ARRB1 Protects Intestinal Tight Junction through Promoting Mitofusin 2 Transcription to Drive Parkin-dependent Mitophagy in Colitis

doi:10.21203/rs.3.rs-2726544/v1

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ARRB1 Protects Intestinal Tight Junction through Promoting Mitofusin 2 Transcription to Drive Parkin-dependent Mitophagy in Colitis

https://doi.org/10.21203/rs.3.rs-2726544/v1

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Intestinal barrier defect is a hallmark of inflammatory bowel disease (IBD). Mitochondrial dysfunction results in energy deficiency and oxidative stress, which contribute to the pathogenesis of IBD. Arrestin beta 1 (ARRB1) is a negative regulator that promotes G protein-coupled receptors (GPCRs)desensitization, endocytosis, and degradation. Our previous study indicated that ARRB1 was involved in mucosal protection in colitis; however, its role in maintaining the intestinal barrier is still unclear. In the present study, we demonstrated that ARRB1 protected the intestinal tight junction barrier against experimental colitis in vivo. ARRB1 deficiency was accompanied by abnormal mitochondrial morphology, lower ATP production, and severe oxidative stress. In vitro, the knockdown of ARRB1 reduced ATP levels and mitochondrial membrane potential while increasing reactive oxygen species levels and oxidative stress. Upon ARRB1 ablation, mitophagy was inhibited, accompanied by decreased LC3BII, phosphatase and tension homologue induced protein kinase1 (PINK1) and parkin, but increased p62 expression. Mitophagy inhibition via PINK1 siRNA or mitochondrial division inhibitor 1 (Mdivi-1) impaired ARRB1-mediated tight junction protection. Mitofusin2 is a critical ubiquitinated substrate for parkin accumulation in mitochondria. Co-immunoprecipitation and luciferase assays indicated that the interaction of ARRB1 with E2F1 activated mitophagy by enhancing the transcription of mitofusin2. Thus, our results suggest that ARRB1 is critical to maintaining the intestinal tight junction barrier by modulating mitophagy. This finding indicates that ARRB1 might be a potential therapeutic target to prevent IBD progression by maintaining mitochondrial homeostasis.

Health sciences/Diseases/Gastrointestinal diseases/Inflammatory bowel disease

Biological sciences/Cell biology/Mechanisms of disease

ARRB1

mitochondrial dysfunction

oxidative stress

mitophagy

mitofusion2

intestinal barrier

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a multifactorial disease involving immune dysregulation, genetic susceptibility, environmental factors, and microbial dysbiosis [1]. The intestinal epithelial barrier defends against para-cellular permeation of endogenous and exogenous noxious antigens and pathogens in the intestinal lumen. Accumulating evidence indicates that the loss of intestinal barrier integrity contributes to increased intestinal permeability, triggering or accelerating intestinal inflammation [2, 3]. The intestinal barrier comprises several cellular components, including tight junctions (TJs), adherens junctions, and desmosomes [4]. TJs limit the para-cellular permeability of luminal pro-inflammatory molecules [5]. Increased intestinal permeability has been observed in IBD, accompanied by abnormal TJ structure and a down-regulation of several TJ proteins, including claudins and occludin [6]. Recent research has demonstrated that regulation of tight junction integrity ameliorates intestinal inflammation [7, 8]. Thus, modulating epithelial barrier function could be a potential therapeutic strategy for IBD.

Reactive oxygen species (ROS) are generated as oxygen reduction (superoxide or hydrogen peroxide) as well as their secondary reactive products of cellular metabolic activities [9]. Oxidative stress is an essential factor in the pathogenesis of gastrointestinal mucosal diseases [10]. Mitochondria are the primary source of intracellular ROS. Since maintaining intestinal epithelial cells (IECs) junction integrity is energy-dependent, mitochondrial function might be central to the appropriate preservation of epithelial barrier function. It has been shown that the potential factors that induce mitochondrial dysfunction contribute to IBD susceptibility [11, 12]. Mitochondrial abnormal structure and dysfunction have been observed in patients with IBD [13], and mitochondrial reactive oxygen species (ROS) and mitochondrial DNA from damaged mitochondria are pro-inflammatory factors [14]. Mitophagy is a necessary and specific form of autophagy that selectively removes dysfunctional or redundant mitochondria [15]. Mitophagy has been demonstrated to have a neuroprotection effect in Alzheimer's disease [16, 17] and be involved in innate immunity [18, 19]. Compromised mitophagy has further been observed in functional studies of CD-associated risk variants in autophagy-related ATG16L1 and immunity-related GTPase M (IRGM) [20]. Preliminary studies indicate mitophagy as a protective process in IBD by regulating pro-inflammatory cytokine production, IEC viability, and possibly pathogen clearance [21, 22]. Furthermore, mitophagy has become integral to immune cell development, activation, and differentiation [23]. Thus, maintaining mitophagy might play a uniquely protective role in preventing the disease progression of IBD.

Arrestin beta 1 (ARRB1) is a negative regulator of G protein-coupled receptor (GPCR) signaling. It promotes GPCR desensitization, endocytosis, and degradation [24]. ARRB1 has also been demonstrated as a molecular scaffold that regulates cellular function through interactions with other partner proteins and contributes to immune response, inflammation, and tumorigenesis [25]. Mounting evidence also suggests that ARRB1 can translocate from the cytoplasm to the nucleus and modulates the transcription of targeted genes [26]. Nuclear ARRB1 interacts with transcription co-factors, such as p300 and IκBα, to play a vital role in cell growth, apoptosis, and immune function [27, 28]. ARRB1 has been identified as a critical regulator of glucose and energy homeostasis [29]. Pan et al. found that high glucose attenuated the cardioprotective effects of glucagon-like peptide 1 through induction of mitochondrial dysfunction via inhibition of ARRB1 [30]. Nevertheless, ARRB1 has increased mitochondrial oxidative stress in cultured human cardiac fibroblasts [31]. Hence, the role of ARRB1 in mitochondrial function requires further investigation.

