1-Oleoyl lysophosphatidic acid (sodium salt) (LPA; item no. 62215; Cayman Chemical Co., Ann Arbor, MI, USA) and Kil16425 (S1315; Selleck Chemicals, Houston, TX, USA) were dissolved in 4 mM sterile stock solutions and stored at −20°C until use. Phorbol 12-myristate 13-acetate (PMA; S1819; PKA agonist), forskolin (S1612; PKA activator), staurosporine (S1882; PKC inhibitor) and H-89 (S1643; selective inhibitor of PKA) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). CCN2 polyclonal rabbit anti-mouse CTGF primary antibody (ab6992, diluted 1:2,000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit anti-GAPDH primary antibody (ab181602, diluted 1:10,000) were purchased from Abcam (Cambridge, UK).

The MC3T3-E1 cell line (China Center for Type Culture Collection, Wuhan, China) was cultured in α-modified minimal essential medium (α-MEM) containing 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA), 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in a 5% CO₂ atmosphere; the medium was changed to fresh medium at an interval of 3 days. The cells were seeded on 6-well plates or 6 cm dishes to examine gene and protein expression. After the cells reached confluence, the culture medium was replaced with α-MEM and 1% penicillin-streptomycin without FBS for 12 h. The medium was then replaced with α-MEM containing 4% bovine seum albumin (BSA) and varying doses of LPA, PMA or forskolin. Inhibitors, such as staurosporine and H-89, were used to suppress PKC and PKA activity, respectively. After co-incubation was performed for the indicated periods of time (0–12 h), the MC3T3-E1 cells were lysed for the subsequent analysis of RNA and protein expression.

The viability of the MC3T3-E1 cells was determined as previously described (21). Briefly, following incubation of the cells with increasing concentrations of LPA (0–40 µM) for different periods of time (12 to 72 h), cell viability was determined using a cell viability analyzer (Beckman Coulter, Fullerton, CA, USA).

MTT assay was performed as described in a previous study (22). Briefly, the MC3T3-E1 cells were seeded in 96-well plates (3×10³ cells/well) and incubated with increasing concentrations of LPA (0–40 µM). At the end of the incubation period, 20 µl of 5 mg/ml MTT were added; the cells were then further incubated for 4 h. Subsequently, the supernatant was removed, and 150 µl of dimethyl sulfoxide (DMSO) were added to each well. The absorbance of each well was detected at 490 nm. Data are presented as percentage of the control group.

RNA isolation and RT-qPCR were performed as previously described (23). Briefly, total RNA was isolated from the ME3T3-E1 cells in each treatment group using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA using a Revert Aid First-Strand cDNA Synthesis kit (#K1622; Thermo Fisher Scientific, Waltham, MA, USA). The obtained cDNA was then amplified by quantitative PCR (qPCR) using an ABI 7900 HT Real-Time PCR system, (Applied Biosystems, Foster City, CA, USA) and SYBR^®-Green Real-Time PCR Master Mix (Toyobo, Tokyo, Japan), under the following conditions: stage 1, 95°C for 10 min; stage 2, 95°C for 10 sec, 55°C for 20 sec, followed by 40 cycles of 72°C for 15 sec; stage 3, melt curve stage. GAPDH was used for the normalization of gene expression. Relative gene expression was analyzed using the 2^−ΔΔCt method based on normalization to the endogenous control, GAPDH, and calculation of the threshold cycle (Ct) value difference. The results were represented as a fold change of the comparative expression level. The sequences of the forward and reverse primers were as follows: 5′-GCCTACCGACTGGAA GACACATTT-3′ and 5′-TTACGCCATGTCTCCGTACA TCTT-3′ for the CCN2 gene; 5′-ACCACAGTCCATGCCA TCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for the internal control GAPDH gene.

The cells were treated with the indicated stimulants (LPA, LPA + Kil16425, LPA + staurosporine, PMA, LPA + H-89, forskolin, or LPA + forskolin) for 6 h. Subsequently, the cells were then washed thrice with phosphate-buffered saline (PBS) and treated with RIPA lysis buffer. The cell lysates were cleared by centrifugation; protein concentration in the supernatant was measured by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Total proteins (20 µg) were separated through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the proteins were then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% (w/v) nonfat milk and incubated overnight with the primary antibodies (CCN2, PKC or GAPDH) at the specified dilutions in TBST buffer (CW0043; Beijing Conwin Biotech Co., Beijing, China) according to the manufacturer's instructions. Protein expression was detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific). GADPH was used as an internal control protein.

