Protein-Binding Function of RNA-Dependent Protein Kinase Promotes Proliferation through TRAF2/RIP1/NF-κB/c-Myc Pathway in Pancreatic β cells
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Double-stranded RNA-dependent protein kinase (PKR), an intracellular pathogen recognition receptor, is involved both in insulin resistance in peripheral tissues and in downregulation of pancreatic β-cell function in a kinase-dependent manner, indicating PKR as a core component in the progression of type 2 diabetes. PKR also acts as an adaptor protein via its protein-binding domain. Here, the PKR protein-binding function promoted β-cell proliferation without its kinase activity, which is associated with enhanced physical interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. In addition, the transcription of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-dependent survival gene c-Myc was upregulated significantly and is necessary for proliferation. Upregulation of the PKR protein-binding function induced the NF-κB pathway, as observed by dose-dependent degradation of IκBα, induced nuclear translocation of p65 and elevated NF-κB-dependent reporter gene expression. NF-κB-dependent reporter activity and β-cell proliferation both were suppressed by TRAF2-siRNA, but not by TRAF6-siRNA. TRAF2-siRNA blocked the ubiquitination of receptor-interacting serine/threonine-protein kinase 1 (RIP1) induced by PKR protein binding. Furthermore, R/P1-siRNA inhibited β-cell proliferation. Proinflammatory cytokines (TNFα) and glucolipitoxicity also promoted the physical interaction of PKR with TRAF2. Collectively, these data indicate a pivotal role for PKR’s protein-binding function on the proliferation of pancreatic β cells through TRAF2/RIP1/NF-κB/c-Myc pathways. Therapeutic opportunities for type 2 diabetes may arise when its kinase catalytic function, but not its protein-binding function, is downregulated.
Double-stranded RNA-dependent protein kinase (PKR) is well known as a pathogen recognition receptor against virus infection and a tumor suppressor in cell growth (1, 2, 3, 4, 5, 6). During virus infection, PKR is activated and blocks viral protein synthesis through phosphorylation of eIF2α, thus leading to antiviral defense (1,3,7). Recently, it was observed that PKR is involved in insulin resistance in peripheral tissues (8, 9, 10, 11) and antiproliferation activities in pancreatic β cells (12), indicating a novel role of PKR in metabolism regulation and type 2 diabetes mellitus (T2DM). These functions of PKR are attributed to its kinase catalytic activity and an effective therapeutic strategy of pharmacologically targeting PKR was confirmed using small-molecule inhibitors of PKR kinase activity that improved insulin sensitivity and glucose clearance in a mouse model of obesity and insulin resistance (13).
Notably, PKR can function as an adaptor protein via its protein-binding domain, but not via its regulatory dsRNA-binding domain (14,15). Structurally, there are two tumor necrosis factor receptor-associated factor (TRAF)-binding domains with one TRAF-interacting motif P/S/A/T × Q/E E in the regulatory domain and another, P × Q × S/T/D, in the kinase domain (14). In unstimulated cells, the two motifs are embedded in the closed conformation of PKR that is mediated by inhibitory intramolecular interactions (16,17). Upon ligand binding, dimerization of PKR is induced and conformation occurs to expose the TRAF-binding domains. TRAF family members, including TRAF2, TRAF5 and TRAF6, combine with PKR upon dimerization in some cells (14). Despite the existence of TRAF-interacting motifs on PKR, physical interaction between PKR and TRAF proteins in pancreatic β cells remains elusive.
TRAF family members originally were identified as signaling adaptors, with a TRAF domain at the C-terminus to interact with upstream recruiter molecules (such as TLR, TNFR, CD40, PKR and a really interesting new gene [RING] finger and several zinc finger motifs in the N-terminal domain) to combine with and activate downstream molecules, including receptor-interacting serine/threonineprotein kinase 1 (RIP1) and other TRAF family members (14,15). TRAF family members play a critical role in signaling transduction forward to the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway and other transcriptional modulations (18, 19, 20). For example, TNFα mediates constitutive NF-κB activation and proliferation in human head and neck squamous cell carcinoma through the TRAF signaling pathway (21).
The aims of this investigation were to determine the effect of the protein-binding domain of PKR on pancreatic β cells and to analyze the underlying mechanism. Under the conditions where the kinase catalytic activity was defective, the protein-binding function of PKR was shown to promote β-cell proliferation, suggesting a role of the PKR protein-binding domain distinct from its kinase activity. If small pharmacological molecules could agonize the protein-binding function to counteract the kinase activity of PKR in vivo, then therapeutic opportunities in T2DM may arise.
