Kisspeptin-10 (KP-10) stimulates osteoblast differentiation through GPR54-mediated regulation of BMP2 expression and activation

Kisspeptin-10 (KP-10) acts as a tumor metastasis suppressor via its receptor, G-protein-coupled receptor 54 (GPR54). The KP-10-GPR54 system plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. Osteoblast differentiation is controlled by a range of hormones and cytokines, such as bone morphogenetic protein (BMPs), and multiple transcription factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Distal-less homeobox 5 (Dlx5). In the present study, KP-10-treatment significantly increased the expression of osteogenic genes, including mRNA and protein levels of BMP2, in C3H10T1/2 cells. Moreover, KP-10 induced BMP2-luc activity and increased phosphorylation of Smad1/5/9. In addition, NFATc4 specifically mediated KP-10-induced BMP2 gene expression. However, KP-10 treatment did not induce expression of the BMP2 and Runx2 genes in GPR54−/− cells. To examine whether KP-10 induced secretion of BMP2 to the culture medium, we used the conditioned-medium (C.M) of KP-10 treated medium on C3H10T1/2 cells. Dlx5 and Runx2 expressions were higher in GPR54−/− cells treated with C.M than in those treated with KP-10. These results demonstrate that BMP2 protein has an autocrine effect upon KP-10 treatment. Taken together, these findings suggest that KP-10/GPR54 signaling induces osteoblast differentiation via NFATc4-mediated BMP2 expression.

expression, C3H10T1/2 cells were treated with various doses of KP-10 for various durations. RT-PCR and real-time PCR analysis showed that KP-10 significantly increased the BMP2 mRNA level in a dose-and time-dependent manner ( Fig. 2A and B). KP-10 also significantly increased BMP2-luc activity in a dose-dependent manner (Fig. 2C). Western blot analysis revealed that KP-10 increased the BMP2 protein level (Fig. 2D). Following extracellular secretion of BMP2, BMP2 dimers bind to BMP receptors in the cell membrane, which induces phosphorylation of Smad1/5/9 33 . KP-10 increased phosphorylation of Smad 1/5/9 in a time-dependent manner (Fig. 2E). This indicated that KP-10 increased BMP2 expression and activation. Taken together, these results suggest that KP-10 increased osteoblast differentiation by increasing expression and activation of BMP2 in C3H10T1/2 cells. KP-10 regulates BMP2 expression through NFATc4. KP-10 induces binding of dephosphorylated NFAT to the BMP7 promoter and thereby regulates BMP7 expression during kidney branching 13 . We investigated whether KP-10 induced expression of NFAT family members in osteoblasts. RT-PCR analyses demonstrated that KP-10 significantly increased expression of NFACTc4, whereas there were slight or no changes expression of NFATc1, NFATc3, and NFATc2 (Fig. 3A). KP-10 increased the NFATc4 mRNA level in a time-dependent manner (Fig. 3B). In addition, BMP2 and osteogenic genes expression was dramatically increased by NFATc4 overexpression using pCMV-NFATc4 (Fig. 3C). BMP2-luc activity was also increased by NFATc4 and/or KP-10 ( Fig. 3D). To examine the effects of NFATc4 on mineralized nodule formation in osteoblasts alizarin red S staining performed. Overexpressed NFATc4 increased matrix mineralization in C3H10T1/2 cells. But, in GPR54 −/− cells was not affected (Fig. 3E). We constructed three types of NFATc4 siRNA primers (siNFATc4I, II and III). Among them, effect of siNFATc4II and III appeared, and next analysis was using the siNFATc4II and III. Interestingly, knockdown of NFATc4 did not increased BMP2 expression by KP-10 in C3H10T1/2 cells (Fig. 3F and G). In addition, siNFATc4 suppressed KP-10-induced BMP2-luc activity, as determined by a luciferase activity assay (Fig. 3H). Taken together, these results suggest that KP-10-induced the expression of the BMP2 gene via NFATc4.

GPR54 deficiency suppresses KP-10-induced osteoblast differentiation. To investigate whether
GPR54 is involved in KP-10-induced BMP2 expression, we analyzed BMP2 protein expression in wild-type and GPR54 −/− cells. KP-10 treatment increased BMP2 protein expression in wild-type cells, but not in GPR54 −/− cells (Fig. 4A). In addition, Runx2 (a downstream target gene of BMP2) expression was also not induced by KP-10 treatment in GPR54 −/− cells, whereas it was significantly increased in wild-type cells (Fig. 4B). Next, to examine whether KP-10 induces autocrine activity of BMP2 (BMP2 protein secreted to the media, and turn on the intracellular signaling by stimulation of specific receptor), we used the conditioned-medium (C.M) of C3H10T1/2 cells treated with KP-10 for 12 h. Expression of Dlx5 and Runx2 was increased in GPR54 −/− cells treated with C.M, but not in those treated with KP-10 (Fig. 4C). These results demonstrate that BMP2 protein has an autocrine effect upon KP-10 treatment. In summary, GPR54 mediated KP-10-induced BMP2 expression, and BMP2 increased expression of osteogenic genes such as Dlx5 and Runx2 via a KP-10-induced autocrine effect in C3H10T1/2 cells.

