FGF-7 Dictates Osteocyte Cell Processes Through Beta-Catenin Transduction

It is well recognized that osteocytes communicate with each other via gap junctions and that connxin43 (Cx43) shows its great potential in gap junction for the contribution enabling transmission of small molecules and operating in an autocrine/a paracrine manner. Fibroblast growth factors (FGFs) play significant roles in new bone formation and adult bone remodeling, and FGF signaling is regulated by the precise spatiotemporal approaches. However, the influence of FGF7 on osteocyte cell processes is not well elucidated. In this study, we aimed to examine the impact of FGF7 on osteocyte cell processes by characterizing the expression of Cx43 and to reveal the underlying mechanism regulating this cell process. We first found that the mRNA level of FGF7 was higher relative to other FGF family members both in osteocytes cell line (MLO-Y4) and bone tissue. We then demonstrated that FGF7 could increase the expression of Cx43 in osteocytes and promote the cell processes in the form of gap junctions between osteocytes. This modulation was due to the FGF7-induced cytoplasmic accumulation and resultant nuclear translocation of β-catenin. Our results could help us to further understand the importance of FGF7 on bone cell behavior and bone physiology and even pathology.

kinases, and furthermore, they are shared by the same core sequence conservation and structure from the point of structural biology 21 , we are interested in whether the profound morphological shift of osteocytes will occur by the induction of FGF7.
In this study, based on the previous reports which showed that nuclear β-catenin activated through the inactivation of glycogen synthase kinase 3 (GSK-3) by PGE2-induced phosphoinositide-3 kinase (PI3K)/Akt and cAMP-PKA signaling stimulates Cx43 expression and gap junction communication between osteocytes 20 .
We aimed to explore the role of FGF7 in osteocyte cell processes and further its potential regulatory mechanisms.

Materials and Methods
Reagents. All  RNA isolations and first-strand cDNA synthesis. Osteocytes RNA samples were isolated using the RNeasy Plus MiniKit with a genomic DNA eliminator. Total RNA samples of bone tissue were isolated from the femur and tibia tissue of 4-6 weeks C57BL/6J mice. Isolated RNA samples were dissolved in RNase-free water and quantified by measuring the absorbance at 260 nm with a spectrophotometer. The RNA samples were then treated with DNase I, and cDNA was prepared from each sample, using 0.5 mg of total RNA and the cDNA synthesis kit in a final volume of 20 μl.
Semi-quantitative PCR. To evaluate the expression levels of FGF members normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), semi-quantitative polymerase chain reaction (PCR) was performed with a PCR kit, using a thermo-cycler (Bio-Rad, Hercules, CA, USA). Selected sets of primers are shown in Table 1. Basic local alignment search tool (BLAST) was used to search for all primer sequences to ensure gene specificity. Semi-quantitative PCR were performed in a 25 μl volume containing a 1 μl cDNA sample. cDNAs of osteocyte and bone tissue are amplificated in two ways: positive reverse transcription (+RT) and mock reverse transcription (−RT) to eliminate the genomic DNA contamination. The PCR procedure consisted of a 30 s denaturation cycle at 94 °C, a 30 s annealing cycle at 55-65 °C and 72 °C, a 30 s elongation cycle, 25-38 amplification cycles. The products were electrophoresed by 2% agarose gel and visualized by staining with ethidium bromide (EB). Quantitative real-time PCR. Quantitative real-time PCR (qPCR) was performed with a QuantiTect SYBR Green PCR Kit using iCycler (Bio-Rad, Hercules, CA, USA) according to operation procedure. qPCR reactions were performed at 0.5 μ mol for each primer in a 25 μl volume containing 1 μl cDNA sample. The reaction was initiated by activating the polymerase with a 5-min pre-incubation at 95 °C. Amplification was achieved with 45 cycles of 15 s denaturation at 94 °C, 15 s annealing at 64 °C and 15 s elongation at 72 °C. The procedure was concluded by the melting curve analysis. All experiments were performed in triplicates. The copy numbers of each gene were determined by cycle threshold (ΔΔCt) methods. Means of the copy numbers of GAPDH were used as internal controls to normalize the data. The standards for establishing standard curves of all primers were prepared from total normal RNA, amplified by qPCR and cloned by TOPO II TA cloning kit, according to the manufacturer's recommendations. FGF7 treatment. Osteocytes were seeded onto six-well plates at 5 × 10 5 cells per well (85~95%) confluence.
Osteocytes allowed to equilibrate for 12 h. Culture media were then replaced with 2% FBS DMEM for a 12 h starvation. Then the osteocytes were divided into the two groups. In the treatment group, the medium was replaced with fresh 1% FBS DMEM containing different concentrations (0.5, 5 and 20 ng/ml) of FGF7. For inhibition experiments, osteocytes were pre-treated with XAV-939 (20 μ mol) for 8 h, then cultured with fresh DMEM containing 20 ng/ml of FGF7 or not. At the protein level, cell lysate samples were collected at 48 h in the control and the treatment groups for western blot.
Immunofluorescence. Osteocytes were seeded onto 35 mm Glass Bottom Dish and allowed to equilibrate for 12 h. Osteocytes were then cultured for 24 h with fresh 10% FBS DMEM containing either 20 ng/ml FGF7 or PBS solution. After the treatments, the cells were washed 3 times for 2 min and fixed with 4% paraformaldehyde for 10 min, followed by three rinsed with PBS. Then they were permeabilized with 2.5% Triton X-100 for 2 min,  Bioinformatics. The information about Genebank ID, gene sequences, the promoter sequences were all from NCBI resources (https://www.ncbi.nlm.nih.gov/) and BioGPS (http://biogps.org/#goto=welcome). The binding site location was achieved through online tool -PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) as previously described 22 . The detailed binding site information was supplemented in Supplementary Material-1.

