Recombinant human ET-1, selective ETAR antagonist, BQ123, and selective ETBR antagonist, BQ788 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The human OSM promoter/luciferase reporter and LightSwitch Luciferase Assay kit were purchased from SwitchGear Genomics (Shanghai, China). Mutant Osx promoter/luciferase reporter constructs were generated by polymerase chain reaction (PCR) and confirmed by sequencing. Life Technologies Lipofectamine 2000 transfection reagent, TRIzol reagent and SuperScript II reverse transcriptase were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Ets-1 (cat. no. sc-29309-V) and control (cat. no. sc-108080) shRNA lentiviral particles, selective phosphatidylinositol 3-kinase (PI3K) inhibitor, BKM120 (cat. no. sc-364437A), mouse monoclonal anti-OSM (E-4; cat. no. sc-365136), rabbit polyclonal anti-Ets1 (C-20; cat. no. sc-350) and mouse monoclonal anti-GAPDH (6C5; cat. no. sc-32233) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The OSM human ELISA kit (cat. no. ab100619) was purchased from Abcam (Shanghai, China). Putative transcription factor binding sites in the human OSM gene promoter sequence were identified using the online software, PROMO (http://alggen.lsi.upc.es) (11,12).

Primary human OA osteoblasts and growth medium kit (cat. no. 406OAK-05a) were purchased from Cell Applications Inc. (San Diego, CA, USA). Cells were treated with 1, 5, 10, 20 or 30 nM ET-1 for 0.5, 1, 2, 3 or 4 h in the presence or absence of BQ123 (1 µM), BQ788 (1 µM) or BKM120 (10 µM). Cells were subjected to further analysis at 26–29.5 h after the treatment, making the total experimental time 30 h. The medium remained unchanged.

RNA was prepared from cells using the TRIzol reagent, and cDNAs were synthesized using SuperScript II reverse transcriptase. RT-qPCR was performed on an ABI PRISM 7700 Sequence Detection System, with fluorescent dye from the SYBR Green Master Mix (both Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. PCR amplification condition were as follows: 20 sec at 95°C followed by 40 cycles of 3 sec at 95°C and 30 sec at 60°C. The primers used in the current study were as follows: Forward, 5′-AGAGTACCGCGTGCTCCTT-3′ and reverse, 5′-AGCTTGCGCTGAAAAGCAT-3′ for OSM; forward, 5′-GGGTGACGACTTCTTGTTTG-3′ and reverse, 5′-GTTAATGGAGTCAACCCAGC-3′ for Ets-1; forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′ for GAPDH. Relative quantification of the mRNA level was determined using the 2^ΔΔCq method (13), which normalizes the expression levels of OSM or Ets-1 against that of GAPDH in the same samples. The mRNA level of treated cells was demonstrated as the fold changes to that of untreated control cells (designated as 1). Each experiment was repeated three times in duplicate.

The secreted OSM levels in cell culture supernatants were determined using an ELISA kit according to the manufacturer's instructions, normalized against cell number (per 10⁵ cells), and demonstrated as the fold changes to that of the untreated control cells (designated as 1). Each ELISA experiment was repeated three times in duplicate. For western blot analysis, human osteoblasts were lysed by three rounds of 3-sec sonication on ice, with a hypotonic buffer, containing 2% Nonidet-P and a protease inhibitor cocktail (Sigma-Aldrich). The supernatant obtained following centrifugation at 2,000 × g for 15 min at 4°C was used to determine the protein concentration with the Coomassie Blue staining (Thermo Fisher Scientific, Inc.). Equal quantities of proteins were separated by 10% SDS-polyacrylamide gel (Thermo Fisher Scientific, Inc.) at 100 V for 2 h and blotted onto a polyvinylidene difluoride microporous membrane (EMD Millipore, Billerica, MA, USA). Membranes were incubated for 1 h with a 1:1,000 dilution of the primary antibodies, washed thrice for 5 min and incubated for 1 h at room temperature with 1:5,000 dilution of secondary antibodies, including bovine anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP; cat. no. sc-2370) or bovine anti-mouse IgG-HRP (cat. no. sc-2371; both Santa Cruz Biotechnology, Inc.). Peroxidase activity was revealed using an ECL kit (GE Healthcare Life Sciences, Shanghai, China). Three independent experiments were performed.

