The IL-12R complex consists of two heterodimer chains named IL-12Rβ1 and IL-12Rβ2. The shared IL-12Rβ1 could also form heterodimer complex with IL-23R chain that is specifically recognized by IL-23. Since IL-12Rβ2 is a tumor suppressor gene, it might be true that IL-23R also possesses antiproliferative and proapoptotic effects. To test this assumption, CCK8 assay was performed to measure the cell growth ability affected by IL-23R. The 293ET cells transfected with different doses of IL-23R were collected at different time points. Figure 1 clearly demonstrates that the numbers of viable cells were significantly reduced as the doses of IL-23R increased after 24, 48 and 72 h post-transfections, indicating that the proliferation potentials of IL-23R transfected cells were markedly inhibited.

The next question that we tried to ask was whether IL-23R over-expression could affect cell survival. To this end, 293ET cells were transiently transfected with IL-23R. After 48-h posttransfection, the cells were collected and subjected to Annexin V and PI double staining. Subsequent flow cytometric analysis showed a marked increase in the population of Annexin V+/PI+ double positive cells in responding to higher dose IL-23R delivery (Figure 2a). Statistical analysis showed that higher dose delivery of IL-23R significantly increased the population of late apoptotic cells (Figure 2b). Since 293ET cell line is a cell line transformed by both SV40 large T antigen and EB virus nuclear antigen EBNA1, the biological properties of 293ET cells might not behave completely like normal cells. The non-cancer character of 293ET cell line could not exclude the possibility that normal cells/tissues are still not sensitized to IL-23R mediated apoptosis.

To further test its pro-apoptotic effect, the plasmid IL-23R DNAs were also transiently transfected into human cervical cancer cell line HeLa cells. Consistently, flow cytometric analysis shows that the Annexin V+/PI+ double positive HeLa cells were dramatically increased as the delivered dose of IL-23R increased (Figure 3a). In addition, the activities of caspase 3/7 effectors were also directly detected in transfected HeLa cells. The caspase 3/7 activities are monitored by aminoluciferin which is coupled with caspase 3/7 substrate cleavage site followed by luciferase detection. Indeed, a significant enhancement in caspase 3/7 activity was observed as the dose of transfected IL-23R increased (Figure 3b), indicating that IL-23R is able to activate the key effectors in the apoptotic pathway. Overall, our data indicates that IL-23R is indeed an apoptotic inducer that could activate the apoptotic processes in both 293ET and HeLa cells.

Since IL-23R was over-expressed in the targeted cells, IL-23R induced cell apoptosis might be associated with the activation of intrinsic mitochondria pathway. To test this hypothesis, cell lysates prepared from IL-23R transfected 293ET cells were first probed with anti-Bcl-xL antibody. Bcl-xL belongs to BCL2 family and exerts its anti-apoptotic function by preventing the oligomerization of BAX/BAK proteins at the mitochondrial outer membrane [20]. Indeed, over-expression of IL-23R in 293ET cells inhibited the expression levels of Bcl-xL proteins in a dose-dependent manner (Figure 4a). Further study indicated that increased delivery of IL-23R into 293ET cells also markedly promoted Apaf-1 expression and procaspase 9 cleavages that subsequently lead to the activation of the executioner caspase 3 (Figure 4b). In addition, a marked increase in the production of the cleaved PARP-1 was also detected as the doses of IL-23R increased. Thus, the data strongly indicates that IL-23R mediated apoptosis is associated with intrinsic mitochondrial pathway in 293ET cells.

To further confirm the result, the same assay system was also applied to HeLa cells. In agreement with the results of 293ET cells, overexpression of IL23R significantly decreased Bcl-xL gene products in a dose dependent manner (Figure 5a). The dose dependent changes were also true for the increased Apaf-1 expression, and promoted caspase 9 and caspase 3 cleavages (Figure 5b). Therefore, the results strongly indicate that IL-23R mediated activation of intrinsic mitochondrial pathways might be a common mechanism responsible for the induction of cell apoptosis in mammalian cells.