We previously demonstrated that ARRB1 mediated mucosal protection by COX-1/PGE2/EP4 in colitis [32]. However, it has not yet been established whether ARRB1 regulates epithelial barrier function in inflammatory bowel disease. Our study demonstrated that ARRB1 deficiency impaired the intestinal tight junction barrier through mitochondrial dysfunction accompanied by lower ATP production and increased oxidative stress. Mechanistically, ARRB1 enhanced mitophagy to maintain mitochondrial function through activating mitofusin2 transcription. These results provide a novel link between ARRB1 and mitophagy, which has clinical significance as a potential treatment strategy for IBD.

2.1 ARRB1 upregulates intestinal barrier function by protecting tight junction during inflammation.

Consistent with our previous study [32], H&E staining showed that DSS induced more colonic mucosal injury in ARRB1 KO mice (Fig. 1A). It is known that the intestinal epithelial barrier defect contributes to the pathogenesis and progression of IBD. Next, we addressed whether the susceptibility of ARRB1 deficiency to colitis was due to an intestinal epithelial barrier defect. Intestinal permeability was evaluated by the FITC dextran (4 000 MW, FD4) test. The serum concentration of FD4 in ARRB1 KO mice was significantly higher in experimental colitis than in WT mice, indicating that ARRB1 deficiency contributed to increased intestinal permeability (Fig. 1B). The integral membrane components of tight junction proteins regulate the selective permeability between epithelial cells. According to scanning electron microscopy, the tight junction between cells on the apical side of the colonic epithelium was significantly more damaged in ARRB1 KO mice than in WT mice with colitis (Fig. 1C). Immunofluorescence staining showed that claudin 1 and occludin, two principal components of tight junctions, were markedly decreased in ARRB1 KO mice compared with WT mice (Fig. 1D). This result was confirmed by Western blot analysis (Fig. 1E and F). These results indicated that ARRB1 deficiency impaired the integrity of the intestinal tight junction barrier during colitis. Afterward, we further investigated the potential protective effect of ARRB1 on the tight junction barrier function of HCoEpic cells. Compared with the negative control (Vector), ARRB1 overexpression (Lv-ARRB1) increased the mRNA and protein levels of claudin 1 and occludin (Fig. 1G and H). In the presence of TNF-α, the concentration of FD4 in the Lv-ARRB1 group increased at a much slower rate, indicating a protective role of ARRB1 on trans-epithelial permeability (Fig. 1I). Together, ARRB1 protects the intestinal barrier function by upregulating the expression of tight junction proteins during inflammation.

2.2 ARRB1 deficiency exacerbates mitochondrial dysfunction and oxidative stress in the colonic epithelium of experimental colitis.

Emerging evidence has emerged that several genetic risk factors associated with mitochondrial function contribute to intestinal epithelial cell dysfunction, thus confirming the importance of mitochondria in IBD [33, 34]. Mitochondrial dysfunction is characterized by abnormal mitochondrial morphology, impaired ATP synthesis, and reduced antioxidant capacity [35]. As indicated by swelling of the mitochondria, fracture of the inner or outer membranes, and rupture of the mitochondrial crest, transmission electron microscopy (TEM) showed that the mitochondria of intestinal epithelial cells from colitis in ARRB1 KO mice were more severely damaged than those of WT mice in colitis (Fig. 2A). Then we analyzed the number, length and width of mitochondria in a double-blinded manner. There were fewer mitochondria and a higher ratio of round mitochondria (length/width less than 2) in ARRB1 KO mice (Fig. 2B). These results indicated that the ARRB1 deficiency resulted in a decreased number and severe morphological abnormalities of mitochondria. Cytochrome c oxidase IV (COX IV), a mitochondrial marker, was reduced more in ARRB1 KO mice (Fig. 2C). The mRNA expressions of mitochondrial cytochrome c oxidase II (MT-CO2) and mitochondrial cytochrome B (MT-CYB), mitochondrial DNA encoded-mitochondrial complexes subunits, were remarkably reduced in ARRB1 KO mice (Fig. 2D). These results indicated that colitis induced more mitochondrial damage in ARRB1 KO mice. Next, we investigated the effects of ARRB1 on mitochondrial function. The mitochondria are prominently responsible for producing antioxidants and ATP in the cell. The antioxidant capacity was assessed by measuring glutathione (GSH) [36]and malondialdehyde (MDA) [37]. In ARRB1 KO mice, colitis induced significant exhaustion of GSH and elevation of MDA, which indicated a deficit in antioxidant capacity (Fig. 2E and F). Similarly, ATP production in the colon tissues of ARRB1 KO mice was significantly lower than that in WT mice (Fig. 2G). These results indicate that ARRB1 knockout exacerbates mitochondrial dysfunction and oxidative stress in the colonic epithelium of experimental colitis.