Cells were transfected with human OSM promoter/luciferase reporter plasmids using Lipofectamine 2000 transfection reagent. The luciferase assays were performed 30 h after transfection using the LightSwitch Luciferase Assay kit according to the manufacturer's instructions. The pRL-CMV plasmid (Promega Corporation, Madison, WI, USA) encoding Renilla reniformis luciferase (at one-fifth molar ratio to test plasmids) was co-transfected with test plasmids in each transfection as an internal control for data normalization. Each experiment was repeated three times in duplicate.

Nuclear extracts were prepared as previously described (14). EMSA was performed with 32P-labeled double-stranded oligonucleotides that were incubated with nuclear extract in EMSA buffer [10 mM Tris (pH 7.5), 5% glycerol, 1 mM EDTA (pH 7.1), 50 mM NaCl, 1 mM DTT, 1 mM EDTA and 0.1 mg/ml poly(dI-dC); Sigma-Aldrich]. For the oligonucleotide competition analysis, a 100-fold molar excess of unlabeled competitor oligonucleotide was added to the mixture and incubated at room temperature for 30 min. For the antibody supershift assays, monoclonal Ets-1 antibody (1 µl) was added to the mixture. The reaction was then incubated on ice for 1 h. Protein-DNA complexes and free DNA were fractionated on 5% polyacrylamide gels in 1X Tris-glycine EDTA buffer (both Sigma-Aldrich) at 4°C and were visualized with a Cyclone Plus Phosphor Imager (C431200; Perkin Elmer, Inc., Waltham, MA, USA).

The Ets-1 shRNA lentiviral particles contained expression constructs encoding target-specific 19–25 nt (plus hairpin) shRNA designed to specifically knockdown the Ets-1 gene expression. The control shRNA lentiviral particles contained a scrambled shRNA sequence that would not lead to degradation of any cellular mRNA, and served as a negative control for the Ets-1 shRNA lentiviral particles. Lentiviral transduction was performed in the cells 24 h prior to western blotting and EMSAs, according to the manufacturer's protocol.

Statistical analyses were performed with SPSS software for Windows, version 19.0 (IBM SPSS, Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Comparisons of means among multiple groups were performed with one-way analysis of variance, followed by post-hoc pairwise comparisons using Tukey's test. Tests were two-tailed and P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential effect of ET-1 on OSM expression in OA osteoblasts, primary human OA osteoblasts were treated with ET-1 (1, 5, 10, 20 or 30 nM) for 0.5, 1, 2, 3 and 4 h. The OSM mRNA and secreted protein levels were determined 26–29.5 h subsequent to treatment, making the total experimental time 30 h. As demonstrated in Table I, 1–20 nM ET-1 treatment significantly increased the OSM mRNA levels within 3 h in a dose- and time-dependent manner, and this effect was suppressed by the ETAR antagonist, BQ123 and PI3K inhibitor, BKM120, however not the ETBR antagonist, BQ788. ELISA assays demonstrated that the data trend for the secreted OSM level of the cell culture supernatants was similar to that of the OSM mRNA expression level (Fig. 1).

As demonstrated in Fig. 2, western blot analysis verified that intracellular protein levels of OSM in OA osteoblasts treated with ET-1 (1, 5, 10, 20 or 30 nM) for 4 h were consistent with the corresponding OSM mRNA levels (Table I) and secreted protein levels (Fig. 1). The results suggested that ET-1 may elevate the secreted OSM levels of OA osteoblasts by inducing OSM mRNA expression in an ETAR- and PI3K-dependent manner.

To examine whether ET-1 induced OSM mRNA expression by trans-activating the OSM gene promoter, the human OSM gene promoter activities were investigated using a commercial human OSM promoter/luciferase reporter, which contained 1,064 bp of 5′-untranslated region directly upstream of the OSM gene translation start codon (Fig. 3A). This gene promoter sequence was inserted in frame with the luciferase cDNA and termed pOSM1064. The luciferase reporter without the inserted promoter sequence served as a basic luciferase activity control. Luciferase assays demonstrated that compared with the basic control, the OSM promoter was constitutively active in OA osteoblasts. ET-1 treatment markedly increased the OSM promoter activity, which was suppressed by BQ123 and BKM120, but not BQ788 (Fig. 3B). The results indicate that ET-1 trans-activates the OSM promoter in OA osteoblasts in an ETAR- and PI3K-dependent manner.

