Up-Regulation of Fibroblast Growth Factor 23 Gene Expression in UMR106 Osteoblast-like Cells with Reduced Viability

Fibroblast growth factor 23 (FGF23) controls vitamin D and phosphate homeostasis in the kidney and has additional paracrine effects elsewhere. As a biomarker, its plasma concentration is associated with progression of inflammatory, renal, and cardiovascular diseases. Major stimuli of FGF23 synthesis include active vitamin D and inflammation. Antineoplastic chemotherapy treats cancer by inducing cellular damage ultimately favoring cell death (apoptosis and necrosis) and causing inflammation. Our study explored whether chemotherapeutics and other apoptosis inducers impact on Fgf23 expression. Experiments were performed in osteoblast-like UMR106 cells, Fgf23 gene expression and protein synthesis were determined by qRT-PCR and ELISA, respectively. Viability was assessed by MTT assay and NFκB activity by Western Blotting. Antineoplastic drugs cisplatin and doxorubicin as well as apoptosis inducers procaspase-activating compound 1 (PAC-1), a caspase 3 activator, and serum depletion up-regulated Fgf23 transcripts while reducing cell proliferation and viability. The effect of cisplatin on Fgf23 transcription was paralleled by Il-6 up-regulation and NFκB activation and attenuated by Il-6 and NFκB signaling inhibitors. To conclude, cell viability-decreasing chemotherapeutics as well as apoptosis stimulants PAC-1 and serum depletion up-regulate Fgf23 gene expression. At least in part, Il-6 and NFκB may contribute to this effect.


Introduction
Cells that make up bone, osteoblasts, and osteocytes produce fibroblast growth factor 23 (FGF23), a protein with classical endocrine, but also paracrine effects [1,2]. As a hormone, it targets renal sodium phosphate co-transporter NaP i IIa, the main phosphate transporter of the proximal tubule, thereby enhancing urinary elimination of phosphate [3]. Moreover, FGF23 down-regulates CYP27B1, the renal key enzyme for the activation of vitamin D [4]. Therefore, FGF23 lowers the plasma concentration of active vitamin D (1,25(OH) 2 D 3 ), which itself is a major regulator of phosphate metabolism [5]. Further endocrine effects of FGF23 are effective in the parathyroid gland, where FGF23 reduces parathyroid hormone (PTH) expression and secretion [6]. These classical endocrine effects require a complex of a FGF receptor (FGFR) and co-receptor αKlotho, a transmembrane protein with high expression in the kidney and parathyroid gland [7][8][9]. A certain motif with FGF23-independent endocrine and paracrine effects can be released from αKlotho upon cleavage, called soluble Klotho (sKl) [7,10]. The correct interplay of FGF23 and αKlotho in the regulation of phosphate and vitamin D metabolism is critical: mice deficient for FGF23 or αKlotho age rapidly and exhibit premature aging-associated diseases with death at young age, whereas overexpression of αKlotho extends life span by about 30% [11][12][13].
To study cell proliferation, cells were trypsinized after 24 h or 48 h, respectively, and counted on a Neubauer hemocytometer.

Quantitative Real Time PCR
Total RNA was isolated from UMR106 cells using RNA-Solv reagent (Omega Bio-Tek, Norcross, GA, USA), and 1.2 µg thereof was used for cDNA synthesis with the GoScript Reverse Transcription System and random primers (Promega, Mannheim, Germany) on a Biometra TAdvanced thermal cycler (Analytik Jena, Jena, Germany).

Enzyme Linked Immunosorbent Assay (ELISA)
Cell culture supernatants were collected and concentrated using Vivaspin ® 2 ultrafiltration columns (Sartorius, Göttingen, Germany). C-terminal FGF23 protein concentration was then determined by ELISA according to the manufacturer's protocol (Immutopics, San Clemente, CA, USA).

