| Part One Expression of proton-sensing G protein-coupled receptors in endplate chondrocytes of the intervertebral disc (IVD) and OGR1 is participated in acid-induced apoptosis and metabolism of endplate chondrocytes in the IVDObjective:This objective of the study is to demonstrate whether the expression of G protein-coupled receptors in endplate chondrocytes of intervertebral disc and OGR1 activation leads to apoptosis and metabolism of endplate chondrocytes induced by extracellular acid in the rat IVD.Methods:Rat endplate chondrocytes were obtained from 4-week-old male Sprague-Dawley rats weighing 160-200 g. A sterile ophthalmic scissor was used to chop the slices into 1 mm3 pieces, and these were transferred to a 50-mL sterile screw cap test tube. Tissues were digested with 0.25% trypsin at 37 ℃ for 30 min followed by 0.2% collagenase at 37 ℃ for 2 h. After centrifugation 400 ×g for 15 min at 37 ℃, the supernatant fluids was discarded, and then the tissue suspension was washed 3 times with DMEM with 10% fetal bovine serum (FBS). The released cells were cultured in DMEM with 10% FBS and plated at a density of 2 × 104 cells/well in 96-well plates. The morphologicalchanges of endplate chondrocytes were observed using a phase contrast microscope. The cells in vitro were judged to be endplate chondrocytes by toluidine blue staining of glycosaminoglycan (GAG) biosynthesis and immunohistochemistry staining of type II collagen. To examine whether proton-sensing G protein-coupled receptors are involved in acid-induced apoptosis of endplate chondrocytes, we first determined the mRNA levels of OGR1, G2A, GPR4, and TDAG8 in endplate chondrocytes. Changes in mRNA levels of proton-sensing GPR receptors in response to acidosis were examined by Real-time PCR. We next investigated the expression of OGR1 protein using Western blotting analysis. We used immunofluorescence to determine cellular and subcellular distributions of OGR1 in endplate chondrocyte. To verify whether activation of OGR1 is involved in apoptosis of endplate chondrocytes of IVDs in response to acidic circumstances, an experiment with RNA interference (RNAi) was performed. OGRl expression was reduced upon transfection with OGR1 shRNA. We examined whether modulation by OGRl-RNAi of acid-induced apoptosis of endplate chondrocytes is associated with changes in intracellular calcium [Ca2+]i. [Ca2+]i was analyzed with laser scanning confocal microscopy by incubating with 4 μM Fluo-3-AM and 0.02% Pluronic F-127 (Biotium). Induction of apoptosis was examined with a Cellular DNA fragmentation ELISA kit quantified to determine the release of apoptotic DNA fragments into cytoplasm due to nucleolytic cleavage after treatment with an extracellular solution at pH 6.4 or 7.4. Apoptosis was analyzed by using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining kit in accordance with the manufacturer’s instructions. Briefly, endplate chondrocytes were treated with extracellular solution (pH 6.4 or 7.4) for 1 h. After acid incubation for 18 h, Flow cytometry analysis was performed. Then endplate chondrocytes were washed in cold PBS, and stained with 5 μL of AnnexinV-FITC and 5 μL of PI in 500 μL binding buffer for 10 min in the dark at room temperature. Stained cells were quantified by flow cytometry. The coverslips were stained with 1% aqueous uranyl acetate, and examined with a transmission electron microscope for stduying apoptosis of endplate chondrocytes. The glycosaminoglycan (GAG) content of endplate chondrocyte culture supernatant was measured using a dimethyl-methylene blue spectrophotometric assay, while hydroxyproline (HYP) content was demonstrated by a chloramine T assay. Relative mRNA and protein expression levels of MMP-2, MMP-9, TIMPs-1, and TIMPs-2 was examined using Real-time polymerase chain reaction (PCR) and ELISA, respectively. Moreover, Expression of type Ⅱ collagen (CO Ⅱ) and aggrecan at mRNA and protein levels was determined.Results:The primary culture rat endplate chondrocytes were polygonal or round shape. After 24h of primary culture, most rat endplate chondrocytes of IVDs grew