The Effect And Mechanism Of Interleukin-17 Inducing Osteoclast-like Cells Differentiation Via Osteoblasts

Background and ObjectiveAlveolar bone resorption is the first step of bone remodeling during orthodontic treatment and mature osteoclast (OC) is the only cell to be responsible for bone resorption. OB mediate pOC diffentiation and activation by multiple factors and ways..Those factors were called bone resorption-relative factors. Mechanical stress induces OB to produce such facorrs. Among them, Receptor activator of NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are the two key factors inducing OC differentiation and function. Inflammatory mediators released from periodontal tissue trigger the biological process of alveolar bone resorption. Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-αare known as born resorption-related-inflammatory cytokines and play important roles to stimulate OC differentiation and activity. Higher levels of prostaglandin E2 (PGE2) were found in the gingival crevicular fluid of teeth undergoing orthodontic movement. PGE2 is able to mediate the inflammatory response and to induce bone resorption through the stimulation of OC differentiation. In addition, the application of compressive force induces bone formation by increasing levels of PGE2 in OB. These reports suggest that PGE2 plays an important role in bone remodeling affecting bone resorption and formation via autocrine action. We recently reported that compressive force induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE2 in OB. IL-17 is a kind of inflammatory cytokines.It originally was cloned from activated T cells (Thl7 cells) but is well known until resently. It shares little or no homology with other proteins and interleukins. At least six members of the family and five subtypes of receptors (IL-17Rs) are in the human and mouse genomes. IL-17 induced inflammation and affect osteoclastic resorption indirectly via osteoblasts. Some reports indicate that IL-17 shows a synergistic effect with IL-10 and TNF-a and induces production of IL-6 in fibroblasts. In OB, there were reported that IL-17 enhanced the synthesis of TNF-a-stimulated IL-6 synthesis and prostaglandin F2a-stimulated IL-6. IL-17 stimulates bone resorption by inducing RANKL expression. So we hypothesize that IL-17 involves in and mediates the process of alveolar bone resorption via OB.However, the expression of IL-17 in OB induced by orthodonic force has not been reported.Therefore, in the present study, we examined the effect of compressive force on the expression of IL-17 and their receptors in OB. We also examined the effect of different concertration of IL-17 on OC differentiation via OB and the indirect effect of IL-17 inducing OC formation, and investigate in vitro the roles of IL-17 inducing OCL formation via OB. This study will contribute to a better understanding of the molecular biology mechanism of orthodontic tooth movement and alveolar bone remodeling.Methods1. Measure the expression of mRNA and protein of IL-17s and their receptors in osteoblast during compressive force.MC3T3-E1 cells from a mouse calvarial cell line were used as osteoblast-like cells. MC3T3-E1 cells were maintained in a-minimal essential medium containing 10% (v/v) heat-inactivated fetal bovine serum at 37℃in a humidified atmosphere of 95% air and 5% CO2. MC3T3-E1 cells were placed into 96-well tissue plates at a density of 2.0 x 104 cells/cm2 and cultured for 7-10 days in a-MEM containing 50 mMβ-glycerophosphate and 50μg/ml ascorbic acid. The presence of mineralized nodules was determined by staining with alizarin red S. For being applied the compressive force, MC3T3-E1 cells were seeded in 100-mm cell culture dishes at a density of 2.0×104 cells/cm2 and incubated until 90% confluent. The cells were then compressed continuously using a uniform compression method similar to those described previously. The cells were subjected to 1.0,2.0 or 3.0 g/cm2 (98,196 or294 Pa, respectively) compressive force for 1,3,6,9,12, or 24 h. The cell growth was measured by Cell Counting Kit after being subjected to the compressive force. Then 1.0 and 2.0g/cm2 compressive force were chosen for experiment. The expression of mRNA of IL-17s, their receptors and IL-la, IL-6 was measured by Real time RT-PCR. IL-17A protein expression after 9h was measured by immunofluorescence and ELISA.MC3T3-E1 cells were seeded onto 100-mm cell tissue culture dishes at a density of 2 x 104cells/cm2. After overnight incubation, the cells were cultured for 6h,24h or 72 h in a-MEM with 0 or 10ng/ml IL-17A. The expression of mRNA of IL-17Rs was measured by Real time RT-PCR.2. Measure the effect of IL-17A on PGE2 and cytokines expression in osteoblast.MC3T3-E1 cells were seeded onto 100-mm cell tissue culture dishes at a density of 2 x 104cells/cm2. Then change the medium with 1% FBS for 12h after 80% confluent.