Western Blot
UMR106 cells were seeded into T25 cell culture flasks (Greiner Bio-One) and cultured for 24 h under standard conditions, then treated with 10 µM cisplatin or vehicle for another 24 h. Next, cells were lyzed using RIPA buffer (Cell Signaling Technology, Frankfurt, Germany) supplemented with protease and phosphatase inhibitor cocktail and EDTA (Halt, Thermo Scientific), total protein concentration measured by Bradford assay (Bio-Rad), and 30 µg of total protein subjected to 10% SDS-PAGE and standard Western Blotting. The following antibodies were used: anti-phospho-p65-NFκB (Ser536; 93H1), anti-GAPDH (D16H11), and anti-rabbit IgG HRP-linked antibody (all from Cell Signaling Technology). For visualization, membranes were incubated for 2 min with Westar Nova 2.0 (GAPDH) or Westar Supernova (phospho-p65-NFκB) ECL substrate (both from Cyanagen, Bologna, Italy). The densitometrical analysis was performed on a C-Digit ® Blot scanner (Li-Cor, Lincoln, NE, USA) and phospho-p65-NFκB bands were normalized to GAPDH bands using the Image Studio™ software (Li-Cor).

Statistics
Data are shown as arithmetic means ± standard error of the mean (SEM) with n representing the number of independent experiments. Normal distribution was tested using Shapiro-Wilk normality test. Effects on cell number and viability and western blots were analyzed with one-sample t-test or one-sample Wilcoxon signed rank test, respectively. Two groups were analyzed with student's t-test, Welch's test, or Mann-Whitney U test. More than two groups were analyzed with one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test, Dunnett T3 test, or with nonparametric Kruskal-Wallis test followed by Dunn-Bonferroni post hoc test. Differences were considered significant if p < 0.05. Statistics were made using IBM SPSS Statistics (Version 27.0; Armonk, NY, USA).

Results
To investigate whether chemotherapeutics impact on Fgf23 expression, we performed experiments in UMR106 osteoblast-like cells. In a first series of experiments, the cells were treated with platinum derivative cisplatin, an antineoplastic drug used in the treatment of a variety of malignancies, and Fgf23 transcript levels were determined by qRT-PCR. As demonstrated in Figure 1A, cisplatin enhanced Fgf23 gene expression in UMR106 cells in a dose-dependent manner within 24 h. By the same token, exposure to cisplatin reduced number ( Figure 1B) and viability ( Figure 1C) of UMR106 cells following a 24-h exposure.