2.3 Mitochondrial dysfunction-induced ROS mediates down-regulation of tight junctions in vitro.

It is unknown whether mitochondrial dysfunction disrupts the intestinal tight junction. We used carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, as an inducer of mitochondrial dysfunction [38]. Upon CCCP treatment, we observed a decrease in the viability of the cells (Fig. 3A) and a reduction in the production of GSH and ATP (Fig. 3B and C).In response to CCCP-induced mitochondrial dysfunction, the mRNA and protein expressions of claudin 1 and occludin were reduced (Fig. 3D-E). Intriguingly, the corresponding reduction in ARRB1 expression suggested that ARRB1 might be involved in maintaining mitochondrial function (Fig. 3D-E). Mitochondria are the primary source of cellular ROS. N-Acetylcysteine (NAC), a ROS scavenging agent, significantly reduced cellular ROS accumulation following CCCP treatment (Fig. 3F). NAC did not rescue the decreased viability induced by CCCP (Fig. 3G). The CCCP-induced downexpression of claudin1 and occludin was reversed by NAC, indicating that ROS was responsible for reducing these proteins induced by mitochondrial dysfunction (Fig. 3H and I).

2.4 Knockdown of ARRB1 reduces mitochondrial membrane potential, antioxidant

activity, and ATP production in vitro.

In order to clarify the effect of ARRB1 on mitochondrial function in vitro, we assessed mitochondrial membrane potential (MMP) by measuring the ratio between red and green fluorescence by JC-1 staining. The knockdown of ARRB1 resulted in a more pronounced reduction of MMP in response to TNF-α exposure (Fig. 4A and B). Sh-ARRB1 cells consistently exhibited faint red and intense green fluorescence under a fluorescence microscope (Fig. 4C). Moreover, the knockdown of ARRB1 increased cellular ROS accumulation (Fig. 4D). As mitochondrial DNA copy numbers can predict the relative number of mitochondria, we extracted it exclusively from the cells and selected MT-CO2 and MT-CYB to represent mitochondrial DNA. We found that TNF-α decreased the levels of MT-CO2 and MT-CYB, but ARRB1 was resistant to this reduction (Fig. 4E). ARRB1 delayed TNF-α-induced mitochondrial quantity reduction as assessed by Mito-Tracker Red CMXRos probe staining (Fig. 4F and G). The knockdown of ARRB1 reduced antioxidant activity with a significant decrease in GSH and an evaluation of MDA level (Fig. 4H and I). The production of ATP was also reduced in sh-ARRB1 cells (Fig. 4J). Thus, these results suggest that ARRB1 knockdown reduces mitochondrial membrane potential and disrupts mitochondrial function in vitro.

2.5 ARRB1 preserves mitochondrial function through promoting PINK1/parkin-dependent mitophagy.

The process of mitophagy helps maintain cell homeostasis by clearing disrupted mitochondria [39]. Mitophagy defects lead to damaged mitochondria accumulation and pathological change. PINK1/Parkin-mediated mitophagy is a significant pathway of mitophagy. Having demonstrated that the absence of ARRB1 disrupted mitochondrial function, we hypothesized that ARRB1 preserved mitochondrial quantity and function through PINK1/parkin-mediated mitophagy. TEM showed an increase in mitochondrial auto-phagosomes in DSS-induced colitis. ARRB1 KO mice exhibited fewer mitochondrial auto-phagosomes than WT mice (Fig. 5A). Western Blotting showed that the mitophagy-associated proteins PINK1, Parkin, and LC3B were significantly upregulated in experimental colitis, but ARRB1 KO inhibited the upregulation of these proteins (Fig. 5B and C). These results demonstrated that the deletion of ARRB1 inhibited mitophagy, which is activated to clear damaged mitochondria in colitis. Next, we examined the effect of ARRB1 on the mitophagic flux in HCoEpic cells. The colocalization of LC3 with COX IV, an indicative mitophagy marker, was less prominent in sh-ARRB1 cells than in sh-NC cells (Fig. 5D). We analyzed the colocalization of LC3 and mitotracker and found that 3-MA treatment inhibited mitophagy flux. CQ treatment increased the colocalization of LC3 and mitotracker because CQ blocks autophagosome degradation. We found that mitophagy was rescued after transfecting parkin to ARRB1 KO cells (Fig. 5E). ARRB1 knockdown significantly downregulated the mRNA expressions of PINK1 and Parkin (Fig. 5F). TNF-α increased the expressions of PINK1, Parkin, and LC3B-II, however, the knockdown of ARRB1 suppressed these up-expressions (Fig. 5G). To confirm that these mitophagy-associated proteins were changed specifically in mitochondria, we isolated mitochondria. We found that TNF-α enriched PINK1, Parkin, and LC3B-II in the mitochondrial fraction, but this effect was suppressed in sh-ARRB1 cells (Fig. 5H). Taken together, ARRB1 promotes PINK1/parkin-mediated mitophagy.

2.6 Inhibition of mitophagy downregulates ARRB1-mediated tight junction protection.

Recent research has demonstrated that mitophagy is the critical mechanism underlying the selective degradation of dysfunctional mitochondria. Subsequently, we validated the importance of dysregulated mitophagy on intestinal tight junctions by ARRB1 knockdown. The silencing of ARRB1 downregulated the expression of claudin 1 and occludin, and NAC reversed this downexpression (Fig. 6A). Thus, the reduction of tight junction proteins by ARRB1 knockdown was due to ROS accumulation. Then, we used PINK1 siRNA to downregulate the mRNA expressions of PINK1 and Parkin (Fig. 6B). In addition, the downregulation of PINK1, Parkin, and LC3B-II indicated that si-PINK1 inhibited mitophagy, undermining ARRB1-mediated claudin 1 and occludin up-regulations (Fig. 6C and D). Transfecting with PINK1 plasmid to sh-ARRB1 cells, we found that claudin 1 and occludin expressions were restored (Fig. 6E). Moreover, we used mitochondrial division inhibitor 1 (Mdivi-1) as a mitophagy inhibitor. Mdivi-1 inhibited mitophagy and reduced the expressions of claudin 1 and occludin (Fig. 6F and G). Furthermore, the inhibition of mitophagy medicated by si-PINK1 and Mdivi-1 significantly reduced GSH levels (Fig. 6H).

