Type 1 diabetes mellitus (T1DM), a chronic autoimmune disease, occurs primarily in childhood and is characterized by hyperglycemia that destroys pancreatic β-cells. T1DM is increasing and affecting millions of people worldwide. It is well known that environmental and genetic factors affect the development of T1DM (1-5). In the early stages of T1DM, the infiltration of inflammatory cells promotes the release of cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ), and promotes cytotoxicity in pancreatic β-cells, which may contribute to the impairment of insulin secretion and lead to β-cell death (6-8). Thus, inhibition of β-cell damage may ameliorate T1DM progression.
Cytokine-induced apoptosis inhibitor 1 (CIAPIN1; originally named anamorsin) is an identified apoptosis-associated protein. Several studies have reported that CIAPIN1 is an anti-apoptotic molecule that has no homology with the anti-apoptotic proteins, such as Bcl-2, caspase, the IAP families, or other signal-transduction molecules; CIAPIN1 is known as a regulator of the RAS signaling pathway (9-12). CIAPIN1 is expressed in various tissues, including metabolic tissues, and has a critical role in various cancers, including gastric cancer, hepatocellular carcinoma, and renal cancer (12, 13). Several previous studies showed that the CIAPIN1 protein inhibits the proliferation of various cancer cells; so this protein could be a new anticancer agent (13-16). In the brain, overexpression of the CIAPIN1 protein reduced dopaminergic neuronal cell death in rat brains. Other studies have reported that this protein is involved in inflammation, ROS, and intracellular signaling-pathway mediators (17-20).
Protein transduction peptides (PTDs) are generally composed of 4-30 basic amino-acid-rich sequences and are promising tools for the delivery of proteins into cells (21, 22). In general, the large molecule of protein prevents them from being deli-vered to cell membranes and the blood-brain barrier (BBB). However, PTD technology permits intracellular delivery of therapeutic molecules without increasing cytotoxicity (23, 24). The Tat PTD is a natural peptide from human immunodeficiency virus type 1 and is commonly used to deliver proteins into cells (25-27). We also have reported that the Tat-CIAPIN1 protein reduced cell deaths in oxidative-stress-induced hippocampal neuronal cells and reduced inflammatory responses in LPS-induced Raw 264.7 cells (28, 29). In addition, many reports have shown that transduced PTD fusion proteins inhibit cell deaths in oxidative stress- or cytokine-induced cells (28-35). In this study, we investigated whether the Tat-CIAPIN1 protein can protect RINm5F β-cells against cytokines-induced cytotoxicity.
Much evidence has demonstrated that a transduced fusion protein has been an effective tool for application of therapeutic proteins (28-35). Although the precise mechanism of transduction of Tat fusion proteins has not been well established, this fusion protein seems to be transduced into cells by either direct translocation or endocytosis (36, 37). This protein delivery has been getting the spotlight as an alternative means for gene delivery (38, 39). To investigate the transductive efficiency of the Tat-CIAPIN1 protein in RINm5F β-cells, we prepared the cell-permeable Tat-CIAPIN1 protein as described previously (28).
Fig. 1A shows purified Tat-CIAPIN1 and CIAPIN1 proteins. To assess the ability for Tat-CIAPIN1 protein transduction, we treated RINm5F β-cells with Tat-CIAPIN1 proteins (0.5-3 μM) for 1 h or with Tat-CIAPIN1 proteins at 3 μM for 15-60 min. As shown in Fig. 1B and 1C, the Tat-CIAPIN1 protein was transduced into RINm5F β-cells in a concentration- and time-dependent manner. In addition, the Tat-CIAPIN1 protein levels remained stable for 12 h after transduction and subsequently disappeared over time (Fig. 1D). These results indicate that the Tat-CIAPIN1 protein was transduced into RINm5F β-cells and was maintained in the cells for at least 12 h. In a previous study, we showed that the Tat-CIAPIN1 protein was transduced into HT-22 and Raw 264.7 cells (28, 29).
It is well known that cytokines promote cytotoxicity in pancreatic β-cells and lead to β-cell death (6-8). Therefore, we first investigated whether the Tat-CIAPIN1 protein inhibits cytokine-induced RINm5F β-cell deaths. As shown in Fig. 2A, transduced Tat-CIAPIN1 protein increased cell survival in a concentration-dependent manner against cytokine-induced cell death. Also, we assessed the toxicity of the Tat-CIAPIN1 protein by measuring the cell viability. As shown in Fig. 2B, the viability of the cells treated with the Tat-CIAPIN1 protein was consistently maintained.
Next, we investigated whether the Tat-CIAPIN1 protein inhibits ROS production and DNA fragmentation (Fig. 2C, D). In cytokine-exposed RINm5F β-cells, ROS production and DNA fragmentation levels were decreased by the Tat-CIAPIN1 protein. These results indicate that the Tat-CIAPIN1 protein can protect against cytokine-induced cytotoxicity. However, the CIAPIN1 protein or Tat peptide alone did not show the protective effects in cytokine-induced RINm5F β-cells.