(1) The cells were cultured for up to 72 h in a-MEM with 0,0.1,1.0, or lOng/ml IL-17A. The expression of mRNA of COX 1, COX2, RANKL, OPG, M-CSF, IL-la, IL-6, TNF-a was measured by Real time RT-PCR. The expression of protein of PGE2, RANKL, OPG, M-CSF, IL-la, IL-6, TNF-a was measured by ELISA.(2) The cells were cultured for up to 24 h in a-MEM with 0 or 1.5 ng/ml PGE2. The expression of mRNA of RANKL, OPG, M-CSF, IL-la, IL-6 andTNF-a was measured at 24h by RT-PCR.(3) The cells were cultured for up to 72 h in a-MEM with 0, or 10 ng/ml IL-17A alone or in the presence of 10μM celecoxib. The expression of protein of PGE2, RANKL, OPG, M-CSF, IL-la, IL-6, TNF-a was measured by ELISA.3. The effct of IL-17A on osteoclast-like cells differentiation via osteoblast.MC3T3-E1 cells were seeded onto 100-mm culture dishes at a density of 2 x 104 cells cm 2 and left overnight to settle. The cells were then cultured for up to 72 h inα-MEM with 0 or 10 ng mL-1 IL-17A. The cell culture medium was changed to a-MEM without IL-17A, and the cells cultured for a further 24 h. Each sample of culture medium collected was diluted to 30% and supplemented with 50 ng mL-1 of soluble RANKL to be used as conditioned medium. RAW264.7 cells were plated into 96-well microplates at a density of 1.25 x 104 cells cm-2 and left overnight to settle. Conditioned medium plus or minus IL-17A was then added to the cells for up to 10 days. Cells were then fixed and stained on days 3,5,7, and 10 of culture using a TRAP staining kit. RAW264.7 cells were plated into 6-well microplates at a density of 1.25×104 cells cm-2 and cultured for up to 10 days in the conditioned medium. The expression of mRNA and protein of cathepsin K, MMP-9 and CAII was measured by Real time RT-PCR and Western-blot respectively.MC3T3-E1 cells were seeded onto 100-mm culture dishes at a density of 2×104 cells cm-2 and left overnight to settle. The cells were then cultured for up to 72 h in the presence or absence of 10ng/ml IL-17A and/or 10μM celecoxib. The cell culture medium was changed to a-MEM without IL-17A and celecoxib, and the cells cultured for a further 24 h. Each sample of culture medium collected was diluted to 30% and supplemented with 50 ng mL-1 of soluble RANKL to be used as conditioned medium. RAW264.7 cells were plated into 96-well microplates or 6-well microplates at a density of 1.25×104 cells cm-2 and left overnight to settle. Conditioned medium was then added to the cells for 7 days. Osteoclastogenesis was measured by TRAP staining. The expression of protein of cathepsin K and MMP-9 was measured by Western-blot.Results1. The expression of mRNA and protein of IL-17s and their receptors in osteoblast during compressive force.1.1 The expressions of IL-17s mRNA with compressive forceWith or without compressive force, the expressions of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F mRNA increased gradually until 6 h of culture, and decreased gradually after 9 h. In addition, the expression of IL-17A was the highest among IL-17s with or without compressive force. The expressions of IL-17A, IL-17D, and IL-17E mRNA increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 after 3,6,9,12, and 24 h of culture. The expressions of IL-17B, IL-17C, and IL-17F increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 after 6,9,12, and 24 h of culture. The expressions of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F at 3,6, and 9 h of culture increased with the strength of the compressive force compared to the control, whereas the expression at 1.0 g/cm2 after 12 and 24 h of culture indicated a higher value than that at 2.0 g/cm2 compressive force at the same time points.1.2 The expression of lL-17Rs mRNA with compressive forceWith or without compressive force, the expressions of IL-17RA, IL-17RC, and IL-17RD mRNA increased gradually until 9 h of culture, and decreased gradually after 12 h. The expressions of IL-17RB and IL-17RE increased gradually until 6 h of culture, and decreased gradually after 9 h. In addition, the expression of IL-17RC mRNA was the highest among IL-17Rs with or without compressive force. The expressions of IL-17RA and IL-17RE increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 over 6-24 h of culture. The expressions of IL-17RC and IL-17RD increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 over 6-12 h of culture. The expression of IL-17RB increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 over 3-9 h of culture. The expressions of IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE increased with the compressive force strength compared to the control at 3,6 and 9 h of culture, whereas the expressions at 1.0 g/cm2 and 12 and 24 h of culture indicated a higher value than that at 2.0 g/cm2 compressive force at the same time points, excluding IL-17RC at 24 h.1.3 The