Results
To investigate whether chemotherapeutics impact on Fgf23 expression, we performed experiments in UMR106 osteoblast-like cells. In a first series of experiments, the cells were treated with platinum derivative cisplatin, an antineoplastic drug used in the treatment of a variety of malignancies, and Fgf23 transcript levels were determined by qRT-PCR. As demonstrated in Figure 1A, cisplatin enhanced Fgf23 gene expression in UMR106 cells in a dose-dependent manner within 24 h. By the same token, exposure to cisplatin reduced number ( Figure 1B) and viability ( Figure 1C) of UMR106 cells following a 24-h exposure.  To check whether upregulation of Fgf23 gene expression is a stress reaction only observable at 24 h, we extended exposure time in a further series of experiments. According to Figure 1D, also a 48-h exposure of UMR106 cells resulted in dose-dependent upregulation of Fgf23 gene expression. Cell number ( Figure 1E) and viability ( Figure 1F), however, were more strongly reduced upon a 48-h exposure to cisplatin compared to a 24-h incubation ( Figure 1B,C). The next series of experiments was carried out to investigate whether anthracyclines, chemotherapeutic drugs that inhibit topoisomerase and intercalate with DNA [33], are similarly capable of inducing Fgf23 gene expression. UMR106 cells exposed to doxorubicin (0.03-0.3 µM) for 24 h exhibited enhanced Fgf23 gene expression in a dose-dependent manner ( Figure 2A). Similar to cisplatin, doxorubicin also compromised cell proliferation ( Figure 2B) and viability ( Figure 2C). Again, we tested whether a longer exposure similarly up-regulated Fgf23. As a result, incubation of UMR106 cells with doxorubicin for 48 h killed virtually all cells ( Figure 2D). Hence, Fgf23 transcripts were not detectable after 48 h.
To check whether upregulation of Fgf23 gene expression is a stress reaction only observable at 24 h, we extended exposure time in a further series of experiments. According to Figure 1D, also a 48-h exposure of UMR106 cells resulted in dose-dependent upregulation of Fgf23 gene expression. Cell number ( Figure 1E) and viability ( Figure 1F), however, were more strongly reduced upon a 48-h exposure to cisplatin compared to a 24-h incubation ( Figure 1B,C).
The next series of experiments was carried out to investigate whether anthracyclines, chemotherapeutic drugs that inhibit topoisomerase and intercalate with DNA [33], are similarly capable of inducing Fgf23 gene expression. UMR106 cells exposed to doxorubicin (0.03-0.3 μM) for 24 h exhibited enhanced Fgf23 gene expression in a dose-dependent manner ( Figure 2A). Similar to cisplatin, doxorubicin also compromised cell proliferation ( Figure 2B) and viability ( Figure 2C). Again, we tested whether a longer exposure similarly up-regulated Fgf23. As a result, incubation of UMR106 cells with doxorubicin for 48 h killed virtually all cells ( Figure 2D). Hence, Fgf23 transcripts were not detectable after 48 h. Our results indicate that cytotoxic reagents up-regulate Fgf23 gene expression in UMR106 cells. In order to test whether this effect is mimicked by direct stimulation of apoptosis, PAC-1, an activator of apoptosis-initiating executioner caspase 3, was applied. As demonstrated in Figure 3A, similar to chemotherapeutics, PAC-1 dose-dependently up-regulated Fgf23 gene expression in UMR106 cells within 24 h. This effect was paralleled by compromised cell proliferation ( Figure 3B) and viability ( Figure 3C), as well. A 48-h exposure to PAC-1 did not significantly modify Fgf23 transcripts in UMR106 cells ( Figure 3D) while suppressing cell proliferation ( Figure 3E) and viability ( Figure 3F). Our results indicate that cytotoxic reagents up-regulate Fgf23 gene expression in UMR106 cells. In order to test whether this effect is mimicked by direct stimulation of apoptosis, PAC-1, an activator of apoptosis-initiating executioner caspase 3, was applied. As demonstrated in Figure 3A, similar to chemotherapeutics, PAC-1 dose-dependently up-regulated Fgf23 gene expression in UMR106 cells within 24 h. This effect was paralleled by compromised cell proliferation ( Figure 3B) and viability ( Figure 3C), as well. A 48-h exposure to PAC-1 did not significantly modify Fgf23 transcripts in UMR106 cells ( Figure 3D) while suppressing cell proliferation ( Figure 3E) and viability ( Figure 3F).   