Calbiochem (Merck KGaA) and MilliporeSigma provided gallein and fluorescein, respectively. R&D Systems, Inc. supplied PGF2α and mouse OPG (cat. no. MOP00) and IL-6 (cat. no. M6000B) ELISA kits. Cell Signaling Technology, Inc. provided antibodies, including phosphorylated (p-)p44/p42 MAPK (cat. no. 9101), p44/p42 MAPK (cat. no. 9102), p-p38 MAPK (cat. no. 4511), p38 MAPK (cat. no. 9212), p-JNK (cat. no. 4668), and JNK (cat. no. 9252). Santa Cruz Biotechnology, Inc. supplied glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (cat. no. sc-25778). Peroxidase-labeled anti-rabbit IgG antibodies (cat. no. 5220-0336; SeraCare Life Sciences, Inc.) and peroxidase-labeled anti-mouse IgG antibodies (cat. no. 7076; Cell Signaling Technology, Inc.) were used as secondary antibodies. Cytiva provided an ECL western blotting detection system.

The osteoblast-like MC3T3-E1 cells are naturally immortalized and calvaria-derived cell lines (14). Dr M Kumegawa (Department of Dentistry, Graduate School of Dentistry, Meikai University, Sakado, Japan) donated these cells. Cells were maintained in minimal essential medium (α-MEM; MilliporeSigma) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc. and incubated at 37˚C and 5% CO₂ (15). Cells were plated on 90-mm diameter plates (2x10⁵ cells/plate) and 35-mm diameter plates (5x10⁴ cells/plate) in α-MEM supplemented with 10% FBS. The incubation medium was replaced with α-MEM supplemented with 0.3% FBS 5 days after seeding. Cells were used for experiments after 48 h.

Cultured cells were treated with 10 mM of PGF2α or vehicle in 1 ml of α-MEM supplemented with 0.3% FBS for 48 h at 37˚C. These cells were pretreated with 1, 3, and 10 µM gallein or fluorescein for 60 min. The conditioned medium was collected at the end of the incubation, and the OPG and IL-6 concentrations in the medium were measured by mouse OPG and IL-6 ELISA kits following the manufacturer's protocol (16).

Equal counts of cells (5x10⁴ cells/35-mm dish) were seeded and cultured through identical process among all the experiments and used for the experiments 1 week after seeding. Cultured cells were pretreated with 10 µM of gallein for 60 min and then stimulated with 10 µM of PGF2α in α-MEM supplemented with 0.3% FBS for 3 h. TRIzol reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA that was reverse transcribed into cDNA using the Omniscript Reverse Transcriptase kit (Qiagen, Inc.). A Light Cycler system with Fast Start DNA Master SYBR Green I (Roche Diagnostics) was used for RT-qPCR. RNA extraction, cDNA synthesis, and qPCR performed according to the manufacturer's protocols. Samples were subjected to the following PCR thermocycling conditions: Initial denaturation at 95˚C for 10 min, followed by 40 cycles of denaturation at 95˚C for 1 sec, annealing at 60˚C for 5 sec and elongation at 72˚C for 7 sec. Predesigned primer sets for mouse OPG (cat. no. MA026526) and IL-6 (cat. no. MA039013) were obtained from Perfect Real-Time Supporting System (Takara Bio Inc.). For the GAPDH primer, the GAPDH sequence from a previous article was adopted (16) and MilliporeSigma synthesized the GAPDH primer (Forward primer ID: 5852-065, Reverse primer ID: 5852-066) with the same sequence. The primer sequences were as follows: OPG forward, 5'-CAATGGCTGGCTTGGTTTCATAG-3' and reverse, 5'-CTGAACCAGACATGACAGCTGGA-3'; IL-6 forward, 5'-CCACTTCACAAGTCGGAGGCTTA-3' and reverse, 5'-GCAAGTGCATCATCGTTGTTCATAC-3'; and GAPDH forward, 5'-AACGACCCCTTCATTGAC-3' and reverse, 5'-TCCACGACATACTCAGCAC-3'. All measurements were analyzed using the 2^-ΔΔCq method (17). All experiments were performed and analyzed in triplicate.

