Interleukin-1 β and 6-Induced Calcium Channel Current Modulation in MC3T3-E1 Osteoblast Cell Line

Interleukin-1 β (IL-1 β ) and -6 (IL-6) are inflammatory cytokines that are involved in bone resorption under pathological conditions. The cytokines are also involved in bone remodeling under physiological conditions

Osteoblasts are known to play a major role in the bone formation.It is well accepted that osteoblasts express IL-1β and -6 receptors and therefore it plays an important role in bone remodeling (Blanchard et al., 2009;Shen et al., 1990).

Cell Culture
Murine osteoblastic MC3T3-E1 cells were purchased from Summit Pharmaceuticals International Corporation (Tokyo, Japan).Cells were cultured at 37°C in a 5%(v/v) CO 2 atmosphere with α-modified minimal essential medium (α-MEM; Gibco BRL, Grand Island, NY, U.S.A.).Unless otherwise specified, the medium contained 10%(v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin.Cell culture medium was changed every 2-3 days.For patch-clamp experiments, cells were harvested using a 0.05% trypsin/0.02%EDTA solution, when cells reached confluence.Cells were plated at very low density in 35 mm tissue culture dishes.Prior to recordings, the cells were washed at least three times with Krebs solution of the following composition (in mM): 136 NaCl; 5 KCl; 2.5 CaCl 2 ; 0.5 MgCl 2 ; 10.9 glucose; 11.9 NaHCO 3 and 1.1 NaH 2 PO 4 .The pH was 7.3-7.4.Cell culture reagents were purchased from Sigma (Tokyo, Japan).Our recordings were performed on 10-30 μm in diameter, since they were well suited for patch clamp recordings.

Whole-Cell Patch-Clamp Recordings
Voltage-clamp recordings were conducted using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981).Fabricated recording pipettes (2-3 MΩ) were filled with the internal solution of the following composition (in mM): 150 CsCl; 5 EGTA; 10 D-glucose and 10 HEPES.The pH was adjusted to 7.3 with CsOH.After the formation of a giga seal, in order to record VSCCs current (I Ca ) carried by Ba 2+ (I Ba ), the extracellular solution was replaced from Krebs solution to a solution containing the following (in mM): 115 BaCl 2 and 20 HEPES.The pH was adjusted to 7.4 with tetraethylammonium hydroxide.Command voltage protocols were generated with a computer software pCLAMP version 10 (Axon Instruments, Union City, CA, U.S.A.) and transformed to an analogue signal using a DigiData1440A interface (Axon Instruments, Union City, CA, U.S.A.).The command pulses were applied to cells through an L/M-EPC7 amplifier (HEKA Elektronik, Lambrecht, Germany).The currents were recorded with the amplifier and a computer software pCLAMP10 acquisition system.Access resistance (< 15 MΩ) was determined by transient responses to voltage commands.L-1β and -6 were applied by perfusion pressure ejection from perfusion tubes with a tip diameter of 1 mm placed within 10 mm of the cell surface.Although the concentration of IL-1β and -6 actually seen by the cell is certainly lower than that contained in the stock solution, we applied the IL-1β and -6 using same perfusion system.L-1β and -6 were applied by perfusion pressure ejection from perfusion tubes with a tip diameter of 1 mm placed within 10 mm of the cell surface.

Statistical Analysis
All date analyses were performed using the pCLAMP10 acquisition system.Values in text and figures are expressed as mean ± S.E.M. Statistical analysis was done using Student's t test for comparisons between pairs of groups and one-way analysis of variance (ANOVA) followed by Dunnett's test.Probability (p) values of less than 0.05 were considered significant.Modulation of I Ba was calculated with the following formula: Modulation of I Ba (%) = After Application peak current / Before Application peak current   2 ± 4.8 4 -3.5 ± 0.6  5   PD98059  7.5 ± 1.1 4 -4.0 ± 0.9 5

