic mice overexpressing β-arrestin2 showed a 40% improvement in survival rates compared to WT groups. Additionally, the production of pro-inflammatory factors like TNF-α and IL-6 mRNA decreased by 2–3 times, while the levels of anti-inflammatory factors like IL-10 and IL-4 mRNA significantly increased in the brain tissues of β-arrestin2 overexpressing mice. Thus, β-arrestin2 can inhibit the inflammatory response in HSE. In vitro infection of microglia isolated from mouse brain tissue with HSV-1 demonstrated that nuclear staining of NF-κB in microglia from β-arrestin2 overexpressing mice was significantly attenuated compared to WT mice, suggesting that β-arrestin2 may inhibit the release of pro-inflammatory mediators from microglia by suppressing NF-κB activation and mitigating the inflammatory response in the brain [142].

In a PD mouse model, Arrb2^−/− mice exhibited significant activation of microglia in brain tissue, with elevated levels of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and iNOS, while anti-inflammatory factors like Arg1, Ym-1, Mrc1, and CD206 were reduced. These results suggest that β-arrestin2 deficiency may exacerbate the neuroinflammatory response in PD by promoting microglial activation and enhancing the secretion of pro-inflammatory factors [143].

These studies reveal that β-arrestin2 participates in the progression of inflammatory immune diseases by regulating the activation of IIR-associated cells and cytokine secretion in various inflammatory contexts.

Conclusions and prospects

Research has demonstrated that various chemical compounds influence inflammatory immune diseases by upregulating β-arrestin2 expression. The primary active component in tanshinone, Tan IIA, has been shown to suppress TNF-α and IL-6 production in PBMCs of individuals with RA by increasing β-arrestin2 expression, effectively restraining the inflammatory response in RA patients [105]. In a murine sepsis model induced by CLP, penehyclidine hydrochloride increases β-arrestin2 expression, leading to reduced lung damage by decreasing pulmonary microvascular permeability [108]. Additionally, oxytocin reduces macrophage responsiveness to LPS stimulation, decreasing IL-1β, IL-6, and TNF-α expression. Conversely, it enhances sensitivity to IL-4 stimulation by increasing the expression of M2 markers such as chitinase-like 3 (Chil3), Arg1, and CD206. This bidirectional adjustment is partly due to elevated β-arrestin2 levels, ultimately inhibiting NF-κB signaling and enhancing STAT6 phosphorylation [35]. In summary, some Chinese medicinal ingredients or compounds may possess anti-inflammatory and immunoregulatory properties by regulating β-arrestin2 expression, offering therapeutic potential for RA and other inflammatory immune diseases.

β-arrestin2 is ubiquitously expressed in various cells, acting as a multifunctional hub of GPCR and non-GPCR pathways and various signal networks to participate the physiological functions of IIR-associated cells. Evidence has shown that aberrant levels of β-arrestin2 are closely associated with the development of various inflammatory immune diseases, suggesting that modulation of β-arrestin2 may be a potential therapeutic approach. Most of the attention on β-arrestin2 has obviously been centered on the abnormal expression of β-arrestin2 under pathological conditions. However, β-arrestin2 is widely expressed in various cells, which also plays an important role in regulating general physiological functions of individuals. Therefore, it is of great significance to understand and explore the mechanism of regulating β-arrestin2 activity, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation, so as to further affect the cellular localization of β-arrestin2 and interaction with downstream signaling molecules. Currently, progress has been made in the research on the mechanism of β-arrestin2 activity regulation, but there are still some issues such as cross talk between several post-translational modifications still need to be further studied. We believe that these may be the focus of β-arrestin2 in the coming years.

Competing interests

The authors declare no competing interests.