Discussion
In this study, we first demonstrated that KP-10 stimulated osteoblast differentiation through the BMP2 signaling pathway in C3H10T1/2 cells. Our data provide evidence that KP-10/GPR54 signaling induces osteoblast differentiation via NFATc4-mediated BMP2 expression and activation.
KP-10 is important for the regulation of tumor metastasis, puberty onset, and fertility 3,28,29 . KP-10 and its receptor, GPR54, are key components in the regulation of GnRH secretion in humans and other mammals 11,27 . However, the role of KP-10/GPR54-mediated signaling in osteoblast differentiation is unknown. Therefore, we focused on the role of KP-10 in osteoblasts. Osteoblasts express various phenotypic markers, including Dlx5, Runx2, and ALP [23][24][25] . BMPs play an instrumental role in osteoblast differentiation signaling, and their effects are mediated through Smad signaling 30,31 . KP-10 positively regulated osteoblast differentiation in C3H10T1/2 cells. In addition, KP-10 significantly increased expression of mature osteoblast marker genes (Dlx5, Runx2, and ALP) and ALP activity. Moreover, KP-10 increased BMP2 gene and protein expression. These results suggest that KP-10 regulates osteoblast differentiation though BMP2 in C3H10T1/2 cells.
The role of KP-10/GPR54-mediated signaling in the BMP2 signaling pathway is unknown. GPR54 regulates BMP7 expression through NFATc2 and Sp1, and plays an important role in embryonic kidney branching morphogenesis and glomerular development 13 . Interaction of KP-10, estrogen and BMP4 regulates GnRH production in GT1-7 cells 14 . In this study, KP-10 regulated BMP2 expression through NFATc4 while osteoblast differentiation. The transcription factor NFATc4 is involved in BMP2 transcriptional regulation. Activation of GPR54 by KP-10 led to binding of NFATc4 to the BMP2 promoter. In addition, the KP-10/GPR54 interaction was important for osteoblast differentiation.
Alizarin red S staining. Alizarin red S (Sigma-Aldrich) was solubilized with distilled water. For mineralization analysis, C3H10T1/2 cells were treated with 50 μM KP-10 or transfected with pCMV-NFATc4 (0.4 g) for 20 days and then fixed with 4% formaldehyde (Duksan Pure chemicals Inc.) for 5 min. After washing with distilled water, cells were stained with 300 μg/mL of Alizarin red S solution for 30 min at room temperature. And washing with distilled water. Staining was then documented with an Epson perfection V37 scanner (Seiko Epson, Suwa, Japan).
Western blot analysis. Total cells were harvested using an EzRIPA Lysis kit (ATTO Technology, Tokyo, Japan) and then centrifuged at 12,000 g for 10 min at 4 °C. Total proteins were quantified using the Bradford assay, separated by SDS-PAGE, and transferred to a PVDF membrane. After blocking in 5% skimmed milk prepared in Tris-buffered saline containing Tween 20, the membrane was incubated with specific primary antibodies (1:1000). Signals were detected using ECL reagent (Advansta, Menlo Park, CA). Densitometric analysis of the blotted membrane was performed using a FUSION solo analyzer system (Vilber Lourmat, Eberhardzell, Germany). CRISPR/Cas9 plasmid for GPR54. The Cas9-expressing plasmid was purchased from Addgene (Cambridge, MA). The sgRNA plasmid, which expresses crRNA and tracrRNA under the control of the hU6 promoter was subcloned from the pCLIIP-ALL-EFS-Puro cloning vector (TransOMIC technologies, Huntsville, AL) into the minimal PUC18 backbone plasmid. To knock out the GPR54 gene, oligonucleotides containing target sequences for exon1 were synthesized (Bioneer, Daejeon, Korea) and inserted into the sgRNA plasmid that had been digested with BsmBI.
Transfection and the T7E1 assay. Cells were transfected using the 4D-nucleofector system (Amaxa, Koeln, Germany) at a molecular weight ratio of 1:2 (plasmid encoding Cas9: plasmid encoding sgRNA) and 700 cells were spread over a 100 mm culture dish to form single cell-derived colonies. Then, 5-10 cells from a colony were collected and lysed. Mutant colonies were selected via PCR and the T7E1 assay. Nested-PCR to amplify the GPR54 gene including the exon1 region was performed with DNA-specific primers (forward, 5′-CAGGACACAATCCTTGAAGG-3′; reverse (1), 5′-GTAGGAAAGTGACGTCTGTG-3′; and reverse (2), 5′-CTCGCTTCGTTCCTGACTTG-3′). PCR products were denatured at 95 °C and re-annealed by reducing the temperature to randomly generate heteroduplex DNA, which was treated with 5 units of T7 endonuclease 1 (New England Biolabs, Beverly, MA) for 1 h at 37 °C and analyzed using 2% agarose gel electrophoresis.

Statistical analysis.
All experiments were repeated at least three times. Statistical analysis was performed using the Student's t-test or analysis of variance, followed by Duncan's multiple comparison tests. P values of < 0.05 were considered significant. Results are expressed as the mean ± SEM of triplicate independent samples.