IL-6 Inhibited I Ba
In addition to facilitation, IL-6-induced inhibition of I Ba could be observed.Figure 3a and b shows that 50 nM IL-6 inhibited I Ba as shown with raw current traces obtained at +10 mV from -80 mV in the absence and presence of IL-6.I Ba was evoked every 5 s with a 100 ms depolarizing voltage step to -10 mV from a holding potential of -80 mV.As shown in Figure 3a, application of 50 nM IL-6 inhibited I Ba from -328 pA to -236 pA (28.0%inhibition) in this cell.
To investigate the voltage dependency of facilitation of I Ba by 50 nM IL-6, we analyzed the current-voltage relations in the absence and presence of 50 nM IL-6.The current-voltage relations measured before and during application of 50 nM IL-6 are shown in Figure 3c.From a holding potential of -80 mV, I Ba was activated with a peak current amplitude at 20 mV.IL-6 did not alter the current-voltage relationship.These results suggest that 50 nM IL-6 inhibited I Ba in MC3T3-E1 cells.

IL-1β and -6 Modulated I Ba Depends On Concentration
The concentration-response relationship in the IL-1β and -6-induced modulation of I Ba is shown in Figure 4a and b, respectively.
Application of 50 pM-50 nM IL-1β facilitated I Ba .To generate a concentration-response curve, IL concentrations were applied randomly, and each MC3T3-E1 cells were exposed to only a single concentration.Figure 4a shows that progressive voltage-dependent facilitations in IL-1β concentrations resulted in a progressively greater facilitation of I Ba .
In addition, application of 50 pM-5 nM IL-6 facilitated I Ba .In contrast to above results, 50 nM IL-6 inhibited I Ba in MC3T3-E1 cells.

Characterization of Second Messengers in 5 nM IL-6-Induced Facilitation of I Ba
To evaluate the possible contribution of adenylate cyclase (AC) to the IL-6-induced facilitation of I Ba , the effects of IL-6 on I Ba in cells treated with SQ22536 (an AC inhibitor) were investigated.Treatment with SQ22536 (10 μM for 30 min) did not attenuate the IL-6-induced facilitation of I Ba .
To evaluate the possible contribution of protein kinase C (PKC) to the IL-6-induced facilitation of I Ba , the effects of IL-6 on I Ba in cells treated with GF109203X (a selective PKC inhibitor) were investigated.Treatment with GF109203X (10 μM for 30 min) did not attenuate the IL-6-induced facilitation of I Ba .
To evaluate the possible contribution of STAT3 to the IL-6-induced facilitation of I Ba , the effects of IL-6 on I Ba in cells treated with staffic (a selective signal transducers and activators of transcription 3, STAT3, inhibitor) were investigated.Treatment with staffic (20 μM for 2 min) did not attenuate the IL-6-induced facilitation of I Ba .
To evaluate the possible contribution of MAPK to the IL-6-induced facilitation of I Ba , the effects of IL-6 on I Ba in cells treated with PD98059 (a selective MAPK inhibitor) were investigated.Treatment with PD98059 (10 μM for 2 min) attenuated the IL-6-induced facilitation of I Ba .
These results suggest that IL-6 facilitates VDCCs involving MAPK pathways in MC3T3-E1 cells.

Characterization of Second Messengers in 50 nM IL-6-Induced Inhibition of I Ba
To evaluate the possible contribution of AC to the IL-6-induced inhibition of I Ba , the effects of IL-6 on I Ba in cells treated with SQ22536 (an AC inhibitor) were investigated.Treatment with SQ22536 (10 μM for 30 min) did not attenuate the IL-6-induced inhibition of I Ba .
To evaluate the possible contribution of protein kinase C (PKC) to the IL-6-induced inhibition of I Ba , the effects of IL-6 on I Ba in cells treated with GF109203X (a selective PKC inhibitor) were investigated.Treatment with GF109203X (10 μM for 30 min) did not attenuate the IL-6-induced inhibition of I Ba .
To evaluate the possible contribution of STAT3 to the IL-6-induced inhibition of I Ba , the effects of IL-6 on I Ba in cells treated with staffic (a selective STAT3 inhibitor) were investigated.Treatment with staffic (20 μM for 2 min) attenuated the IL-6-induced inhibition of I Ba .
To evaluate the possible contribution of MAPK to the IL-6-induced inhibition of I Ba , the effects of IL-6 on I Ba in cells treated with PD98059 (a selective MAPK inhibitor) were investigated.Treatment with PD98059 (10 μM for 2 min) attenuated the IL-6-induced inhibition of I Ba .
These results suggest that IL-6 inhibits VDCCs involving STAT3 and MAPK pathways in MC3T3-E1 cells.