adhering with the paste wall and displayed long spindle and round shape, cytoplasm was translucent. After 5 days, rat endplate chondrocytes of IVDs spreaded whole surface of dish. The cells in vitro were evaluated to be rat endplate chondrocytes by using toluidine blue staining of glycosaminoglycan (GAG) biosynthesis and immunohistochemistry staining for type Ⅱ collagen (CoⅡ). To measure whether proton-sensing G protein-coupled receptors are involved in acid-induced apoptosis of rat endplate chondrocytes, we first evaluated OGR1, G2A, GPR4, and TDAG8 mRNA. RT-PCR analysis revealed high levels of OGR1 mRNA and low levels of G2A and TDAG8 mRNA in rat endplate chondrocytes. However, GPR4 mRNA was not found. The mRNA levels of OGR1 increased in response to acidosis (P< 0.01), whereas those of the other receptors in endplate chondrocytes were unchanged. This indicates that OGR1 is responsive to pH in endplate chondrocytes.We next investigated the expression of OGR1 protein using Western blot analysis, and found that OGR1 protein (41 kDa) was expressed at readily detectable levels in rat endplate chondrocytes of IVDs, which was consistent with data from a positive control peptide. We used immunofluorescence to determine cellular and subcellular distributions of OGR1 in endplate chondrocytes; OGR1 protein was mostly located in the cytoplasm. Altogether, these results suggest that OGR1 was expressed in the endplate chondrocytes of the rat IVDs. OGR1 expression was decreased upon transfection with OGR1 shRNA, showing that this strategy was effective in specifically knocking down OGR1 expression. We used extracellular acid (pH 6.4) to induce rat endplate chondrocytes in the presence of pharmacological inhibitors. As shown by confocal micrographs and representative traces, addition of acid induced a transient increase in [Ca2+]ⅰ in endplate chondrocytes. Transient [Ca2+]ⅰ elevation was attenuated when the cells were transfected with OGR1 shRNAs. However, the negative control shRNA had little effect on acid-induced increases in [Ca2+]i under the same pH. Thus, OGRl may be the mediator in acid-induced increases in [Ca2+]ⅰ in the endplate chondrocytes of IVDs. To investigate whether OGR1 is involved in apoptosis of rat endplate chondrocytes induced by extracellular acid (pH 6.4) in the rat IVD, the endplate chondrocytes were stimulated at pH 6.4 or pH 7.4. Cell apoptosis was measured via DNA fragmentation assays at various time points. A 1-h incubation at pH 6.4 induced a time-dependent increase in apoptosis of endplate chondrocytes, compared with endplate chondrocytes treated with pH 7.4 medium; continuous treatment at pH 6.4 induced greater apoptosis. Flow cytometry with Annexin V-FITC/PI double staining and electron microscopy were used to further confirm the induction of apoptosis. Consistent with the DNA fragmentation assays, Annexin V-FITC/PI double staining and electron microscopy showed similar increases in cell apoptosis induced by incubation for 1 h at pH 6.4 compared with pH 7.4. Treatment of endplate chondrocytes with OGR1 shRNAs attenuated the increase in apoptosis induced by incubation with pH 6.4 extracellular solution. However, the negative control-shRNAs had little effect on the apoptosis of endplate chondrocytes under the same pH conditions. This implies that OGR1, when exposed to extracellular acidification, is associated with apoptosis of endplate chondrocytes in IVDs. A 1-h incubation at pH 6.4 induced a decrease of MMP-2, MMP-9, TIMPs-1, and TIMPs-2 of endplate chondrocytes, compared with endplate chondrocytes treated with pH 7.4 medium; while induced an increase of MMPs/TIMPs expression in endplate chondrocytes. Blocking OGR1 could significantly suppress the reduction of MMP-2, MMP-9, TIMPs-1, and TIMPs-2 levels induced by extracellular acidification, while the effect of MMPs/TIMPs expression was decreased. We also found that the acid-induced expression levels of type Ⅱ collagen and aggrecan were increased by the OGR1 shRNAs.Conclusion:OGR1 was expressed in endplate chondrocytes of rat IVDs and have an important role in the regulation of chondrocyte