Materials and Methods
Roswell Park Memorial Institute (RPMI) 1640, Dulbecco modified Eagle medium and fetal bovine serum (FBS) were Gibco products (Thermo Fisher Scientific Inc., Waltham, MA, USA). TRIzol and Lipofectamine 2000 reagent were Invitrogen products (Thermo Fisher Scientific). The Reverse Transcription Kit and the SYBR Green PCR Master Mix were bought from Takara (Otsu, Shiga, Japan). Type V collagenase, Histopaque-1077, MTT and coumermycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). TNFα was purchased from Pepro-Tech (Rocky Hill, NJ, USA). The Cell-LightEdU DNA Cell Proliferation Kit was purchased from RiboBio (Guangzhou, China). TRAF2-siRNA, TRAF6-siRNA, RIP1-siRNA, c-Myc-siRNA and antibodies against β-actin, PKR, eIF2α, p-eIF2α, TRAF2, TRAF6, RIP1 and ubiquitin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Cyclin D1, Cyclin D2, Cdk2, Cdk4 and c-Myc were from Cell Signaling Technology (Boston, MA, USA). Antibodies against P21, P27 and P53 were purchased from Bioss (Beijing, China). The NF-κB-dependent reporter construct pGMNFκB-Lu was purchased from Genomeditech (Shanghai, China). Bay11-7082, ortho-Nitrophenyl-β-galactoside, RIPA lysis buffer, nuclear and cytoplasmic protein extraction kit and BCA kit were obtained from Beyotime Inc (Shanghai, China). Plasmids encoding PKR-K296R, GyrB-PKR, GyrB-PKR-K296H and pSG5 (22) were provided by Tom Dever (National Institutes of Health, Bethesda, MD, USA).
Cell Culture and Transfection
The MIN6 cell line was grown in Dulbecco modified Eagle medium (4.5 g/L glucose) containing 15% (v/v) FBS, 121 mol/L 2-mercaptoethanol (23). Lipofectamine 2000 (Invitrogen [Thermo Fisher Scientific]) was used for transfection of siRNAs and plasmid constructs, according to the manufacturer’s instructions.
Islet Isolation, Culture and In Vitro Transfection
All animal studies were performed according to guidelines established by the Research Animal Care Committee of Nanjing Medical University (Nanjing, China). Ten-week-old (20–25 g) male imprinting control region (ICR) mice were purchased from the Model Animal Research Center of Nanjing University. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals (24). Islet isolation and culturing techniques have been described previously (25).
At 2-d postisolation, the isolated islets were transferred to and cultured in serum-free transfection medium (Ca2+-containing Krebs-Ringer-HEPES medium) and in vitro transfection was conducted as described previously (26).
Protein Isolation and Western Blotting
The protein concentrations were determined using a BCA kit (Beyotime Inc., China). Denatured samples were prepared for Western blot analysis using various primary antibodies as indicated. Protein signals were detected using secondary antibodies against rabbit or mouse IgG.
The same amounts (400 µg) of cell lysates were incubated with 1 to 2 µg antibody overnight at 4°C. Protein A/G-agarose spheres (Santa Cruz Biotechnology) were added to the samples and stored at 4°C. After 2 h, the samples were centrifuged at 14,000g for 2 min at 4°C. The samples were then washed three times with lysis buffer and 20 µL 5× SDS loading buffer was added before boiling for 10 min. Denatured samples were kept at −20°C for Western blotting (27).
Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) Analysis
Total RNA was isolated using the TRIzol reagent. By using a reverse transcription kit, 1 µg of total RNA was converted into first-strand cDNA. SYBR Green and the 7300 Real-Time PCR system (Applied Biosystems [Thermo Fisher Scientific]) were used to carry out the qRT-PCR analysis. All data were analyzed using β-actin gene expression as an internal standard.
For MTT measurement, MIN6 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and then subjected to the indicated treatments. Thereafter, 20 µL of 5 mg/mL MTT was added to each well and incubated for 4 h. The supernatant was removed and the formazan crystals were dissolved in dimethyl sulfoxide. Cell viability was assessed by measuring the absorbance at 490 nm using a microplate reader (12).