Discussion
The present study investigated the effects of IL-1β and -6 on I Ba in MC3T3-E1 cells.Application of 50 pM-50 nM IL-1β facilitated I Ba .Application of 50 pM-5 nM IL-6 facilitated I Ba involving MAPK pathways.In contrast, 50 nM IL-6 inhibited I Ba involving STAT3 and MAPK pathways in MC3T3-E1 cells.In this study, we used 50 pM-50 nM IL-1β and IL-6 to modulate I Ba .These IL concentrations are quite high.In normal state, serum IL-1β concentration is 0.9 pg/ml approximately (Chang et al., 2013).In the electrophysiological study and the immunocytochemistry study, however, several study demonstrated that high concentration, i.e. 0.57 nM and 4.5 nM, of IL was used to modulate ion channel activity and protein expression in single neuron (Zhou et al., 2006;Islam et al., 2009).IL-1β and -6 were applied by perfusion pressure ejection from perfusion tubes with a tip diameter of 1 mm placed within 10 mm of the cell surface.Although the concentration of IL-1β and -6 actually seen by the cell is certainly lower than that contained in the stock solution, we applied the IL-1β and -6 using same perfusion system.We also observed that I Ba were recovered to values of peak I Ba after IL wash out using time course graph (Figures. 1b,2b and 3b).Therefore, it can be considered that 50 pM-50 nM IL-1β and IL-6 had no effect on the I Ba .
In this study, we have shown that 50 pM-50 nM IL-1β facilitated I Ba .It has been demonstrated that local treatment with IL-1β (0.5 ng/h) for 72 h resulted in increased osteoblasts concentration (Olmedo et al., 1999).Furthermore, reports of IL-1β promoting osteoblast proliferation during acute bone repair (Lange et al., 2009) and inducing differentiation of human mesenchymal stem cells into osteoblasts (Sonomoto et al., 2012).
Increased DNA synthesis in response to IL-1β in vitro has been observed in osteoblast (Evans et al., 1989) and especially MC3T3-E1 cells (Ikeda et al., 1998).In contrast, however, it has been reported that IL-1β inhibits the stimulation of osteocalcin synthesis and has no influence on proliferation (Taichman et al., 1992).IL-1β also increases both osteoprotegerin protein release and mRNA levels (Hofbauer et al., 1998;Hofbauer et al., 1999).
Recent study demonstrated that treatment with IL-1β resulted in biphasic effects on osteoblast differentiation.Short-term exposure (2 days) to IL-1β early in culture induces differentiation.Longer term exposure (6 days) to IL-1β inhibits osteoblast differentiation (Bellido et al., 1997).
In this study, we have shown that 50 pM-5 nM IL-6 facilitated I Ba involving MAPK pathways.In contrast, 50 nM IL-6 inhibited I Ba involving STAT3 and MAPK pathways.What is the physiological relevance of IL-6-induced dual modulation of VSCCs, i.e. both facilitation and inhibition?Several studies indicated that IL-6 enhanced in vitro differentiation on osteoblasts (Bellido et al., 1997;Bellido et al., 1998;Franchimont et al., 2005;Itoh et al., 2006;Liu et al., 2006;Wong et al., 2003).In contrast to above reports, other studies have shown an inhibitory effect of IL-6 on bone formation (Malaval et al., 2001;Malaval et al., 2005).It can be considered that such dual effects are depends on IL-6 receptor's intracellular signals transduction mechanisms.There are three major MAPK subgroups identified, including the extracellular signal-regulated kinases (ERK1/2), p38 MAPK and c-jun-NH 2 -terminal kinases (JNKs).The signal transduction induced by IL-6 is mediated by homo-or heterodimerization of the gp130 receptor, subsequent activation of JAK, and phosphorylation of STAT (Heinrich et al., 2003).It has been demonstrated that activation of STAT3 is necessary for osteoblast differentiation and bone formation induced by IL-6 (Itoh et al., 2006).On the other hand, PKCδ and ERK1/2 are implicated in IL-6's inhibitory effect on bone formation (Chipoy et al., 2004).STAT3 plays multiple roles depending on the context or condition of cells.In some conditions, STAT3 is involved in growth arrest and differentiation, and even in cell death (Nakajima et al., 1996;Hirano et al., 1997;Hirano et al., 2000).In other conditions, STAT3 is involved in proliferation and cell survival (Hirano et al., 1997;Hirano et al., 2000).It is also possible that these dual effects depend on the conditions or differentiation stage of the osteoblast.IL-6 would stimulate the first stages of differentiation but on more mature cells, they would prevent further stimulation (Malaval et al., 2001;Malaval et al., 2005).Interestingly, VSCCs serve as growth of bone, regulate proliferation and differentiation, as mentioned above.