Figure 4A). Again, the effect was paralleled by decreased proliferation ( Figure 4B) and viability ( Figure 4C) of UMR106 cells. The stimulatory effect of serum depletion on Fgf23 transcripts was followed by enhanced secretion of C-terminal FGF23 protein into the cell culture supernatant ( Figure 4D). Also, 48 h serum depletion up-regulated Fgf23 gene expression ( Figure 4E), an effect again paralleled by reduced proliferation ( Figure 4F) and viability ( Figure 4G).
Downstream signaling of pro-inflammatory stimuli may eventually result in the activation of transcription factor complex NFκB, an important driver of FGF23 production [30]. Further experiments, therefore, focused on the involvement of NFκB in the stimulation of Fgf23 by cisplatin. Within 24 h, treatment of UMR106 cells with 10 μM cisplatin resulted in enhanced Rela expression, the gene encoding p65 subunit of NFκB ( Figure 6A). As detected by Western Blotting, cisplatin (10 μM, 24 h) significantly stimulated phosphorylation of p65 ( Figure 6B). Moreover, treatment with doxorubicin (0.3 μM, 24 h) enhanced Rela expression ( Figure 6C). Hence, cisplatin and doxorubicin induced NFκB activity in UMR106 cells. A last series of experiments explored whether NFκB activity is required for the effect of cisplatin on Fgf23. To this end, UMR106 cells were treated with and without cisplatin and NFκB inhibitors wogonin or withaferin A for 24 h. As depicted in Figure 6D, wogonin significantly attenuated the cisplatin-induced effect on Fgf23 gene expression. Similarly, withaferin A blunted cisplatin-induced up-regulation of Fgf23 (Figure 6E). : Arithmetic means ± SEM of Fgf23 transcript levels relative to Tbp in UMR106 cells treated without (ctr) or with 10 µM cisplatin in the presence or absence of 1 µM Il-6 signaling inhibitor SC144 (n = 9; Kruskal-Wallis followed by Dunn-Bonferroni test) for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significant differences from vehicle-treated cells (1st bar); # p < 0.05 indicates significant difference from absence of SC144 (2nd bar vs. 4th bar). a. u., arbitrary units; ctr, control.
Downstream signaling of pro-inflammatory stimuli may eventually result in the activation of transcription factor complex NFκB, an important driver of FGF23 production [30]. Further experiments, therefore, focused on the involvement of NFκB in the stimulation of Fgf23 by cisplatin. Within 24 h, treatment of UMR106 cells with 10 µM cisplatin resulted in enhanced Rela expression, the gene encoding p65 subunit of NFκB ( Figure 6A). As detected by Western Blotting, cisplatin (10 µM, 24 h) significantly stimulated phosphorylation of p65 ( Figure 6B). Moreover, treatment with doxorubicin (0.3 µM, 24 h) enhanced Rela expression ( Figure 6C). Hence, cisplatin and doxorubicin induced NFκB activity in UMR106 cells. A last series of experiments explored whether NFκB activity is required for the effect of cisplatin on Fgf23. To this end, UMR106 cells were treated with and without cisplatin and NFκB inhibitors wogonin or withaferin A for 24 h. As depicted in Figure 6D, wogonin significantly attenuated the cisplatin-induced effect on Fgf23 gene expression. Similarly, withaferin A blunted cisplatin-induced up-regulation of Fgf23 ( Figure 6E). ; Kruskal-Wallis test followed by Dunn-Bonferroni test) or 500 nM withaferin A ((E); n = 9; Kruskal-Wallis test followed by Dunn-Bonferroni test). * p < 0.05, ** p < 0.01 indicate significant difference from vehicle-treated cells (1st bar). # p < 0.05 indicates significant difference from the absence of NFκB inhibitors wogonin and withaferin A, respectively (2 nd bar vs. 4 th bar). a. u., arbitrary units; ctr, control.