To identify the potential ET1-responsive cis-DNA element in the OSM promoter, a deletional analysis of the OSM promoter activity was conducted. As demonstrated in Fig. 4A, a series of 5′-end deletion constructs of the human OSM promoter/luciferase reporter were established. As demonstrated in Fig. 4B, the luciferase assays suggested that the OSM promoter region between -664 and -464 bp contained potential ET1-responsive cis-DNA elements. Further luciferase assays suggested that the cis-DNA element was between -514 and -464 bp in the OSM promoter (Fig. 4C). In addition, a mutational analysis of the OSM promoter activity was performed. A series of mutant OSM promoter reporters were generated by introducing mutations into the OSM promoter region between -514 and -464 bp in pOSM1064 with an EcoRI linker (GAATTC) at 6-bp intervals. As demonstrated in Fig. 5, mutations in the promoter region between -496 and -485 bp suppressed the trans-activating effect of ET-1 on the OSM promoter. Analysis of this region with the online software, PROMO (11,12) revealed a consensus Ets-1 binding site between -494 and -488 bp (Fig. 6A).

EMSAs were conducted to examine whether Ets-1 specifically binds to the -494/-488 putative Ets-1 binding site. Oligonucleotide WT504/475 corresponding to the human OSM promoter sequence between -504 and -475 bp was radiolabeled and used as the probe to incubate with OA osteoblast cell nuclear extract in the EMSAs (Fig. 6A). Unlabeled WT504/475, and Mut496/491 and Mut490/485, two oligonucleotides with the same sequence as WT504/475 except for the EcoRI linker (GAATTC) mutations at -496/-491 and -490/-485, respectively, served as competitors to the probe (Fig. 6A). As demonstrated in Fig. 6B, nuclear extract from ET-1-treated cells indicated a markedly stronger binding activity with the probe compared with that of the untreated control cells. A 100-fold molar excess of unlabeled WT504/475 (but not the mixture of 100-fold molar excess of each of Mut496/491 and Mut490/485) suppressed the binding activity (Fig. 6B), suggesting specific protein binding at the -494/-488 putative Ets-1 binding site. Furthermore, the control serum exerted no effect, whereas the anti-Ets1 antibody supershifted the major protein-DNA complex to a higher position (Fig. 6B). The results indicate that ET-1 may markedly increase specific binding of Ets-1 to the -494/-488 region in the OSM promoter in OA osteoblasts. In addition, the Ets1-DNA complex was abolished by Ets-1 shRNA, and not the scramble control shRNA, further confirming the specificity of the Ets1-DNA complex.

To determine the functional role of Ets-1 in the ET1-induced expression of OSM, the activity of Ets-1 in OA osteoblasts was knocked down with lentiviral shRNA. As demonstrated in Fig. 7A, the protein levels of Ets-1 were markedly increased by ET-1, compared with the Ets-1-shRNA group. However, transduction of Ets-1-shRNA significantly decreased the Ets-1 protein level by ~80%, in the presence and absence of ET-1 (P<0.05). As demonstrated in Fig. 7B, Ets-1 knockdown suppressed the ET1-induced expression of OSM. The results indicate that Ets-1 is an important mediator of the inducing effect of ET-1 on OSM expression in OA osteoblasts.

OA osteoblasts were treated with ET-1 (10 and 30 nM) for 4 h to assess the effect of ET-1 on the Ets-1 mRNA and protein expression levels. As demonstrated in Fig. 8, ET-1 treatment (10 and 30 nM) significantly increased the expression of Ets-1 at the mRNA and protein levels compared with the control group (P<0.05). This effect was significantly suppressed following co-treatment with BQ123 or BKM120, compared with the control and ET-1 only groups (Fig. 8; P<0.05). However, co-treatment with BQ788 significantly increased the expression of Ets-1 compared with the control and ET-1 only groups (Fig. 8; P<0.05). These findings indicate that ET-1 may increase Ets-1 expression levels in OA osteoblasts in an ETAR- and PI3K-dependent manner.