Cell Proliferation Assay by 5-Ethynyl-2′-Deoxyuridine EdU Labeling
For the EdU incorporation assay, MIN6 cells were cultured in 24-well plates on coverslips. After treatment, EdU was added to the culture medium (50 µmol/L) for 2 h and cell proliferation was determined according the manufacturer’s instructions. For the isolated mouse islets, media supplemented with 20 µmol/L EdU was added to the plates. Isolated mouse islets are not adherent and could not easily be made adherent, therefore, an alternative protocol for suspended cells was used and centrifugation (5 min, 4°C, 268 × g) was required for every step.
Immunofluorescence Assay (IFA)
IFA was used to observe changes in the localization of NF-κB (p65) or the level of PKR, as described previously (12). After transfection and pharmaceutical treatment, MIN6 cells or isolated mouse islets were subjected to IFA. Antibodies against insulin were applied to mark pancreatic β cells of islets. Isolated mouse islets in suspension were subject to centrifugation (5 min, 4°C, 268 × g) for every step (23).
Flow Cytometric Analysis
MIN6 cells were digested in a trypsin-EDTA solution, collected by centrifugation at 500g for 5 min, washed three times with PBS and at last fixed in cold 75% ethanol at 4°C overnight. The percentages of cells in G0/G1, S and G2/M phases were determined by flow cytometry following propidium iodide (PI) staining.
Luciferase Reporter Assay
The luciferase reporter construct pGMNF-κB-Lu was cotransfected transiently with pSG5 or GyrB-PKR-K296H into MIN6 cells grown in 24-well plates, using the lipofectamine 2000 reagent according to the manufacturer’s instructions. A plasmid expressing the gene-encoding β-galactosidase driven by the cytomegalovirus (CMV) promoter (Clontech Laboratories, Palo Alto, CA, USA) was cotransfected simultaneously as an internal control. The medium was replaced 6 h after transfection. Twenty-four hours after transfection, the cells were treated with the specific ligand coumermycin for an additional 24 h and harvested for luciferase reporter assays, as described previously (28). Similar protocol for PKR-K296R-induced MIN6 cells was conducted.
All data were representative of at least three experiments. Results are expressed as the mean ± SEM. Comparisons were performed using the Student t test for two groups or analysis of variance (ANOVA) for multiple groups. P values < 0.05 were considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
A Model of PKR with Its Protein-Binding Function but without Its Kinase Catalytic Activity Was Built into Pancreatic β cells
Protein-Binding Function of PKR Improved Both Cell Viability and Proliferation of Pancreatic β cells
Protein-Binding Function of PKR Upregulated Transcription of c-Myc in MIN6 Cells
NF-κB Is Required for PKR Protein-Binding Domain-Induced c-Myc Upregulation and Cell Proliferation in MIN6 Cells
NF-κB Was Activated by PKR Protein Binding in MIN6 Cells
TRAF2, but Not TRAF6, Plays a Critical Role in PKR Protein-Binding-Induced NF-κB Activation and Proliferation in MIN6 Cells
RIP1 Is Located Downstream of TRAF2 and Implicated in PKR Protein-Binding-Mediated NF-κB Activation and Proliferation in MIN6 Cells
PKR Protein Binding, Which Can Remit the Deleterious Effect of TNFα on MIN6 Cell Proliferation Also Was Evoked in Response to Glucolipitoxicity and TNFα
Considering that T2DM is characterized by relative insufficiency of insulin production by β cells (12,23,27,30,53,54), how to increase the β-cell mass by expending β cells and enhancing insulin secretion are of great importance. In adult mammals, proliferation of mature β cells plays a critical role in the maintenance of the β-cell mass (55). Hence, inducible proliferation of pancreatic β cells in pathophysiological settings is proposed to improve T2DM. Our investigations suggested that pancreatic β-cell proliferation would be promoted by elevating the protein-binding function of PKR in the absence of its kinase catalytic activity.
PKR acts as an adaptor protein via its protein-binding domain, whose biological function may be quite different from its catalytic activity (14,56,57). A pitfall in this investigation is that the β-cell line MIN6 and primary islet β cells used were not devoid of PKR; therefore, endogenous PKR potentially would be activated upon stimulus by poly(I:C), BEPP, glucolipitoxicity or proinflammatory cytokines (8,12,29,30,58) when kinase-defective mutant PKR-K296R was utilized to establish a model of elevated PKR protein-binding function. Fortunately, GyrB-PKR-K296H was able to avoid such a pitfall, as demonstrated by the fact that the specific activator coumermycin (14,22) had no influence on endogenous PKR activity (see Figure 1A). As depicted in Figure 1B, the PKR binding function was greatly upregulated after treatment by coumermycin in GyrB-PKR-K296H-induced β-cell line, accompanied by enhanced physical interactions between PKR and TRAF2 and TRAF-6. These observations indicated prospective roles of TRAF2 and −6 in PKR protein-binding-mediated signaling pathways.