2.7 ARRB1 protects tight junction through MFN2-mediated mitophagy.

Next, we intended to investigate the mechanisms by which ARRB1 modulated mitophagy. Mitofusin2 (MFN2), located on the outer mitochondrial membrane, is not only the regulator of mitochondrial fusion but also involved in the progression of mitophagy. MFN2 deficiency has been linked to decreased mitophagy and the accumulation of defective mitochondria [40, 41]. By analyzing two GEO data (GSE87466 and GSE 107499), we discovered that MFN2 was significantly down-regulated in UC (Fig. 7A). Then, we found that silencing MFN2 with siRNA reduced ATP production and GSH levels, indicating that MFN2 deficiency contributed to mitochondrial dysfunction (Fig. 7B and C).

We analyzed GSE87466 and GSE107499 data and revealed a significant positive correlation between MFN2 and ARRB1 mRNA (Fig. 7D). Thus, we hypothesized that ARRB1 regulated mitophagy via MFN2. We demonstrated that the knockdown of ARRB1 reduced MFN2 mRNA and protein expression (Fig. 7E and F). Subsequently, we detected MFN2 expression in colonic tissue. MFN2 expression was reduced in colitis, and ARRB1 deficiency exacerbated this decrease (Fig. 7G). Immunohistochemistry presented consistent results (Fig. 7H).

Furthermore, the knockdown of MFN2 abrogated the ARRB1-mediated mitophagy activation (Fig. 7I) and claudin 1, occludin upexpression (Fig. 7J). Transfecting with PINK1 plasmid to sh-ARRB1cells, we found that claudin 1 and occludin expressions were restored(Fig. 7K). These results indicate that MFN2 is involved in ARRB1-upregulated mitophagy.

2.8 Interaction of ARRB1 with E2F1 enhances MFN2 transcription.

We then investigated how ARRB1 regulated MFN2 expression. It is known that ARRB1 translocates from the cytoplasm to the nucleus, interacting with transcription co-factors such as p300 and NF-kappaB to modulate targeted gene transcription [27, 28]. A previous study demonstrated that E2F1 activates MFN2 expression by binding to its promoter [42]. Consistent with this finding, we found that the knockdown of E2F1 inhibited ARRB1-mediated MFN2 upexpression (Fig. 8A), and the transfection of E2F1 restored the repression of MFN2 by the knockdown of ARRB1 (Fig. 8B). Co-immunoprecipitation showed an interaction between ARRB1 and E2F1 (Fig. 8C and D). These results indicated that ARRB1 might regulate MFN2 expression by interaction with E2F1. However, E2F1 worked as a transcriptional factor in the nucleus. To identify whether nuclear translocation of ARRB1 regulates the expression of MFN2, we performed transient transfection with ARRB1 mutant Q394L, in which glutamine 394 has been mutated to leucine to create a nuclear export signal [43]. The ARRB1 mutant Q394L failed to rescue MFN2 expression in sh-ARRB1 cells, suggesting that nuclear translocation of ARRB1 was required for transcriptional regulation of MFN2 (Fig. 8E). The Luciferase activity assay also showed that the mutant Q394L did not enhance E2F1- mediated transcription from the MFN2 promoter (Fig. 8F). Previous studies have shown that amino acids 1-163 of ARRB1 are required to bind to E2F1 [44]. Thus, ARRB1 1-163 could competitively inhibit the interaction of ARRB1 WT and E2F1. The luciferase activity assay showed that ARRB1 1-163 inhibited the ARRB1-mediated MFN2 upexpression by competitive binding to E2F1 (Fig. 8F). Consistent with the luciferase activity assay, ARRB1 WT increased the MFN2 mRNA levels, but not ARRB1 mutant Q394L. Moreover, the ARRB1-mediated upregulation of MFN2 was inhibited by ARRB1 1-163 (Fig. 8G). As a result, ARRB1-E2F1 interaction enhanced MFN2 transcription.

ARRB1 was initially discovered and described as a negative regulator of G protein-dependent signaling in the GPCR signaling pathway [24]. Further research has found that ARRB1, as a scaffolding protein, interacts with several effector proteins [45]. ARRB1 regulates fundamental biological processes such as proliferation and apoptosis [46]. Our previous study found that ARRB1 is involved in COX-1/PGE2/EP4-mediated mucosal protection in colitis [32]. However, the effect of ARRB1 on colitis remains unclear. Due to the contribution of intestinal epithelial barrier impairment to the initiation and progression of IBD, we sought to investigate the role of ARRB1 in the intestinal barrier. We demonstrated that ARRB1 KO mice appeared susceptible to DSS-induced colitis with increased intestinal permeability. As a crucial component of the physical barrier, the tight junctions between intestinal epithelial cells regulate paracellular permeability. ARRB1 deficiency reduced the expression of claudin 1 and occludin, which are essential components of the intestinal tight junction barrier. Therefore, ARRB1 plays a critical role in maintaining intestinal tight junction barrier function.