Cell apoptosis is frequently associated with the alterations of cell survival signaling pathways. To detect the influence of IL-23R mediated apoptosis on the activation of major mitogenic signaling pathways, western blot analysis was conducted to assay the phosphorylation levels of STAT1, STAT3, Akt and ERK in 293ET cells. Figure 6a shows that the activation of ERK was inhibited as the doses of IL-23R increased. Interestingly, the activations of STAT3 were inversely related to the protein levels of inactivated STAT3 which were upregulated with the increased doses of IL-23R (Figure 6b). However, no significant alterations were observed when the phosphorylation levels of both STAT1 and Akt were assayed (Figure 6a). The data indicates that IL-23R could markedly inhibit both RAS/MAPK and STAT3 signaling pathways but not STAT1 and Akt signaling pathways in 293ET cells.

To further elucidate the molecular mechanisms responsible for IL-23R mediated apoptosis, the cell survival signaling pathways were also assayed in transfected HeLa cells. Consistent with the previous results, the increased delivery of IL-23R markedly reduced the phosphorylation levels of both ERK and STAT3, demonstrating that IL-23R inhibited both ERK and STAT3 pathways in a dose-dependent manner (Figure 7a). However, no significant alteration in Akt phosphorylation could be detected as the dose of IL-23R increased (Figure 7b). Interestingly, increased delivery of IL-23R into HeLa cells did not reduced but markedly enhanced the phosphorylation level of STAT1, indicating that activation of STAT1 might be associated with cell apoptosis in HeLa cells (Figure 7b). Overall, the data strongly indicates that inhibition on both ERK and STAT3 signaling pathways might be the common mitogenic signaling pathways associated with IL-23R mediated apoptosis in mammalian cells.

To assay the endogenous expressions of IL-23 complex in responding to IL-23R overexpression, total RNAs were extracted from IL-23R transfected 293ET and HeLa cells. RT-PCR analysis revealed that no significant alterations in the expression of IL-23α subunit were observed as the delivered doses of IL-23R increased in 293ET cells, while no expression of IL-12β mRNA could be detected in the same condition (Figure 8a). Similarly, in IL-23R transfected HeLa cells, no significant enhancement of either IL-23α or IL-12β mRNAs could be detected (Figure 8b). In contrast, western blotting analysis demonstrates that increased delivery of IL-23R into either 293ET or HeLa cells significantly promoted IL-12Rβ1 expressions (Figure 8c,d). To further detect the driving force of IL-23R mediated apoptosis, cell supernatants were collected and assayed for the presence of IL-23 ligand by ELISA analysis. Figure 8e demonstrates an inverse correlation between the levels of extracellular IL-23 ligands versus the increased delivery of IL-23R inside the transfected cells, indicating that overexpression of IL-23R does not promote IL-23 production in HeLa cells. Collectively, the data indicates that overexpression of IL-23R might induce cell apoptosis in a ligand independent manner.

Some Bcl-2 family members, for instance Bcl-xL, could function as a negative regulator to counteract the cellular apoptotic process. Under certain circumstances, enhanced expressions of Bcl-xL promote tumor growth and subsequently lead to drug resistance that in turn affects the efficacy of chemotherapy. Therefore, Bcl-xL has long been considered as an ideal target for certain cancer treatments [20]. The expression level of Bcl-xL was regulated by many transcription factors such as Ets, NFκB, STAT3 and AP-1 [21], whereas ERK1/2 pathway could be the upstream regulator to be responsible for the activations of these transcriptional factors. Emerged evidence has strongly indicated that chemical mediated inhibition on ERK1/2 signaling pathway can greatly improve the efficacy of tumor chemotherapy by down-regulating the expression of Bcl-xL that often leads to the activation of intrinsic apoptotic pathway [22–26]. Therefore, it is likely that IL-23R could be developed as a new therapeutic target for certain cancer treatment.

Human embryonic kidney cells expressing SV40T and EBNA1 (293ET) and human cervical cancer cell line (HeLa) were derived from the Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Mouse anti-Myc, rabbit anti-caspase 3, rabbit anti-STAT3, rabbit anti-p-STAT3 goat anti-Akt1/2, rabbit anti-p-Akt1/2/3, rabbit anti-ERK1, rabbit anti-p-ERK and mouse anti β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-PARP1 was purchased from Epitomics Inc. (Burlingame, CA, USA). Rabbit anti-Apaf1 antibody was purchased from Beijing Biosynthesis Biotechnology (Beijing, China). Rabbit anti-STAT1 and Rabbit anti-p-STAT1 were purchased from Bioworld Technology Inc. (Bioworld, CA, USA). The peroxidase-conjugated secondary antibodies were from Zhongshan Biotechnology (Beijing, China). The full length of human IL-23R cDNA was isolated from the total RNAs of PBMC [10], and subcloned into the SalI/KpnI sites of pCMV-Myc vector to generate the eukaryotic expression vector pCMV-Myc-IL-23R whose sequence was subsequently confirmed by DNA sequencing analysis.