Cultured cells were preincubated with 10, 30, and 50 µM of gallein and stimulated with 10 µM of PGF2α in α-MEM containing 0.3% FBS for the indicated periods. Cells were rinsed with phosphate-buffered saline and lysed with lysis buffer that contained 62.5 mM of Tris/HCl, 50 mM of dithiothreitol, 2% sodium dodecyl sulfate (SDS), and 10% glycerol for western blot analysis. Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride (PVDF) membrane, followed by western blot analysis (18). Although the mass of protein per lane was not quantified, the same number of cells (2x10⁵ cells/dish) was seeded in each dish. The days of treatment, conditions, and lysis buffer dose were also the same for each dish. Lysates (10 µl) were applied per lane of all SDS-PAGE gels. The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline-Tween (TBS-T; 20 mM Tris-HCl, pH7.6, 137 mM NaCl, 0.1% Tween20) at room temperature for 2 h before incubation with primary antibodies. The membrane was then incubated with primary antibodies (1:1,000) against p38 MAPK, p-p38 MAPK, p44/p42 MAPK, p-p44/p42 MAPK, JNK, p-JNK and GAPDH in 5% fat-free dry milk-TBS-T at 4˚C overnight. The membrane was incubated with secondary antibodies (1:1,000) in 5% fat-free dry milk-TBS-T at room temperature for 1 h. The same cell lysate was used for phosphorylation, total, and GAPDH in the present study. The peroxidase activity on the PVDF sheet was visualized on X-ray film using the ECL western blotting detection reagents (cat. no. RPN2109, Cytiva).

A scanner and image analysis software (ImageJ version 1.48; National Institutes of Health) was used for densitometric analysis of western blots. Phosphorylated protein levels were calculated as follows: The background-subtracted signal intensity of each phosphorylation signal was normalized to the respective intensity of total protein and plotted as a fold increase compared to unstimulated control cells.

Mini StatMate (version 2.01; ATMS) was used for all data analysis. Analysis of variance followed by the Bonferroni significance test was used for multiple comparisons between the two groups. All measurements were conducted and analyzed in triplicate from dependent cell preparations. Data were expressed as mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The present study first investigated the effect of gallein on PGF2α-induced OPG secretion using an osteoblastic MC3T3-E1 cell line. The result revealed that gallein did not affect OPG release in MC3T3-E1 cells, but significantly increased PGF2α-induced OPG secretion. The effect of gallein on OPG secretion increased dose-dependently up to 10 µM (Fig. 1A). The association of fluorescein, a gallein-like compound that does not bind Gβγ (13), with PGF2α-induced OPG secretion was also investigated. The result indicated that fluorescein at concentrations of 1-10 µM demonstrated no effect on OPG secretion with or without PGF2α stimulation (Fig. 1B).

The present study next investigated the effect of gallein on PGF2α-induced IL-6 secretion using the osteoblastic MC3T3-E1 cell line. The result revealed that gallein did not affect IL-6 release in the MC3T3-E1 cell line, but it significantly increased PGF2α-induced IL-6 secretion. The effect of gallein on IL-6 secretion increased dose-dependently at 1, 3, and 10 µM (Fig. 2A). In addition, the association of fluorescein with PGF2α-induced IL-6 secretion was investigated. The result revealed that 1, 3, and 10 µM of fluorescein did not affect IL-6 secretion with or without PGF2α stimulation (Fig. 2B).

The aforementioned results reveal that gallein improves OPG and IL-6 protein secretion; thus, the association of gallein with PGF2α-induced OPG and IL-6 mRNA expression levels was then investigated using the MC3T3-E1 cell line. The results revealed that 10 µM of gallein significantly improved PGF2α-induced OPG and IL-6 mRNA expression levels (Fig. 3).

Regarding the signaling mechanism in osteoblasts, the authors previously used osteoblast-like MC3T3-E1 cells to demonstrate that PGF2α activates the phosphorylation of three major MAPKs, including p44/p42 and p38 MAPKs and JNK (10). The present study investigated how gallein affected p44/p42 and p38 MAPKs and JNK phosphorylation in these cells with or without PGF2α stimulation. The result was that gallein alone at 10, 30, and 50 µM did not affect p44/p42 and p38 MAPKs and JNK phosphorylation as well as PGF2α-activated p44/p42 and p38 MAPKs and JNK phosphorylation (Fig. 4).