PKC activity was assessed by determining the translocation of PKC from the cytosol to the membrane. After the cells were treated with the indicated stimulants for the indicated periods of time, PKC in the cytosol and in the membrane of MC3T3-E1 cells was extracted using a Mem-PER Plus Membrane Protein Extraction kit (Thermo Fisher Scientific). PKC expression in the cytosol and membrane was then determined by western blot analysis. The membrane translocation of PKC was also investigated by immunofluorescence. Briefly, the MC3T3-E1 cells were plated on cover slips and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. The cells were then washed thrice with PBS buffer, permeabilized with PBS buffer containing 0.1% Triton X-100 and blocked with 1% BSA. The cells were then incubated with PKC antibody (P5704; Sigma, St. Louis, MO, USA) for 2 h at 37°C. The cells were washed again with PBS buffer thrice and incubated with PE-labeled secondary antibody (CW0113; Beijing Conwin Biotech Co.). The fluorochrome dye, 4′,6-diamidino-2-phenylindole (DAPI), was used to visualize the cell nuclei. Images were captured using a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Data were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 software. Data are expressed as the means ± standard error of the mean (SEM) of 3 independent experiments. A value of P<0.05 was considered to indicate a statistically significant difference.

We initially examined the effects of LPA on the viability and proliferation of the MC3T3-E1 cells. The results of cell viability assay demonstrated that LPA did not exert cytotoxic effects on the MC3T3-E1 cells (Fig. 1A). A previous study reported that LPA induced an increase in DNA synthesis in rat osteoblasts in vitro (24). However, our results from MTT assay revealed that increasing concentrations of LPA did not affect MC3T3-E1 cell proliferation in vitro (Fig. 1B).

The MC3T3-E1 cells were stimulated with 20 µM LPA and 4% BSA for 0.5, 1, 2, 4, 6, 8 and 12 h, in order to examine the effects of LPA on CCN2 expression in osteoblasts. The CCN2 mRNA expression levels were measured by RT-qPCR. The results revealed that LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation. The mRNA expression levels of CCN2 subsequently decreased (Fig. 2A). The results from western blot analysis revealed that the CCN2 protein expression levels was similarly enhanced following 6 h of stimulation with LPA (Fig. 3D).

LPA elicits its functions through receptors on plasma membranes; MC3T3-E1 cells express LPA-specific receptors with the following relative abundance pattern: LPA₁ > LPA₄ > LPA₂ > LPA₃ (2). Thus, in this study, we wished to determine whether Kil16425, a specific inhibitor of LPA_1/3 (25), is was capable of antagonizing the effects of LPA on CCN2 expression in MC3T3-E1 cells. We found that pre-treatment with Ki16425 (20 µM) significantly reduced the LPA-induced increase in the mRNA expression of CCN2 in the MC3T3-E1 cells (Fig. 2B). Kil16425 also blocked the LPA-induced increase in the protein expression of CCN2 in the MC3T3-E1 cells (Fig. 3D).

The effects of LPA on CCN2 expression may be attributed to LPA₁ due to the very low expression of LPA₃ (2). Thus, LPA induces an increase in CCN2 expression through the LPA₁ receptor in MC3T3-E1 cells.

LPA receptors belong to the GPCR family (9). The LPA-induced activation of GPCR induces an increase in a variety of second messengers, such as Ca²⁺ in the cytoplasm of osteoblasts (2,3,8,26). Ca²⁺ is essential for PKC membrane translocation and activation (27). The activation PKC is manifested by the membrane translocation of PKC (28) and PKC activity may be thus enhanced by LPA. In this study, the MC3T3-E1 cells were stimulated with LPA for 15 min; PKC membrane translocation was observed by immunofluorescence staining (Fig. 3A). Western blot analysis revealed that LPA increased the ratio between membrane-derived PKC and cytosolic PKC (Fig. 3B). These results indicate that LPA induces PKC membrane translocation and enhances PKC activity in osteoblasts.

Staurosporine and PMA, an inhibitor and an agonist of PKC, respectively, were used to determine whether PKC is involved in the effects of LPA on CCN2 expression. Staurosporine (20 nM) markedly impaired the ability of LPA to enhance CCN2 expression in the MC3T3-E1 cells, whereas PMA (1 µM) mimicked LPA and induced the expression of CCN2 (Fig. 3C and D).

Cyclic AMP (cAMP), another second messenger, accumulates in cells expressing LPA_4,5 receptors (29,30) in the presence of LPA; the accumulation of cAMP increases PKA activity. We thus examined the role of PKA in the expression of CCN2 in LPA-treated MC3T3-E1 cells. The MC3T3-E1 cells were pre-incubated with H-89 (10 µM), a selective inhibitor of PKA, for 30 min; LPA (20 µM) was then added or the cells were treated with the PKA activator, forskolin, alone for 2 and 6 h. The mRNA and protein expression levels of CCN2 in the LPA-H-89 group were enhanced compared with those of the LPA group (Fig. 4). Pre-treatment with forskolin (10 µM) significantly decreased the mRNA and protein expression levels of of CCN2 which had been increased by LPA in the MC3T3-E1 cells. No significant effects were observed in the cells treated with forskolin alone compared to the cells also treated with LPA (Fig. 4).