Cells were cultured in DMEM containing 10% fetal calf serum at 37 °C supplemented with 5% CO₂. Transient transfection was performed using Vigofect (Vigorous Inc, Beijing, China) with the indicated plasmid DNAs. Cells seeded onto 6 well culture plates or 35 mm² culture plates were received 0, 3 and 6 μg transfected pCMV-Myc-IL-23R DNAs, while cells plated onto 12 well culture plates were delivered with the doses of 0, 0.5 and 2 μg of pCMV-Myc-IL-23R DNAs. Then the transfected cells were continuously incubated for 48 h before harvesting.

Cell proliferation was monitored by CCK-8 assay. Briefly, cells were cultured to a density of 5 × 10⁴ cells/mL and then certain amount cells were released and transferred into a 96-well plate after 24, 48 or 72 h post-transfection. Cell-counting kit-8 (Dojindo, Kumamoto, Japan) solution was added into each well. After 2.5 h incubation, the absorbance at 450 nm was measured using a microplate reader.

Cell apoptosis was detected by Annexin V-FITC apoptosis detection kit (Beijing RuiBang XingYe Science & Technology Co., LTD, Beijing, China). After 48 h transfection, cells were released and washed with pre-cooled 1× PBS. Then the cell pellet was resuspended with 50 μL binding buffer followed by adding 5 μL Annexin V-FITC and 5 μL Propidium Iodide (PI). Then the cell suspension was incubated away from light for 15 min at room temperature. Another 150 μL binding buffer was added into the mixture before flow cytometric analysis by Accuri C6 (BD Biosciences, San Jose, CA, USA).

Caspase-3/7 activity was detected using Caspase-Glo 3/7 assay kit (Promega, Madison, WI, USA). After 48 h transfection, cells were released and washed with pre-cooled 1× PBS. Then cells were transferred into a 96-well plate and the same amount of Caspase-Glo 3/7 Reagent was added to each well. The mixture was incubated at room temperature for 1 h. The luminescence of each sample was measure in a plate-reading luminometer.

Total RNAs were extracted from the cultured cells with TRIzol (Invitrogen, Carlsbad, CA, USA). The primers used in the RT-PCR reactions were listed in Table 1. One microgram of total RNAs was subjected to RT-PCR analysis using PrimeScript One Step RT-PCR Kit (Takara Biotechnology, Dalian, China). The reverse transcription was conducted at 42 °C for 35 min. After being denatured at 94 °C for 5 min, the reaction products were subsequently PCR amplified at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 40 s for 25 cycles before extension at 72 °C for 10 min. The level of β-actin gene expression was served as internal control.

The transfected cells were lysed with gentle rotation in a lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 200 μM NaVO₄, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 1 μM PMSF). Cell lysate for each sample was resolved on 12% SDS-PAGE followed by blotting onto Hybond nitrocelluar membrane (Amersham Biosciences, Freiburg, Germany). The transferred membrane was then probed with primary antibodies followed by relevant secondary antibodies conjugated to horseradish peroxidase. Detection was enhanced by chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The secretion of IL-23 ligand was monitored by human interleukin 23 (IL-23) ELISA Kit (Beijing Gersion Biotechnology Co. Ltd, Beijing, China). Briefly, after 48 h transfection, 10 μL of culture supernatant together with 40 μL of sample diluent were added into the testing samples that were well pre-coated with hIL-23 antibody. Then, 100 μL of HRP-conjugated reagent were added into each well and incubated at 37 °C for 60 min. After aspirating and washing, 50 μL of chromogen A and B were added into each well and incubated away from light at 37 °C for 15 min. Finally, about 50 μL of stopping solution was added to each reaction before detection at 450 nm using a microtiter plate reader.

All values were calculated as mean ± standard deviation (SD) from four independent experiments. The statistical difference between the assayed group and the standard group was subject to student’s t test (two-tailed, unpaired). The calculated difference was considered significant as the p value < 0.05 or < 0.01.