expression of lL-17A protein with compressive forceIL-17A protein was not detected after 9h of compressive force stimulation in control group. Red fluorescence was detected in the cytoplasm of part of cells in the compressive force stimulating group.IL-17A protein level is 7.125pg/ml With 1.0 g/cm2 compressive force and 9.019pg/ml with 2.0 g/cm2 compressive force.1.4 The expression of IL-17 Rs protein with IL-17A stimulation MC3T3-E1 cells were cultured with or without 10 ng/ml IL-17A for up to 72 h. Compared to the control, the gene expression of IL-17RA, IL-17RB, IL-17RC, and IL-17RE increased significantly in the presence of 10 ng/ml IL-17A after culture for 6, 24, or 72 h, whereas the expression of IL-17RD was not affected.1.5 The expression of lL-1αand IL-6 mRNA with compressive forceWith or without compressive force, the expressions of IL-la and IL-6 mRNA increased gradually. The expressions of IL-la mRNA increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 after 6,9,12, and 24 h of culture(P<0.01). The expressions of IL-6 increased significantly compared to the control at 1.0 and/or 2.0 g/cm2 after 9,12, and 24 h of culture (P<0.05或P<0.01)2. The effect of IL-17A on PGE2 and cytokines expression in osteoblast.2.1 Effect of IL-17A on the expression of RANKL, M-CSF and OPG mRNA and proteinCompared to the control, the expression of RANKL and M-CSF increased significantly in the presence of 0.1,1.0, and/or lOng/ml IL-17A after culture for 24 and/or 72 h. On the other hand, the expression of OPG decreased significantly in the presence of 0.1,1.0, and 10ng/ml IL-17A after 72 h. The expression increased by 1.3-1.8-fold on RANKL (P>0.05 or P>0.01), by 1.6-1.9-fold on M-CSF (P>0.01) and decreased by 0.8-0.9 on OPG (P>0.05).With the addition of IL-17A after 72h, the production of RANKL and M-CSF increased and OPG decreased significantly compared to control levels:the expression increased by 1.9-2.4-fold on RANKL (P>0.05), by 1.2-1.4-fold on M-CSF (P>0.05) and by 0.7-0.8 on OPG (P>0.05).2.2 Effect of IL-17A on the expression of inflammatory cytokines mRNA and proteinIn both the presence and absence of IL-17A, IL-la expression decreased gradually until 48 h of culture, whereas TNF-a expression increased gradually until 72 h of culture. The expression of IL-6 increased gradually until 24 h of culture and decreased after 48 h. With the addition of IL-17A, the production of each cytokine increased significantly compared to control levels at 6,12,24,48 and/or 72 h of culture:the 0xpression increased by 1.3-4.3-fold on IL-la (P<0.05 or P<0.01),1.3-2.5-fold on IL-6(P<0.05 or P<0.01) and 1.2-3.5-fold on TNF-a(P<0.05 or P<0.01).With the addition of IL-17A after 24h or 72h, the production of IL-1α, IL-6 and TNF-a increased significantly compared to control levels:the expression increased by 1.3-1.5-fold on IL-la (P<0.05 or P<0.01), by 2.1-2.6-fold on IL-6 (P<0.01) and by 1.5-fold on TNF-a (P<0.01).2.3Effect of IL-17A on the expression of COXs mRNAIn both the presence and absence of IL-17A, COX-2 expression decreased gradually until 48 h of culture. With the addition of IL-17A, COX-2 expression increased significantly compared to control levels at 6,12 and 24 h:the expression increased by 1.2-2.4-fold. In contrast, COX-1 expression was not affected by the addition of IL-17A and was changed depending on the culture days.2.4 Effect of IL-17A and/or celecoxib on PGE2 productionWith the addition of IL-17A, PGE2 production at 6,24, and 72 h of culture increased by 1.06-2.19,1.57-2.05, and 2.75-3.50-fold, respectively, in a dose-dependent manner in the presence of IL-17A. When celecoxib was present in the culture media, the stimulatory effect of IL-17A on PGE2 production was inhibited since the levels of PGE2 production remained similar to those in the untreated controls. These data suggest that the indirect stimulatory effect of IL-17A on PGE2 levels is dependent on COX-2 activity.2.5 Effect of PGE2 on the production of osteoclast differention-related factorsWith the addition of PGE2 after 24h, the production of RANKL and M-CSF increased significantly compared to control levels (P<0.01), whereas the production of OPG decreased significantly compared to control levels (P<0.05).With the addition of PGE2 after 24h, the production of IL-la and IL-6 increased significantly compared to control levels (P<0.01), but the production of TNF-a was not affected.2.6 Effect of lL-17A and celecoxib on the expression of osteoclast differention-related factors proteinWhen celecoxib was present in the culture with IL-17A, it appeared to block the stimulatory effect of IL-17A on the production of RANKL, M-CSF and OPG; the levels of these productions in cells treated with both IL-17A and celecoxib were similar to that in the controls without IL-17A.When celecoxib was present in the culture with IL-17 A, it appeared to block