In contrast to the kinase activity of PKR, the PKR protein-binding function exhibited a proliferative role on pancreatic β cells, indicating a kinase-free biological function of PKR. Analogous to PKR, other kinases such as RIP, IRAK, JNK1 and Tyrk2 also are able to mediate certain biological functions independent of their kinase activity when their binding domains are ready for alignment with associated partners and trigger signals (56,57,59,60). Therefore, the exact biological function of PKR is determined mainly by the predominance of related domains in certain settings. In fact, the proliferative role of PKR in cells is not infrequent, with high expression and activation levels of PKR found in various cancers (4,61, 62, 63). Further support for our data comes from the evidence that PKR participates in TNFα-induced proliferation in mouse embryo fibroblast (MEF) (33). The impact of PKR in cellular processes of different cells or even of the same cell type, from antiproliferation to proliferation, might be separate from its kinase catalytic activity and protein-binding function.
In the present study, the PKR protein-binding domain-dependent proliferation seemed to correlate with reduced β cells at G1 phase and augmented levels at S phase in pancreatic β-cell line (Figure 2). This was associated with the substantial increase in the positive regulator c-Myc and was further confirmed by the antiproliferative role of c-Myc-siRNA (Figure 3). Generally, c-Myc hyperactivates cyclin/Cdk or antagonizes the activity of cell cycle inhibitors, such as P21 and P27, to drive cell cycle progression and promote cell proliferation (64). As demonstrated, modest overexpression of c-Myc is able to drive proliferation in normal rat and human β cells (34). In addition, c-Myc mRNA was upregulated and contributed to adaptive proliferation of pancreatic islets during rat pregnancy (55). Hence, it is feasible that PKR protein-binding-induced proliferation might be closely linked with elevated levels of c-Myc proteins in pancreatic β cells.
Correlating with previous data that c-Myc is modulated by NF-κB transcriptional activity (38,42), there was a significant boost in NF-κB-dependent luciferase activity after increased PKR protein-binding function, as well as proteolytic degradation of IκBα and nuclear translocation of p65 in pancreatic β-cell line (Figure 5). Most importantly, activated NF-κB in this investigation obviously promoted β-cell proliferation in pancreatic β cells (see Figure 5). Notably, the proliferative role of NF-κB was observed previously in NIT-1 cells when stimulated with a low dosage of LPS (40). Furthermore, the PKR-facilitated prosurvival NF-κB pathway in cholangiocarcinoma cell lines is significantly associated with neoplastic progression in human cancers (61,63). However, previous studies also suggested that the PKR-triggered NF-κB pathway could cause cell death or apoptosis when eIF2α was phosphorylated and activated by PKR (65). The data was in line with a previous hypothesis that PKR-mediated activation of NF-κB has two distinctive functions decided by the presence of p-eIF2α and inhibition of protein synthesis (29).
As well-characterized adaptor proteins, TRAFs have an important role in assembling active NF-κB signaling scaffolds in response to extracellular or intracellular stimuli (19,43). To date, only TRAF2 and TRAF6 have been identified in pancreatic β cells (66). In the immunoprecipitation experiments, these two proteins could bind with dimerized PKR (see Figure 1B), implying a possible association of TRAF2 and TRAF6 with the PKR protein-binding-dependent NF-κB pathway. Human PKR interaction with TRAFs in HeLa and 293T cells after treatment with IFN-α/β reinforces our hypothesis (14). Nevertheless, PKR protein-binding function-accelerated β-cell proliferation was reversed only by si-TRAF2, whereas si-TRAF6 produced no effect in β-cell line. We also observed abrogated NF-κB activation and reduced c-Myc expression after gene silencing of TRAF2 (Figure 6). Thus, we concluded that TRAF2, but not TRAF6, links PKR with the proliferative pathway of NF-κB when recruited by PKR via the TRAF-interacting motif at the C-terminus (14). This conclusion was in accordance with previous evidence showing that an increase in the TRAF2 level correlated with improved cell viability of MIN6 (67), whereas TRAF6 was reported to participate in the early stages of cytokine-induced pancreatic β-cell death (68).