Statistical analysis.
All experiments were performed in triplicate and reproduced at least three separate times. Statistical analysis of the data was performed with SPSS 16.0 using independent sample t-test analysis to determine whether differences existed. The critical significance level was set to p < 0.05.

Results mRNA level of FGF7 is higher compared with other members of FGF family both in vitro cell line and in vivo bone tissue.
We screened twenty-two family members of FGFs (FGF1-23, wherein FGF15 is the mouse ortholog of human FGF19) which have been identified in C57BL/6J mice (Figs 1 and 2). In order to establish methods for detecting mRNAs encoding known FGFs, the primer pairs of the entire FGF family members in mice were designed (as listed in Table 1). Here, we first determined the expression of the mRNA levels of the entire FGF family in osteocyte cell line MLO-Y4 cells with 22 unique PCR primer pairs (Figs 1 and 2). Using quantitative real time PCR, the transcripts of the whole FGF family members were detected (Fig. 1A). Resultantly, there was a significant expression of mRNA level of FGF7 in vitro osteocytes. We then used RNA samples isolated from the femur and tibia tissue of 4-6 weeks C57BL/6J mice to confirm mRNA levels of the entire FGF family. We found that the FGF7 gene showed a relative lower mRNA level in bone tissue than in osteocyte cell line but still exhibited a higher level compared with other members of the entire FGF family (Fig. 1B). This broad array of expressed FGFs reflects the fetal stage of the bone tissue. FGFs can be divided into three subfamilies: canonical, hormone-like, and intracellular. Using semi-quantitative PCR, compared with quantitative real time PCR, we demonstrated that among the entire FGF family, FGF7 and 10, were the predominant FGF members expressed both in osteocyte cell line and bone tissue, However, FGF16 and 23, were weakly detectable ( Fig. 2A-C). Taken together, we identified FGF7 which was predominantly expressed in osteocyte cell line and bone tissue and thus may serve as the autocrine or paracrine physiologic ligand for FGF receptors.  the cell processes (Fig. 3A). And the quantification further showed the number of osteocyte processes increased. (Fig. 3B). Furthermore, after treatments of FGF7 from 0 (control), 0.5, 5, to 20 ng/ml for 48 h, the expression of Cx43, which is regarded as a protein marker of gap junction, was increased in a dose-dependent manner by western blot (Fig. 3C), and the quantification further confirm the increase (1.23, 1.52, and 1.69-fold) with the increased concentrations of FGF7, respectively (Fig. 3D).

FGF7 regulates CX43 expression and promotes the elongation of osteocyte cell processes.
In order to further indicate the distribution change of Cx43 in osteocytes after FGF7 induction, we used CLSM to confirm its variation. Briefly, osteocytes were cultured with FGF7 at concentration 20 ng/ml. After 24 h of in vitro induction, we found FGF7 increased Cx43 and further modulated cell processes compared with the control group (Fig. 4A). The diameter of lengths (Fig. 4B) and widths (Fig. 4C) of the cell processes were increased to 334%, 360%, respectively. To explore whether FGF7 plays a role on the formation of the gap junction, we cultured the osteocytes with FGF7 (20 ng/ml) as we previously described. After 24 h of in vitro induction, we found FGF7 promoted the new gap-junction formation compared with the control group (Fig. 5A), and more apparently in the magnified boxed area (Fig. 5B). The quantification further showed the length and width of per cell gap junction were increased to 175% and 267%, respectively (Fig. 5C). Herein we proved that FGF7 signaled in a paracrine manner on osteocytes to modulate cell processes.