Discussion
According to our study, two cytotoxic drugs with different cellular targets used in the treatment of several malignancies as well as apoptosis inducers PAC-1 and serum depletion stimulated Fgf23 gene expression in UMR106 osteoblast-like cells within 24 h. The effect was paralleled by a reduction in cell viability and proliferation as deduced from cell number. ; Kruskal-Wallis test followed by Dunn-Bonferroni test) or 500 nM withaferin A ((E); n = 9; Kruskal-Wallis test followed by Dunn-Bonferroni test). * p < 0.05, ** p < 0.01 indicate significant difference from vehicletreated cells (1st bar). # p < 0.05 indicates significant difference from the absence of NFκB inhibitors wogonin and withaferin A, respectively (2 nd bar vs. 4 th bar). a. u., arbitrary units; ctr, control.

Discussion
According to our study, two cytotoxic drugs with different cellular targets used in the treatment of several malignancies as well as apoptosis inducers PAC-1 and serum depletion stimulated Fgf23 gene expression in UMR106 osteoblast-like cells within 24 h. The effect was paralleled by a reduction in cell viability and proliferation as deduced from cell number.
UMR106 osteoblast-like cells were chosen for our study because under physiological conditions, bone is the major site of FGF23 production [45] and these cells are a versatile tool employed in many studies to unravel the regulation of FGF23 [25,[46][47][48][49].
Incubation of UMR106 cells with cisplatin or in serum-depleted medium for 48 h also resulted in enhanced Fgf23 expression. Prolonged incubation with doxorubicin, however, killed all cells. In contrast to 24 h, 48-h exposure of the cells to PAC-1 did not significantly modify Fgf23 expression, possibly because PAC-1-dependent apoptosis induction occurs much earlier and late apoptotic cells cannot up-regulate Fgf23 gene expression any longer.
Cisplatin, doxorubicin, PAC-1 as well as serum depletion have in common that they cause cellular damage reducing cell number and viability, which may ultimately result in cell death. Cisplatin is effective by interfering with DNA replication [50], doxorubicin inhibits topoisomerase and intercalates with DNA [51], PAC-1 directly stimulates apoptotic cell death through executioner caspase 3 [35], whereas serum depletion favors apoptotic cell death due to lack of essential growth factors [36]. Although the mechanism of cell damage is different, the up-regulation of Fgf23 gene expression is consistent for all four inducers of cellular injury. This important finding may point to a role of FGF23 in cellular stress, cell death, and survival. Indeed, FGF23-Klotho signaling favors cell proliferation and inhibits apoptosis, elicited by vitamin D, through phosphoinositide-3 kinase (PI3K) signaling [52]. Moreover, FGF23 exerts many effects through serum and glucocorticoiddependent kinase 1 (SGK1) [53]. SGK1 is an important mediator of pro-survival signaling inhibiting apoptosis [54]. Moreover, in acute kidney injury (AKI), FGF23 has turned out to stimulate cell proliferation promoting regeneration of injured tubules through influencing SDF-1/CXCR4 signaling [55]. In tumor cells, namely prostate cancer, FGF23 similarly stimulates cell proliferation [56]. According to these studies, FGF23 has pro-survival/antiapoptotic properties. Hence, up-regulation of FGF23 in cell stress as demonstrated in our study may help the cell activate pro-survival signaling. Alternatively, FGF23 may not only be a disease biomarker, but Fgf23 gene expression may also indicate injury on cellular level or even serve as a marker for moribund cells. Definitely, further research is required to elucidate this.
In UMR106 cells, basal Fgf23 expression is low unless the cells are pretreated with 1,25(OH) 2 D 3 which strongly up-regulates Fgf23 expression [24]. Therefore, it must be kept in mind that although Fgf23 transcripts significantly increased upon treatment with cisplatin, doxorubicin, or PAC-1, yet the cellular FGF23 protein concentration remained below the detection limit of ELISA. Serum depletion experiments were accomplished in the presence of 10 nM 1,25(OH) 2 D 3 , hence, C-terminal FGF23 protein in the cell culture supernatant could be detected by ELISA and was significantly up-regulated in serumdepleted cells compared to control cells.
Chemotherapy is known to induce inflammation [37]. We demonstrated that both cisplatin and doxorubicin induce pro-inflammatory cytokine Il-6 within 24 h. Importantly, Il-6 is a stimulator of FGF23 [28]. In line with this, Il-6 signaling inhibitor SC144 significantly blunted cisplatin-induced Fgf23 gene expression. Moreover, expression and phosphorylation of NFκB subunit p65 were up-regulated by cisplatin. Accordingly, wogonin and withaferin A, inhibitors of NFκB, significantly blunted cisplatin-induced up-regulation of Fgf23 expression. This is in line with the pivotal role of NFκB and generally inflammation for the stimulation of FGF23 production. Importantly, cisplatin is a powerful inducer of NFκB activity [57], which may also contribute to treatment resistance [58] or nephrotoxicity [59]. Doxorubicin also induces inflammation by activating NFκB [60,61]. Hence, it appears likely that chemotherapy-induced inflammation involving Il-6 and NFκB is a major contributor to the up-regulation of Fgf23 expression. In our experiments, wogonin and withaferin A tended to decrease Fgf23 transcript levels in untreated cells, a difference, however, not reaching statistical significance. Presumably, the effect of NFκB inhibition on Fgf23 is smaller in cells with low basal Fgf23 expression in the absence of 1,25(OH) 2 D 3 stimulation than in cells pre-treated with 1,25(OH) 2 D 3 to up-regulate Fgf23 expression [30].
Direct executioner caspase-3-activator PAC-1 also up-regulated Fgf23 gene expression. The same holds true for serum depletion, which favors apoptosis through growth factor deficiency [62]. However, caspase 3 activation and subsequent apoptosis are rather associated with decreased NFκB activity and not with a pro-inflammatory response [63]. Hence, additional mechanisms elucidated by future studies can clearly be expected to be also involved in the up-regulation of Fgf23 expression of injured cells.
Taken together, the induction of cellular injury through cytotoxic drugs, serum depletion, or caspase 3 activation resulting in decreased proliferation and viability leads to the up-regulation of Fgf23 gene expression. This effect can in part, but not fully, be explained by IL-6 up-regulation and NFκB activation.