Recent evidence has placed mitochondria as the gatekeeper of intestinal epithelial cell homeostasis [34]. Mitochondria supply energy and essential metabolites for cell activities. It has been shown that dysregulated mitochondrial signaling and function contribute to the pathogenesis of IBD [47]. Two recent studies proved that Paneth cells are highly susceptible to mitochondrial dysfunction in IBD. Prohibitin (PHB), a substantial component protein of the inner mitochondrial membrane, is crucial for the optimal assembly and function of the respiratory chain. Mice lacking PHB in IECs developed Paneth cell abnormalities and spontaneous ileitis preceded by mitochondrial dysfunction [48]. In another study, Dirk Haller and colleagues demonstrated that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggered a metabolic imbalance and drove intestinal stem cells to transition into aberrant Paneth cells [49]. We found that CCCP-induced ROS accumulation resulted in tight junction impairment. However, the role of ARRB1 in mitochondrial function is still unclear. High glucose induces mitochondrial dysfunction by inhibiting ARRB signaling to attenuate the cardio-protective effects of glucagon-like peptide 1(GLP-1) [30]. Knockdown of ARRB1 inhibited isoproterenol-induced mitochondrial ROS production [50]. In another study, the deletion of ARRB1 did not affect cerebral ischemia-induced inflammation and oxidative stress but markedly suppressed autophagy and induced neuronal apoptosis/necrosis [51]. This study found ARRB1 deficiency-induced mitochondrial dysfunction, accompanied by increased ROS production and severe oxidative stress.

Recent research has demonstrated the association between ARRB1 and autophagy. ARRB1 regulates BECN-dependent auto-phagosome formation to mediate neuroprotection in cerebral ischemia [51]. Lei et al. indicated that HBx induced hepatocellular carcinogenesis through ARRB1-mediated autophagy [52]. Mitophagy is protective in eliminating dysfunctional or redundant mitochondria to maintain mitochondrial homeostasis [53]. Mitophagy impairment perturbs mitochondrial function and causes progressive accumulation of defective organelles, leading to cell and tissue damage [54]. Recent studies have revealed mitophagy regulatory pathways mediated by ubiquitin-dependent or ubiquitin-independent pathways [55]. The phosphatase and tensin homolog (PTEN) induced putative kinase 1 (PINK1)-parkin pathway regulates ubiquitin-dependent mitophagy [56]. PINK1 transported into the inner mitochondrial membrane under physiological conditions is degraded by several proteasomes. Once damaged mitochondria induce the membrane potential dissipation, PINK1 is activated, leading to parkin translocation to the mitochondrial surface. PINK1-dependent phosphorylation alters parkin conformation, promoting its association with triggering E3 ligase activity. Parkin mediates a feed-forward mechanism generating poly-Ub chains, thereby amplifying mitophagy. In addition, Ub-independent mitophagy involves serval mitophagy receptors, which target dysfunctional organelles directly to autophagosomes for degradation. Mitophagy receptors interact directly with LC3 and GABARAP autophagosome membrane proteins through their LIR motifs, mediating mitochondrial elimination. The outer mitochondrial membrane (OMM) proteins, such as NIP3 like protein X (NIX), BCL2 interacting protein 3 (BNIP3), and FUN14 domain-containing protein 1 (FUNDC1), are mitophagy receptors that fine-tune mitochondrial populations in response to various stimuli [57, 58]. Mitophagy plays a vital role in intestinal inflammation. Vincent et al. found that NIX deficiency aggravated colitis via mitophagic inhibition, which induced the inability to clear damaged or dysfunctional mitochondria [59]. Consistently, we found mitophagy was involved in ARRB1-mediated tight junction protection. ARRB1 deficiency suppressed PINK1-parkin dependent mitophagy and induced severe oxidative stress and ROS accumulation.

Mitofusins are preferred targets at the OMM and are ubiquitylated by E3 ligases [60]. Parkin-dependent proteasomal turnover of mitofusins abolishes mitochondrial fusion, isolating defective organelles from the healthy mitochondrial network [61]. MFN2 is not only a regulator of mitochondrial fusion but is also associated with mitophagy. Upon mitophagy induction, ubiquitination of MFN2 targets them for degradation by proteasomes, quickly leading to the abrogation of mitochondrial fusion events and resulting in mitochondrial fragmentation. Thus, MFN2 acts as a pro-mitophagic receptor. It has been demonstrated that loss of MFN2 is associated with defects in autophagosome or autophagosome-lysosome formation, two events of mandatory nature for mitophagy [62]. Consistently, depletion of MFN2 in murine cardiomyocytes caused an accumulation of defective mitochondria. MFN2 deficiency inhibited mitophagy and increased apoptosis [63]. MFN2-induced mitophagy improved disease prognosis in gastric cancer [64]. In this work, we found that ARRB1 inhibited mitophagy through regulating MFN2 expression. Down-expression of MFN2 leads to suppression of PINK1/parkin-dependent mitophagy, subsequently hindering the elimination of dysfunctional mitochondria. Furthermore, ARRB1 regulates MFN2 transcriptionally. ARRB1 not only acts as a scaffold protein in multiple signaling pathways but also plays a co-factor role in gene transcription regulation. Zecchini and colleagues demonstrated that nuclear ARRB1 induced pseudohypoxia and cellular metabolism reprogramming in prostate cancer via regulation of HIFA transcription activity [65]. Another study indicated that ARRB1 bound to E2F1 target genes that modulate epithelial-mesenchymal transition in nicotine-induced growth of lung tumors [43]. In this study, we demonstrated that ARRB1 regulates MFN2 transcription by cooperating with E2F1. This finding explains how ARRB1 functions as a transcriptional regulator in cell growth, apoptosis, and mitochondrial function modulation.