FGF7 modulates osteocyte cell processes through beta-catenin transduction.
To explore the possible downstream effectors of FGF7-FGFRIIIb activated signaling involved in the regulation of Cx43 expression, the effect of FGF7 on beta-catenin signaling and its downstream Lef-1 were examined. We found that the expression of both beta-catenin and active beta-catenin were increased (Fig. 6B,C). After the treatments of FGF7 (20 ng/ml) for 24 h in vitro induction, immunofluorescence also confirmed the accumulation of cytoplasmic beta-catenin and partial nuclear translocation (Fig. 6A). As the Cx43 serves as a potential target of beta-catenin signaling 23 , we deduced that the Wnt signaling pathway can regulate Cx43 expression to some extent. And then we used the inhibitor of β-catenin, XAV-939, to inhibit the β-catenin expressions and found that β-catenin inhibition reduced the Cx43 level. In the presence of FGF7, β-catenin inhibition partially reduced the Cx43 in relative to FGF7 induction group (Fig. 6D,E). Furthermore, after treatments of FGF7 from 0 (control), 0.5, 5, to 20 ng/ml for 48 h, the expression of Lef-1, the downstream protein of β-catenin, was increased by western blot (Fig. 6F,G). We finally found that Lef1 has the binding sites in the promoter of Gja1, the gene name of Cx43, by bioinformatics (Fig. 6H). This provides the potential and direct role of β-catenin in modulating gap junction formation.