apoptosis and cell metabolism in response to extracellular acid stimulation. Therefore, OGR1 played a pivotal role in CEP degeneration, and may be a original target for development of new therapies for IVDs.Part Two The mechanism of proton-sensing receptor OGRl-mediated calcium influx on endplate chondrocytes apoptosis of the intervertebral discObjective:The mechanism of proton-sensing receptor OGR1-mediated calcium influx on endplate chondrocytes apoptosis of the intervertebral disc.Methods:Rat endplate chondrocytes were obtained from 4-week-old male Sprague-Dawley rats weighing 160-200 g. A sterile ophthalmic scissor was used to chop the slices into 1 mm3 pieces, and these were transferred to a 50-mL sterile screw cap test tube. Tissues were digested with 0.25% trypsin at 37 ℃ for 30 min followed by 0.2% collagenase at 37 ℃ for 2 h. fter centrifugation 400 ×g for 15 min at 37 ℃, the supernatant fluids was discarded, and then the tissue suspension was washed 3 times with DMEM with 10% fetal bovine serum (FBS). The released cells were cultured in DMEM with 10% FBS and plated at a density of 2 × 104 cells/well in 96-well plates. The morphological changes of endplate chondrocytes were observed using a phase contrast microscope. To investigate whether activation of OGR1 is involved in acid-induced apoptosis of rat endplate chondrocytes in IVDs, an experiment with RNA interference (RNAi) was performed. OGR1 expression was reduced upon transfection with OGR1 shRNA. We examined whether modulation by OGR1-RNAi of acid-induced apoptosis of endplate chondrocytes is associated with changes in [Ca2+]i. Intracellular calcium ([Ca2+]i) was analyzed with laser scanning confocal microscopy by incubating with 4 μM Fluo-3-AM and 0.02% Pluronic F-127 (Biotium). Induction of apoptosis was examined with a Cellular DNA fragmentation ELISA kit quantified to determine the release of apoptotic DNA fragments into cytoplasm due to nucleolytic cleavage after treatment with an extracellular solution at pH 6.4 or 7.4. Moreover, we examined that BAPTA-AM, cell-permeable cytosolic calcium chelator, but not the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA), suppressed acid-induced increases in [Ca2+]i. Moreover, we examined that the acid-induced rise of [Ca2+]ⅰ was inhibited by the PLC inhibitor U73122. We further evaluated the role of [Ca2+]ⅰ, calcium-sensitive proteases, and their downstream signaling in acid-induced apoptosis of endplate chondrocytes by treatment with BAPTA-AM, EGTA, U73122, calpeptin, cyclosporine A, and KN-93 as well as whether modulation by OGR1-RNAi. The protein level of calpain, calcineurin, Bid, Bad, Caspase 3, and cleavage of poly (ADP-ribose) polymerase (PARP) in rat endplate chondrocytes of the IVD was detected by Western blot.Results:It is well accepted that elevation of Ca2+ levels is important for the regulation of apoptosis. We examined whether modulation by OGRl-RNAi of acid-induced apoptosis of endplate chondrocytes is associated with changes in [Ca2+]ⅰ. We used extracellular acid (pH 6.4) to induce rat endplate chondrocytes in the presence of pharmacological inhibitors. As shown by confocal micrographs and representative traces, addition of acid induced a transient increase in [Ca2+]i in endplate chondrocytes. Moreover, we found that BAPTA-AM, but not the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA), suppressed acid-induced increases in [Ca2+]ⅰ. Ca2+ release from intracellular stores often involves phospholipase C (PLC)-mediated synthesis of inositol 1,4,5-trisphosphate. We found that the acid-induced rise of [Ca2+]ⅰ was inhibited by the PLC inhibitor U73122 (10 μM). These results indicated that acid-induced increases in [Ca2+]ⅰ might be attributed to Ca2+ release from intracellular stores in a PLC-dependent manner in endplate chondrocytes. Transient [Ca2+]ⅰ elevation was attenuated when the cells were transfected with OGR1 shRNAs. However, the negative control shRNA had little effect on acid-induced increases in [Ca2+]ⅰ under the same pH. Thus, OGR1 may be the mediator in acid-induced increases in [Ca2+]ⅰ in the endplate