In conclusion, the knockout of ARRB1 resulted in mitochondrial dysfunction and a disrupted intestinal barrier in colitis. Mechanistically, ARRB1 promoted PINK1/parkin-mediated mitophagy to eliminate damaged mitochondria. Silencing of ARRB1 resulted in ROS accumulation and exacerbated oxidative stress. As a transcriptional regulator, ARRB1 interacted with E2F1 to promote MFN2 transcription and enhance cell mitophagy flux. Our findings may open new therapeutic avenues for regulating mitochondrial dysfunction to treat inflammatory bowel disease.

4.1 Mice

All animal procedures were approved by the Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University. C57BL/6 genetic background mice were used. The original Arrb1 heterozygous mice were kindly provided by Dr. R. J. Lefkowitz, Duke University Medical Center, Durham, NC, USA. All colonies were housed in micro isolator cages with 50% humidity and 12-hour light-dark cycles. 6–8 weeks old and sex-matched mice were randomly assigned to groups. 3% dextran sulfate sodium (DSS; MP Biomedicals, LLC, Solon, OH) in drinking water for 7 days was administered to induce acute colitis in mice.

4.2 Cell culture and treatment

The HCoEpic cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C under 5% CO₂. For drug intervention, cells were treated with 40 ng/µl TNF-α (PeproTech, Hamburg, Germany), indicated concentration of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (MedChemExpress (MCE), USA), 10µM mitochondrial division inhibitor 1 (Mdivi-1) (MCE, USA) and 10µM N-acetylcysteine (NAC) (MCE, USA).

4.3 Transient transfection, stable cell line generation and RNA interference

Transient transfection was performed according to the manufacturer’s protocol (jetPRIME transfection agents, Polyplus-transfection, China). Small interfering RNAs, including si-mfn2 (5ʹ-ACUUUGUCACUGCCAAGAA-3ʹ), si-pink1 (5ʹ-ACUUUGUCACUG-CCAAGAA-3ʹ), si-E2F1 (5ʹ-GUCACGCUAUGAGACCU-CA-3ʹ), were used according to the manufacturer’s instructions (Gene Pharma, China).

pLenti-ARRB1-puromycin (GeneChem, China) was used for lentiviral transfection to generate the stable ARRB1-overexpression cell line. ARRB1-shRNA was cloned into the lentiviral vector GV112 (GeneChem, China) to generate the stable ARRB1-knockdown cell line. Stable transfections were selected with respective antibiotics for 2 weeks.

4.4 Plasmid transfection

Plasmid transfection was performed according to the manufacturer’s instructions. Briefly, 70–80% of confluent cells were transfected. Lipofectamine 3000 reagent (Invitrogen, USA) was used to deliver plasmid DNAs into cells growing in serum-free opti-MEM media. Subsequent experiments were completed 24 hours after transfection. The GFP-LC3 and parkin plasmids were kindly provided by Professor Yunfei Qin (Department of The Biological Therapy Center, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China). ARRB1 plasmid was kindly provided by Dr. Gan Pei (Chinese Academy of Sciences, Shanghai). PINK1, MFN2, E2F1, ARRB1 (Q394L), and ARRB1 (1-163) plasmids were created and synthesized by YouminBio (Guangzhou, China). The pcDNA3.0-Vector was transfected as the negative control.

4.5 Hematoxylin and eosin (H&E), Immunohistochemistry (IHC) and immunofluorescence (IF)

As previously described, H&E, IHC, and IF staining were performed [32]. After antigen retrieval was performed, 3 µm-thick paraffin-embedded sections were incubated with the indicated primary antibodies against MFN2 (1:200, Proteintech, 12186-1-AP), COX IV (1:200, Proteintech, 11242-1-AP), claudin 1 (1:200, Abcam, ab15098), and occludin (1:200, Proteintech, 27260-1-AP) overnight at 4°C. For IHC, the sections were incubated with secondary anti-mouse/rabbit IgG (1:300, A0208/A0216, Beyotime, China) at 37°C for 2h and then stained with diaminobenzidine and hematoxylin. For IF staining in tissue samples, the sections were incubated with the related biotin-conjugated secondary antibody and streptavidin Alexa Fluor 488 or 594 (1:300, A-11001/A-11012, Invitrogen, USA) at 37°C for 2h. 4,6-diamidino-2-phenylindole (DAPI) (D1306, Invitrogen, USA) was used to stain nuclei. For IF staining in cell samples, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and incubated with 0.5% Triton X-100 (ST797, Beyotime, China). After washing with PBS three times, cells were incubated with primary antibodies against COX IV (1:200, Proteintech, 11242-1-AP) overnight at 4°C, followed by the corresponding biotin-conjugated secondary antibody and streptavidin Alexa Fluor 488 or 594 at 37°C for 2 h. DAPI was used to stain nuclei.

4.6 Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using TRIzol (15596-018, Invitrogen, USA) and transcribed into cDNA using a High Capacity cDNA Kit (FSQ101, TOYOBO, Japan). Then aliquots of cDNA were amplified using gene-specific primers and ChamQ SYBR qPCR Master Mix (Q441, Vazyme, China) in a real-time PCR system (Bio-Rad, USA). Each sample was tested in triplicate. Relative expression levels were calculated by the 2^−ΔΔCt method and normalized to β-actin. The sequences of the primers are listed in Supplementary Table 1.