Discussion
Osteocytes occupy the lacunar space reside within the mineralized bone matrix and send cell processes through tiny tunnels called canaliculi to form a canalicular network. Of the major cell types in the bone, osteocytes remained unknown for a long time and defined primarily by their morphology and location rather than by their function 24 . In fact, osteocytes have an extensive network through long, dendritic-like cell processes. Numerous dendritic processes connect osteocytes with adjacent osteocytes and with osteogenic cells on the bone surface via connexin43 (Cx43)-containing gap junctions, the result is a functional syncytium of cells throughout the bone 25 . Abundant data suggest that Cx43 and gap-junction contribute to osteoblastic proliferation and differentiation in vitro [26][27][28] . Meanwhile, in vivo studies, Cx43 null mice display impaired intramembranous bone formation also strongly show that Cx43 may be involved in osteoblastic signaling processes 29,30 . Numerous studies have shown that canonical FGFs, such as FGF2, act in bone. Compared with FGF2, other canonical FGFs have not be studied in detail. Meanwhile, previous study showed that treatment of human skin with recombinant KGF leads to the expression of fibrotic markers 31 . The expression of FGF7 but not of FGF6 and FGF8 is detected in primary osteoblast cell model 12 . Previous study identified the interaction of FGF7 with FGFR2IIIb 32 .
The Wnt signaling pathway has been highlighted in the regulation of bone homeostasis 33 . Beta-catenin is the obligatory transducer for canonical Wnt signaling which is reported to accumulate in the nucleus and bind transcription factors of the high-mobility-group (HMG) box Tcf/Lef family and then stimulates downstream gene expression 34,35 . Further, recent studies identified that osteocytes, as central mediators, control the canonical Wnt signaling pathway in bone and thus induce bone anabolism 36 . Previous study has been demonstrated that Cx43 itself is a target of Wnt signaling pathway 37 . The association of beta-catenin with the 5′ promoter region of Cx43 in MLO-Y4 cells was determined by CHIP assay, and moreover the association of the Cx43 promoter with Pol II and LEF1 was demonstrated 23 .
In this study, we found the following: (1) the mRNA level of FGF7 is higher when compared with other members of FGF family both in vitro and in vivo. (2) FGF7 can increase Cx43 expression and promote gap junction elongate, likely independent of intracellular signaling pathways that may involve concomitant FGF7 induced the accumulation of cytoplasmic beta-catenin and partial nuclear translocation.
There are limitations in this study. First, although MLO-Y4 cell line have proved to be a very useful tool for studying osteocytes in vitro, they cannot provide information on the temporal changes in gene expression and with Image-J software 6.0. Data were means of three independent experiments (n = 3); Significant difference with respect to control, *P < 0.05, **P < 0.025, ***P < 0.001. (C) Western blots showed connexin43 expressions after FGF7 induction increased in a concentration-dose (0.5, 5, 20 ng/ml) dependent manner. The cell lysates were collected at 48 h following FGF7 treatments for connexin43 and beta-actin; the images were collected from two gels with the same loading amounts. The blot gels shown are representatives of three independent experiments (n = 3). (D) Quantitative analysis of western blots of (C) with Image-J software 6.0. Data were means of three independent experiments (n = 3); Significant difference with respect to control. *P < 0.05, **P < 0.025.  morphology which occur as an osteoblast differentiates into an osteocyte 24 . As osteocytes are gaining increasing interest as a target of therapeutics to increase bone mass. We hope to further study the effects of FGF7 on primary osteocytes. Second, as far as we know, FGF7 deficient mice developed a somewhat greasy or matted appearance in the hair coat, especially among the male mutant mice. Moreover, FGF7 is not required for the development of many mesenchymal epithelial organs, including salivary glands, kidney, lung, spleen, liver, small intestine, and heart 38 . Later, some studies demonstrated that FGF-7 levels modulate the extent of ureteric bud growth and the number of nephrons in the kidney 39 . And others suggest that FGF7 deficiency impairs inhibitory synapse formation, which results in mossy fiber sprouting and enhanced neurogenesis 40 . At present, there are still lacking studies examining whether deletion of the Fgf7 gene impacts on bone formation and pathology, or the phenotype of Western blot showed beta-catenin and active beta-catenin expressions after FGF7 induction increased in a concentration-dose (0.5, 5 and 20 ng/ ml) dependent manner. Cell lysates were collected at 48 h following FGF7 treatments for beta-catenin, active beta-catenin and beta-actin; the images were collected from two gels with the same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). (C) Quantitative analysis of western blots of (B) with Image-J software 6.0. The data shown are representative of three independent experiments (n = 3). ***P < 0.001, ****p < 0.0001. (D) Western blot showed the inhibitor of β-catenin, XAV-939, reduced the Cx43 level. In the presence of FGF7, β-catenin inhibition partially reduced the Cx43 in relative to FGF7 induction group. The blot gels shown are representatives of three different experiments (n = 3). (E) Quantitative analysis of western blots (D) with Image-J software 6.0. The data shown are representative of three independent experiments (n = 3). *P < 0.05, **P < 0.025. (F) Western blot showed Lef-1 expressions after FGF7 induction increased in a concentration-dose (0.5, 5 and 20 ng/ml) dependent manner. (G) Quantitative analysis of western blots of (F) with Image-J software 6.0. The data shown are representative of three independent experiments (n = 3). ***P < 0.001, ****p < 0.0001, NS, no significant difference. (H) The bioinformatics showed that Lef1 has the binding sites in the promoter of Gja1, the gene name of Cx43.
SCientifiC RepoRts | (2018) 8:14792 | DOI:10.1038/s41598-018-33247-8 FGF deficiency mice may be not very significant due to multiple complicated factors involving in this modulating process. However, in the current study, we show changes of Cx43 in osteocytes in response to recombined Fgf7. This result could provide evidence for the important role of Fgf7 in osteocyte behavior. Third, the intracellular effects of FGF7 activated via complicated signal transduction pathways, we cannot eliminate the involvement of other signaling mechanisms. Many signaling pathways combined with other uncharacterized pathways ultimately lead to the increased expression of Cx43 and the formation of numerous functional gap junction channels to accommodate the effects of FGF7. Fourth, Previous study showed that ERK activity is required for the full effect of Cx43 on osteoblast responsiveness to FGF2 41 . And others suggest that Runx2 is a down-stream transcription factor of ERK signaling involved in FGF7-mediated facilitation of DAG-induced differentiation in mouse ESCs 17 . Furthermore, FGF7 treatment augments mineralization of rat BMSCs with increased expression of osteogenic marker genes and this augmentation is suppressed by inhibitors of JNK and ERK 19 . However, in the current study, we only focused on β-catenin signaling but not elucidated the mechanism of MAPK signaling pathway. It should be investigated in the next work.
Further studies need to clarify the concrete molecular mechanisms between Cx43, beta-catenin, and skeletal homeostasis. More detailed experiments using FGF7 and various osteoblastic cells will be needed to further confirm its exact role in bone formation. Last but not the least, we hope that we can explore bone cell behavior and bone physiology or pathology through an eye of the FGF7 in the future.