chondrocytes of IVDs. Because a rise in [Ca2+]ⅰ is important in apoptosis, we further evaluated the role of [Ca2+]ⅰ in acid-induced apoptosis of endplate chondrocytes. Similar to the effects of OGR1 RNAi, chelation of cytosolic Ca2+ with BAPTA-AM (50 μM) and the PLC inhibitor U73122 (10μM) abolished the enhancement of apoptosis induced by an extracellular solution at pH 6.4, but not that at pH 7.4. These results suggest that extracellular acidic conditions induced Ca2+ influx through OGR1 and thereby induced apoptosis of endplate chondrocytes in IVDs. We next investigated which downstream Ca2+ -activated proteases were activated during the process of acid-induced endplate chondrocyte apoptosis. Calpain is a Ca2+ -activated neutral cysteine protease involved in apoptotic signaling. Here we wanted to know whether acid-induced endplate chondrocyte apoptosis was related to calpain activation. To inhibit calpain activity, we used a membrane-permeable inhibitor that specifically inhibits calpain activation (calpeptin). Interestingly, calpeptin (10 μM) significantly inhibited acid-induced apoptosis, indicating that endplate chondrocyte apoptosis under these conditions was dependent on calpain activation. Calcineurin is a downstream target of Ca2+ which putatively contributes to apoptosis. To investigate the role of the calcineurin pathway in acid-induced rat endplate chondrocyte apoptosis, we investigated the effect of cyclosporine A (1μM), a specific calcineurin inhibitor. Cyclosporine A significantly inhibited acid-induced endplate chondrocyte apoptosis. However, we did not observe any changes in acid-induced endplate chondrocyte apoptosis after treatment with KN-93 (1μM), a specific CaMKII inhibitor. This indicates that acidification increases endplate chondrocyte apoptosis in a CaMKII-independent manner. To directly investigate the involvement of OGR1 in acid-induced activation of calpain and calcineurin, we used Western blot to measure calpain and calcineurin protein levels. OGR1-RNAi markedly inhibited acid-induced calpain and calcineurin protein expression in rat endplate chondrocytes. This indicates that OGR1-mediated Ca2+ influx contributes significantly to acid-induced endplate chondrocyte apoptosis by regulating calcium-sensitive proteases. To assess whether acid-induced apoptotic signals were mediated by elevation of [Ca2+]ⅰ, expression levels of the apoptosis-related proteins Bid, Bad, Caspase-3, and PARP were determined by Western blot. The levels of Bid were significantly higher 4 h after the exposure of endplate chondrocytes to extracellular acid relative to these levels at 0 h; the levels of Bad were significantly increased after 8 h. Caspase 3, a typical execution caspase in the apoptotic process, was clearly activated by extracellular acid since cleavage of Caspase 3 and cleavage of PARP (a substrate of Caspase 3) were distinctly evident after 4 h of incubation under acidic conditions. Moreover, when the cells were pretreated with BAPTA-AM, acid-induced Bid, Bad, and Caspase 3 activation and PARP cleavage were inhibited, with no significant difference between 0 h and 4 h. This suggests that extracellular acid-induced apoptotic signals in endplate chondrocytes were mediated by [Ca2+]i elevation. To investigate the involvement of an OGR1-mediated calcium-induced signal pathway in acid-induced apoptosis of rat endplate chondrocytes, RNAi for OGR1 experiments were performed. Similar to the effects of BAPTA-AM, enhancement of Bid, Bad, and Caspase 3 activation and PARP cleavage via extracellular acid was inhibited by OGR1-RNAi, with no significant difference in the levels of these proteins between 0 h and 4 h. Our results suggest that OGR1 is involved in acid-induced calcium-activated signals in the apoptosis of rat endplate chondrocytes.Conclusion:OGR1-mediated [Ca2+]i increase has a decisional role in apoptosis of rat endplate chondrocytes by adjusting activation of calcium-sensitive proteases and their downstream signaling including calpain, calcineurin, Bid, Bad, Caspase 3 and, PARP. |
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