4.7 Western blot analysis and co-immunoprecipitation

Total protein was either isolated from the intestinal tissues or cells using a lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (78430, Thermo Scientific, USA). Immunoblotting was performed as described previously [32]. Western blotting was performed using antibodies against ARRB1 (1:1000, Abcam, ab32099), SQSTM1/p62 (1:1000, Proteintech, 18420-1-AP), LC3B (1:1000, Sigma, L7543), PINK1 (1:1000, Proteintech, 23274-1-AP), parkin (1:1000, Proteintech, 14060-1-AP), claudin 1 (1:1000, Abcam, ab15098), occludin (1:1000, Proteintech, 27260-1-AP), MFN2 (1:1000, Proteintech, 12186-1-AP), COX IV (1:1000, Proteintech, 11242-1-AP), E2F1 (1:100, Santa Cruz, sc-251), and GAPDH (1:5000, Cell Signaling Technology, 2118S). PVDF membranes with proteins were incubated with primary antibodies at 4°C overnight and corresponding secondary antibodies at room temperature for 2 h. Proteins were visualized using a Bio-Rad ECL machine (Bio-Rad, USA). The protein bands were quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

For immunoprecipitation, cells were lysed in IP Lysis Buffer (P0013, Beyotime, China) with a protease and phosphatase inhibitor cocktail (78430, Thermo Scientific, USA). The lysate was precleared with protein A magnetic beads (1614013, Bio-Rad, USA) at 4°C overnight and then incubated with anti-ARRB1 and anti-E2F1 antibodies for 2 h. Anti-mouse or rabbit IgG antibodies (A7016/A7028, Beyotime, China) from the related species were used as a control. After removing the beads by centrifugation, the boiled samples were subjected to immunoblot analysis.

4.8 Cell viability assay

Cell counting kit-8 (CCK-8, Dojindo Laboratories, JAPAN) assays were used to measure cell viability according to the manufacturer’s instructions. HCoEpic cells (2.5 × 10³/well) were seeded into 96-well plates. After treatment with the indicated chemicals, 10 µl CCK-8/90µl culture medium was added to each well. After incubation at 37°C for 2 h, the absorbance at 450 nm was measured by a spectrophotometer (BioTek-Epoch2, USA). The experiments were performed in triplicate wells and three times independently.

4.9 ATP measurement

The ATP content was measured using the ATP assay kit (S0027, Beyotime, China) following the manufacturer’s instructions. Briefly, cells or tissue samples were washed with ice-cold PBS and then homogenized and sonicated in lysis buffer on ice. After sonication, the lysed cells or tissues were centrifuged at 12 000 g for 5 min to remove debris. Then, ATP was determined using the ATP assay kit based on the luciferin/luciferase assay and normalized for protein content.

4.10 Intracellular reactive oxygen species (ROS) detection

Intracellular ROS levels were measured using an oxidation-sensitive fluorescent probe, 2’-7’dichlorofluorescin diacetate (DCFHDA). According to the manufacturer’s instructions, cells were washed twice in PBS and incubated with 10 µM DCFH-DA at 37°C for 30 min. DCFH-DA was deacetylated intracellularly by nonspecific esterases and further oxidized ROS to the fluorescent compound 2,7- dichlorofluorescein (DCF). DCF fluorescence was detected using flow cytometry (excitation wavelength at 488 nm and emission wavelength at 525 nm).

4.11 Measurement of malondialdehyde (MDA) and glutathione (GSH)

The MDA and GSH levels were measured according to the manufacturer's protocol (Boxbio Science & Technology, Beijing, China). MDA measurement was determined by the reaction of thiobarbituric acid (TBA) with MDA to generate the stable end product of the MDA-TBA adduct. The cells were lysed by sonication, and the tissue samples were homogenized on ice. Then the samples were centrifuged at 8,000 g for 10 min at 4°C, and the supernatants were mixed with the MDA detection working solution and incubated at 100°C for 60 min. After cooling to room temperature, the mixtures were centrifuged at 10 000 g for 10 min, and the supernatants were evaluated using a spectrophotometer (BioTek-Epoch2) at 450 nm, 532 nm, and 600 nm wavelengths. The MDA content was calculated by the difference of the value at 450 nm, 532 nm, and 600 nm.

For GSH measurement, the samples were centrifuged at 10,000 g for 10 min at 4°C, and the supernatants were subjected to the GSH assay kit and mixed with the GSH detection working solution. Then the output was measured immediately at 412 nm by a spectrophotometer (BioTek-Epoch2, USA). The protein concentration of each sample was determined by a bicinchoninic acid (BCA) protein assay kit (23227, Thermofisher, USA). In addition, MDA and GSH levels were normalized according to the protein concentrations.

4.12 Mitochondrial membrane potential (MMP)

MMP was detected with JC-1 staining. When the membrane potential is low, JC-1, as a monomer, emits green excitation light. At higher membrane potentials, JC-1 aggregates increase and emit red light. After treatment with the indicated drugs, cells were incubated with JC-1 (5mg/ml) for 20 min at 37°C, avoiding light and washed twice with PBS. Then, the red and green fluorescence cell ratio was measured by a fluorescence microscope (Zeiss, Oberkchen, Germany) or FACS flow cytometer (BD Biosciences, USA).