TRAF2-mediated NF-κB pathways contain canonical and alternative routes (69, 70, 71). However, in the alternative NF-κB pathway, downregulation of TRAF2 was shown to increase activity of the p52/RelB NF-κB complex through stabilization and activation of NF-κB-inducing kinase (NIK) (72). In this sense, TRAF2 appeared to participate in the assembly of protein complexes necessary for canonical NF-κB activation, where TRAF2 would form multimeric complexes and subsequently facilitate Lys63 ubiquitination of downstream molecules, including cIAP1, RIP1, TANK and TAK1 (73,74). TRAF2-mediated Lys63 ubiquitination of RIP1 is a prerequisite for IKK complex activation and the removal of the central inhibitor IκBα from NF-κB complexes (21,44,45,74,75). In the present study, RIP1 was ubiquitinated by the PKR protein-binding function, and gene silencing of TRAF2 abolished such ubiquitination in β-cell line (Figures 7A, B), confirming the involvement of RIP1 downstream of TRAF2 in PKR protein-binding-triggered signaling. Additionally, Jackson-Bernitsas et al. clearly demonstrated that proliferation in human head and neck cancer cells was in part attributed to RIP1-associated constitutive NF-κB activation (21). In our study, a similar role of RIP1 on proliferation in a pancreatic β-cell line was verified.
We also sought to examine whether PKR protein binding initiated and affected β-cell growth during progression of T2DM. Glucolipitoxicity and TNFα are closely associated with the decompensatory capacity of in vivo β-cell mass and the development of T2DM (12,13,23,46, 47, 48, 49, 50, 51, 52). The catalytic activity of PKR is promoted by glucolipitoxicity and TNFα and implicated in β-cell proliferation inhibition (12). In PKR-K296R-induced β-cell line, PKR binding with TRAF2 and −6 was evoked in response to glucolipitoxicity and TNFα. However, PKR protein binding could not activate NF-κB or c-Myc upregulation under conditions of glucolipitoxicity. Glucolipitoxicity also can trigger endoplasmic reticulum stress, which plays an important role in insulin resistance and the decline in pancreatic β cell mass during T2DM (34,76,77). It is possible that endoplasmic reticulum stress-mediated signals would interfere with the PKR protein-binding domain-induced NF-κB pathway. In a PKR-K296R-induced β-cell line, NF-κB activation did not result in a remarkable increase of c-Myc mRNA level (Figures 8B, C). Since overexpressed PKR-K296R was still able to alleviate the negative impact of TNFα on cells viability (Figure 8D) and proliferation (Figure 8E) of β-cell line, we presumed that a kinase-defective mutant may protect β cells from TNFα-triggered deleterious effect through downregulating c-Myc mRNA level to a reasonable extent. As a vivid example, moderate expression of c-Myc can promote cell proliferation, whereas immoderate overexpression of c-Myc can exert a negative effect on cell growth through apoptosis (34,78,79). The protective effect of overexpressed mutant PKR-K296R may be partially attributed to the PKR protein-binding function. Of course, its powerful competitive capacity against endogenous PKR activity elicited by TNFα also may play a role.
Based on our studies (12), both kinasecatalytic and protein-binding activities of PKR would be induced in the development of type 2 diabetes, despite their opposite effects. While the proliferative effort of the PKR binding function in pancreatic β cells is highlighted, its side effect should not be overlooked. For instance, tumorigenesis might occur if the PKR protein-binding function is elevated remarkably, leading to uncontrollable cell proliferation, and potential issues remain to be further verified.
This study shows that the PKR protein-binding function was inducible upon dimerization and could promote modest β-cell proliferation through the TRAF2/RIP1/NF-κB/c-Myc pathway when its kinase-catalytic activity was suppressed. These results indicated a therapeutic opportunity in T2DM through pharmacological maintenance of the PKR protein-binding function after suppressing kinase activity, since inducible proliferation of β cells plays a pivotal role in the mass increase and compensatory capacity of islets for insulin resistance.
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank Charles Tom Dever for providing plasmids encoding PKR-K296R, GyrB-PKR, GyrB-PKR-K296H and pSG5. The work was supported by grants from the National Natural Science Foundation of China (no. 81170714), the Natural Science Foundation of Jiangsu Province (BK20131110) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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