the stimulatory effect of IL-17A on the production of IL-1 a, IL-6; the levels of these productions in cells treated with both IL-17A and celecoxib were similar to that in the controls without IL-17A. In contrast, TNF-a was not affected by the addition of celecoxib.3. The indirect effect of IL-17A on osteoblast-like cells differentiation via osteoblast.3.1 Indirect effects of IL-17A on TRAP staining of osteoclast-like cellsThe multinucleated cell numbers increased after 3 days of culture. In both conditioned medium from IL-17A-treated and untreated cells, the number of TRAP-positive multinucleated cells increased gradually until day 7 of culture, and decreased from day 7 to 10. TRAP staining of osteoclast-like cells in the conditioned medium from IL-17-treated cells on days 5 and 7 of culture was stronger than the conditioned medium from untreated control cells. The number of TRAP-positive multinucleated cells increased significantly in the conditioned medium from IL-17A-treated cells compared to that of the controls on days 5 and 7 of culture, increasing by 1.45-and 1.34-fold, respectively.3.2 Indirect effects of IL-17A on cathepsin K, MMP-9, and CAⅡmRNA expressionIn both conditioned medium from IL-17A-treated and untreated MC3T3-E1 cells, the expression of cathepsin K and MMP-9 increased gradually until day 10 of culture, whereas CA II expression remained unchanged until day 7, eventually increasing on day 10. Cathepsin K expression in the conditioned medium from IL-17A-treated cells increased significantly by 1.50-and 1.40-fold, on days 7 and 10 of culture, respectively, compared with that of the conditioned medium from untreated control cells. In addition, MMP-9 expression in the medium from IL-17A-treated cells increased significantly by 1.31-fold compared to untreated cells on day 10 of culture. CA II expression remained unaffected by IL-17A treatment until day 10 of culture. 3.3 Indirect effects of IL-17A on cathepsin K, MMP-9, and CAⅡprotein expressionWhen the protein levels of cathepsin K and MMP-9 in the conditioned medium from IL-17A-treated cells were quantified, similar to the data observed from the mRNA analysis, their levels increased by 1.35-and 1.42-fold, respectively, compared with the untreated control cells. CAⅡexpression was again unaffected by indirect IL-17A treatment. Thus, the indirect effects of IL-17A on cathepsin K, MMP-9, and CAⅡmRNA expression levels were correlated with their protein levels in conditioned media.3.4 Indirect effect of IL-17A and/or celecoxib on TRAP staining of osteoclast-like cellsTRAP staining of osteoclast-like cells in the conditioned medium from IL-17-treated MC3T3-E1 cells was significantly stronger than untreated cells. The addition of celecoxib to the IL-17A conditioned media reduced TRAP staining of osteoclast-like cells to levels identical to the untreated cells. In addition, celecoxib blocked the stimulatory indirect effect of IL-17A on the number of TRAP-positive multinucleated cells.3.5 Indirect effects of IL-17A and/or celecoxib on cathepsin K and MMP-9 mRNA expressionThe mRNA levels of both cathepsin K and MMP-9 in the conditioned medium from MC3T3-E1 cells treated with both IL-17A and celecoxib were similar to those in the controls lacking IL-17A. These data suggest that COX-2 inhibition by celecoxib inhibits the ability of IL-17A to stimulate cathepsin K and MMP-9 expression in the conditioned media.Conclusion1. MC3T3-E1 cells express six different IL-17 subtypes and five different IL-17R subtypes at the mRNA level and IL-17A at protein level.2. Compressive force induces the expression of IL-la,IL-6, IL-17s and IL-17Rs in these cells, which suggests that IL-17s may be involved in the alveolar bone resorption during orthodontic tooth movement.3. IL-17A increased the mRNA and protein expression of RANKL and M-CSF, decreased expression of OPG in dose-dependent way. IL-17A also stimulates the expression of bone resorption-related inflammatory cytokines. It indicates that IL-17A in vitro regulates bone resorption-related cytokines expression in osteoblast.4. IL-17A stimulates bone resorption-related cytokines expression through an autocrine mechanism involving celecoxib-blocked PGs by increasing COX-2 expression, mainly PGE2, in osteoblasts.5. IL-17A stimulates not only the differentiation of RAW264.7 cells into osteoclast-like cells but also the expression of cathepsin K and MMP-9 in osteoclast-like cells. It indicates that IL-17A in vitro indirectly stimulates the differentiation and function of osteoclast-like cells via osteoblasts.6. IL-17A stimulates osteoclast-like cells differentiation through an autocrine mechanism involving celecoxib-blocked PGs by increasing COX-2 expression, mainly PGE2, in osteoblasts.7. This study will contrubuteto a better understanding of molecule mechanism of bone remodeling during tooth movement.