4.13 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

Fresh colon tissues were fixed with ice-cold 2.5% glutaral overnight. Electron microscopy samples were obtained at the electron microscopy core lab of Sun Yat-Sen University with the scan and transmission electron microscope (JEOL, Japan).

4.14 Intestinal and transepithelial permeability

Intestinal and transepithelial permeability were detected by FITC dextran (4000 MW, FD4, Sigma Aldrich) as previously described [66]. Mice were administered 0.6 mg/g body weight FD4 in PBS by oral gavage. Blood was collected 4 hours later by retro-orbital bleeding. The serum concentration of FITC-dextran was determined using a microplate reader (Infinite 200 pro, TECAN) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Serial-diluted FITC-dextran was used to generate a standard curve. The transepithelial permeability was assessed by apical to basolateral FD4 transmission in the transwell cultures. Briefly, 5 mg/ml FD4 was added to the up-chamber, and after several hours, the sample of the down-chamber was detected by a microplate reader (Infinite 200 pro, TECAN). The transepithelial permeability was presented as the concentration of FD4 in the basolateral chamber.

4.15 MitoTracker staining

After treatment, cells were incubated with 200 nM MitoTracker Red CMXRos (Beyotime, China) for 30 min at 37°C in the dark. Then the cells were washed with PBS and observed using a laser scanning confocal microscope (Leica, Germany) and a microplate reader (Infinite 200 pro, TECAN).

4.16 Mitochondrial isolation

Mitochondria were extracted using the mitochondrial extraction kit (Beyotime, China). Briefly, cells were rinsed in pre-cold PBS and homogenized in 1 ml ice-cold lysis buffer with a pre-cool Dounce-type glass homogenizer. Then the homogenate was centrifuged 3 times (1000 g for 10 min, 4 ^◦C) to pellet cell debris and nuclei and collected the supernatants. Finally, mitochondria from the supernatant were pelleted by centrifugation at 12 000 g for 10 min at 4 ^◦C.

4.17 Quantification of mitochondrial DNA

Total cellular DNA was extracted using the DNeasy Blood and Tissue kit (Bio-generating, China) and quantified using a NanoDrop2000 spectrophotometer (BioTek-Epoch2). A total of 100 ng DNA was amplified using ChamQ SYBR qPCR Master Mix (Vazyme, China) in a real-time PCR system (Bio-Rad, USA). The target gene content was normalized to β-globin DNA. The primers are listed in Supplementary Table 1.

4.18 Dual-Luciferase Reporter Assay

The Dual-Luciferase Reporter Assay System was used to measure luciferase activity according to the manufacturer’s instructions. Transfection efficiency was normalized to Renilla luciferase activity.

4.19 Statistical analysis

Data were presented as means ± SEM. The statistical significance was analyzed using Student’s t-tests or one-way analysis of variance tests (ANOVA), and all tests were two-tailed. The Pearson correlation coefficient was used to estimate the correlation between the mRNA expression levels of MFN2 and ARRB1. The statistical significance was set at p < 0.05.

Acknowledgments

We thank Professor Robert J. Lefkowitz at Duke University for providing the arrb1-knockout mice, Professor G. Pei at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, and the Chinese Academy of Sciences for providing the ARRB1 plasmids, and Professor Yunfei Qin at the Department of The Biological Therapy Center, The Third Affiliated Hospital of Sun Yat-Sen University for providing the GFP-LC3 plasmids.

Conflict of Interest

The authors declare no conflict of interest.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82070574, 818004580) and the Nature Science Foundation Team Project of Guangdong Province (2018B03031200)

Data Availability Statements

This article's data will be shared with the corresponding author at a reasonable request.

Author Contributions

SW and HL performed the experiments, analyzed the data, and wrote the manuscript. JY and MX helped perform the experiments, and JJ conducted administrative and material support. BW and JT contributed to designing, supervising the study, and revising the manuscript. All the authors read and approved the final version of the manuscript.

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ARRB1 Protects Intestinal Tight Junction through Promoting Mitofusin 2 Transcription to Drive Parkin-dependent Mitophagy in Colitis

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Abstract

Figures

1. Introduction

2. Results

2.1 ARRB1 upregulates intestinal barrier function by protecting tight junction during inflammation.

2.2 ARRB1 deficiency exacerbates mitochondrial dysfunction and oxidative stress in the colonic epithelium of experimental colitis.

2.3 Mitochondrial dysfunction-induced ROS mediates down-regulation of tight junctions in vitro.

2.4 Knockdown of ARRB1 reduces mitochondrial membrane potential, antioxidant

2.5 ARRB1 preserves mitochondrial function through promoting PINK1/parkin-dependent mitophagy.

2.6 Inhibition of mitophagy downregulates ARRB1-mediated tight junction protection.

2.7 ARRB1 protects tight junction through MFN2-mediated mitophagy.

2.8 Interaction of ARRB1 with E2F1 enhances MFN2 transcription.

3. Discussion

4. Materials And Methods

4.1 Mice

4.2 Cell culture and treatment

4.3 Transient transfection, stable cell line generation and RNA interference

4.4 Plasmid transfection

4.5 Hematoxylin and eosin (H&E), Immunohistochemistry (IHC) and immunofluorescence (IF)

4.6 Quantitative RT-PCR (qRT-PCR)

4.7 Western blot analysis and co-immunoprecipitation

4.8 Cell viability assay

4.9 ATP measurement

4.10 Intracellular reactive oxygen species (ROS) detection

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