Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling

Abstract

Ciliary neurotrophic factor (CNTF) was identified as a survival factor in various types of peripheral and central neurons, glia and non-neural cells. At present, there is no available data on the expression and localization of CNTF-receptors in cementoblasts as well as on the role of exogenous CNTF on this cell line. The purpose of this study was to determine if cementoblasts express CNTF-receptors and analyze the mechanism of its apoptotic regulation effects on cementoblasts. OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals). Quantified transcriptional (RT-qPCR) and translational (WB) products of CNTFRα, IL-6Rα (CD126), LIFR, p-GP130, GP130, p-ERK1/2, ERK1/2, Caspase-8, -9, -3 and cleaved-caspase-3 were evaluated. Immunofluorescence (IF) staining was applied to visualize the localization of the CNTF-receptors within cells. The apoptosis ratio was measured with an Annexin-V FITC/PI kit. The ERK1/2 antagonist (FR180204, Calbiochem) was added for further investigation by flow cytometry analysis. The CNTF-receptor complex (CNTFRα, LIFR, GP130) was functionally up-regulated in cementoblasts while cultivated with exogenous CNTF. CNTF significantly attenuated cell viability and proliferation for long-term stimulation. Flow cytometry analysis shows that CNTF enhanced the apoptosis after prolonged duration. However, after only a short-term period, CNTF halts the apoptosis of cementoblasts. Further studies revealed that CNTF activated phosphorylated GP130 and the anti-apoptotic molecule ERK1/2 signaling to participate in the regulation of the apoptosis ratio of cementoblasts. In conclusion, CNTF elicited the cellular functions through a notable induction of its receptor complex in cementoblasts. CNTF has an inhibitory effect on the cementoblast homeostasis. These data also elucidate a cellular mechanism for an exogenous CNTF-triggered apoptosis regulation in a mechanism of ERK1/2 and caspase signaling and provides insight into the complex cellular responses induced by CNTF in cementoblasts.

Keywords: CNTF; CNTF-receptors; ERK1/2; apoptosis; caspase; cementoblasts.

Figure 1

The tripartite CNTF-receptor complex is up-regulated by ciliary neurotrophic factor in cementoblasts. (A,B) WB showed protein expression of the CNTF-receptors (CNTFRα, LIFR and IL-6Rα) in OCCM-30 cells induced by CNTF protein (400 ng/mL) for various periods. Internal β-actin serves as loading control. (C) The expression of mRNAs encoding the CNTF-receptors were quantified by RT-qPCR. The relative mRNA expression of each gene was obtained through normalizing to internal PPIB. The statistical significance was determined by student t-test (n = 3 for each group). (D,E) The CNTF-receptors immunofluorescent localization showed the expression of CNTFRα (red arrow), LIFR (yellow arrow) and IL-6Rα (orange arrow) in CNTF-treated OCCM-30 cells. Nuclei are stained with DAPI (blue). Scale bar: 100 μm (image magnification: 40×); 50 μm (image magnification: 60×). Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005).

Figure 2

Ciliary neurotrophic factor triggers GP130 protein expression and phosphorylated GP130 in cementoblasts. (A,B) The protein expression of GP130 and phosphorylated GP130 were determined by WB. Internal β-actin serve as loading control. The line chart shows the densitometric analysis of p-GP130 expression related to total GP-130 expression. (C) RT-qPCR quantification of GP130 (IL-6st) gene expression in OCCM-30 cells when treated with CNTF (400 ng/mL) for indicated time. The relative mRNA expression was obtained through normalizing to internal PPIB. (D,E) IF staining of subcellular localization of GP130 (white arrow) as well as p-GP130 (grey arrow) in non-stimulated cells (negative control) and CNTF-stimulated OCCM-30 cells. Nuclei are stained with DAPI (blue). Individual and merged images of GP130 and p-GP130 are shown. Scale bar: 100 μm (image magnification: 40×); 50 μm (image magnification: 60×). Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Figure 3

Ciliary neurotrophic factor impairs OCCM-30 homeostasis and activates the expression of ERK1/2 MAPK signaling. (A,B) Immunofluorescence microscopy images show representative proliferation markers Ki-67 expression. OCCM-30 cells were exposed to CNTF (400 ng/mL) and then underwent immunofluorescence staining for Ki-67 to visualize cells in the proliferation stage. The proportion of proliferating cells for each group was quantified according to Ki-67 positive cells (Ki-67⁺)/total cell counting (DAPI). (C) Cell viability assay was performed by MTS assay. IL-6 cytokine served as positive control. (D,E) Representative immunoblot of p-ERK1/2 protein expression in the presence of CNTF (400 ng/mL) at different time points. Internal β-actin serves as loading control. Densitometric immunoblot analysis of bands indicated the enhanced p-ERK1/2 expression relative to that of the control group. Densitometric results are showed as fold change. Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (* p < 0.05; ** p < 0.005; *** p < 0.0005).

Figure 4

Ciliary neurotrophic factor regulates apoptosis rate and triggers the caspases signaling. (A,B) Representative plots from Annexin-V FITC and PI staining by flow cytometry analysis performed in triplicate are shown. Apoptotic cells (Annexin-V FITC⁺/PI⁺) are shown in the upper right quadrant. Graphics show the percentages of apoptotic cells exposed to CNTF (400 ng/mL) at different time periods. (C,D) Representative immunoblot showed that the protein expression of Caspase-8, -9 and -3 as well as cleaved-caspase-3 in response to CNTF (400 ng/mL) in a time-dependent manner. β-actin was loaded as an internal control. (E) mRNA expression of Caspase-8, -9 and -3 in response to CNTF (400 ng/mL) stimulation at indicated time period. Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (ns, no significant difference; * p < 0.05; ** p < 0.005; *** p < 0.0005).

Figure 5

ERK1/2 signal is involved in the regulation of apoptosis of cementoblasts and the caspases pathway. (A,B) Graphics show the percentages of apoptotic cells exposed to ERK1/2 inhibitor (1.0 μg/mL, FR180204) as well as co-stimulation with CNTF (400 ng/mL). (C) The scheme summarizes the mode of CNTF action in cementoblasts: CNTF activated the tripartite CNTF-receptor complex targets and phosphorylated GP130 protein, which recruits ERK1/2 signaling and caspases signaling expression. FR180204 promotes apoptosis in OCCM-30 cells and CNTF addition suppressed the ERK1/2 inhibitor-induced apoptosis within a short-term period. Bar indicates values ± standard deviation (SD) which represent three independent experiments. Statistically significant differences (indicated by asterisks) are shown as follows (** p < 0.005; *** p < 0.0005).