Since pancreatic β-cells contain significantly fewer antioxidant proteins, including superoxide dismutase, catalase, and glutathione peroxide, than do other tissues in the rat model, several studies have reported that cytokines and oxidative stress are major factors for the destruction of pancreatic β-cells. The anti-oxidant proteins play a beneficial role in pancreatic β-cell viability (40-43).
Some studies have shown that NF-κB is a key factor in cytokine-induced pancreatic β-cell damage and have suggested that inhibition of NF-κB may be a novel strategy for delaying the progression of T1DM (44, 45). Sakai
Since it is known that excessive cytokine leads to cell death via the apoptosis signaling pathway and that Bcl-2 and Bax protein expression is associated with apoptosis (47-50), we examined whether the Tat-CIAPIN1 protein regulates apoptotic signaling (Fig. 4). In cytokine-induced RINm5F β-cells, the Tat-CIAPIN1 protein significantly reduced cleaved Caspase-3 and Bax protein expression, whereas Bcl-2 protein expression was significantly increased by this fusion protein. These results indicate that Tat-CIAPIN1 plays a crucial role for cell survival by regulating signaling pathways in cytokine-exposed RINm5F β-cell (Fig. 4). Zhang
In summary, we showed that the Tat-CIAPIN1 protein inhibited pancreatic β-cell death by reducing ROS generation, activating NF-κB and MAPK, and regulating the apoptosis-associated proteins in cytokine-induced RINm5F β-cells. Although the precise function of the CIAPIN1 protein in T1DM remains to be verified, the Tat-CIAPIN1 protein plays a beneficial role in pancreatic β-cells. Therefore, we suggest that Tat-CIAPIN1 may provide a potential therapeutic protein agent for T1DM.
Tat-CIAPIN1 protein was prepared as described previously study (28). The used antibodies were obtained from Santa Cruz Bio-technology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA). 2‘,7‘-Dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cytokines (IL-1, TNF-α, and IFN-γ) were obtained from R&D system (Minneapolis, MN, USA). Fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) were purchased from Gibco (Carlsbad, CA, USA). All other agents were of the highest grade available unless otherwise stated.
Pancreatic β-cells (RINm5F β-cells) were obtained from the ATCC (Manassas, VA, USA) and maintained in RPMI1640 medium containing 10% FBS and 1% antibiotics.
To assess the ability of Tat-CIAPIN1 protein transduction, RINm5F β-cells were treated with 0.5-3 μM or 15-60 min of Tat-CIAPIN1 protein in culture medium. After washing, transduced levels were determined by Western blotting.
Protein concentrations were determined with Bradford assay (52). Equal amount of proteins were loaded onto 12% SDS-PAGE, and transferred to nitrocellulose membrane. The membranes were incubated in 5% milk followed by incubation overnight at 4°C with primary antibodies. After washing, the membranes were incubated with Horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Then, protein bands were visualized using ECL reagents (Amersham, Franklin Lakes, NJ, USA) (28, 33).
Cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After transduced Tat-CIAPIN1 protein (0.5-3 μM) into RINm5F β-cells for 1 h, cytokines (5 ng/ml IL-1β, 10 ng/ml TNF-α, and 10 ng/ml IFN-γ) were exposed the cells for 12 h. Cell viability was determined as described in previous (28).
DNA fragmentation was determined using a Cell Death Detection Kit (Roche Applied Science, Basel, Switzerland). After transduced Tat-CIAPIN1 protein (3 μM) into RINm5F β-cells for 1 h, cytokines (5 ng/ml IL-1β, 10 ng/ml TNF-α, and 10 ng/ml IFN-γ) were exposed the cells for 24 h. Then, DNA fragmentation levels were confirmed as described in previous (28, 33).
Intracellular ROS levels were determined using a DCF-DA staining. After transduced Tat-CIAPIN1 protein (3 μM) into RINm5F β-cells for 1 h, cytokines (5 ng/ml IL-1β, 10 ng/ml TNF-α, and 10 ng/ml IFN-γ) were exposed the cells for 22 h and the cells incubated for 30 min with DCF-DA (20 μM). Then, ROS production levels were confirmed as described in previous (28, 33).
Data represent the mean of three experiments ± SEM. Differences between groups were analyzed by ANOVA followed by a Bonferroni’s post-hoc test using GraphPad Prism software (version 5.01; GraphPad Software Inc., San Diego, CA, USA). P < 0.05 was considered to indicate a statistically significant difference.
This research was supported by Basic Science Research Program (2017R1D1A3B04032007 & 2019R1A6A1A11036849) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education.
The authors have no conflicting interests.