Figure
Figure 1a and b shows that, in the presence of high external Ba 2+ , 5 nM IL-1β facilitated I Ba as shown with raw current traces obtained at +10 mV from -80 mV in the absence and presence of IL-1β.I Ba was evoked every 5 s with a 100 ms depolarizing voltage step to -10 mV from a holding potential of -80 mV.As shown in Figure 1a, application of 5 nM IL-1β facilitated I Ba from -161 pA to -225 pA (39.7% facilitation) in this cell.To investigate the voltage dependency of facilitation of I Ba by IL-1β, we analyzed the current-voltage relations in the absence and presence of IL-1β.The current-voltage relations measured before and during application of IL-1β are shown in Figure 1c.From a holding potential of -80 mV, I Ba was activated with a peak current amplitude at 0 mV.IL-1β did not alter the current-voltage relationship.These results suggest that 5 nM IL-1β facilitated I Ba in MC3T3-E1 cells.

Figure 1 .
Figure 1.(a) Typical superimposed I Ba traces according to the time course graph B. I Ba was evoked from a holding potential of -80 mV by a 100 msec voltage step to -10 mV at 5 sec intervals.(b) Typical time course of 5 nM IL-1β-induced facilitation of I Ba .IL-1β (5 nM) was bath-applied during the times indicated by the filled bar.(c) Current-voltage relationship of I Ba evoked by a series of voltage steps from a holding potential of -80 mV to test potentials between -80 and ＋40 mV in ＋10 mV increments in the absence (opened circles) and presence (filled circles) of 5 nM IL-1β

Figure 2 .
Figure 2. (a) Typical superimposed I Ba traces according to the time course graph B. I Ba was evoked from a holding potential of -80 mV by a 100 msec voltage step to -10 mV at 5 sec intervals.(b) Typical time course of 5 nM IL-6-induced facilitation of I Ba .IL-6 (5 nM) was bath-applied during the times indicated by the filled bar.(c) Current-voltage relationship of I Ba evoked by a series of voltage steps from a holding potential of -80 mV to test potentials between -80 and ＋40 mV in ＋10 mV increments in the absence (opened circles) and presence (filled circles) of 5 nM IL-6

Figure 3 .
Figure 3. (a) Typical superimposed I Ba traces according to the time course graph B. I Ba was evoked from a holding potential of -80 mV by a 100 msec voltage step to -10 mV at 5 sec intervals.(b) Typical time course of 50 nM IL-6-induced inhibition of I Ba .IL-6 (50 nM) was bath-applied during the times indicated by the filled bar.(c) Current-voltage relationship of I Ba evoked by a series of voltage steps from a holding potential of -80 mV to test potentials between -80 and ＋40 mV in ＋10 mV increments in the absence (opened circles) and presence (filled circles) of 50 nM IL-6

Table 2 .
Statistical mean ± S.E.M. for IL-6 treated with each inhibitors.