The production of fibroblast growth factor 23 is controlled by TGF-β2

Transforming growth factor-β (TGF-β) is a cytokine produced by many cell types and implicated in cell growth, differentiation, apoptosis, and inflammation. It stimulates store-operated calcium entry (SOCE) through the calcium release-activated calcium (CRAC) channel Orai1/Stim1 in endometrial Ishikawa cells. Bone cells generate fibroblast growth factor (FGF) 23, which inhibits renal phosphate reabsorption and 1,25(OH)2D3 formation in concert with its co-receptor Klotho. Moreover, Klotho and FGF23 counteract aging and age-related clinical conditions. FGF23 production is dependent on Orai1-mediated SOCE and inflammation. Here, we explored a putative role of TGF-β2 in FGF23 synthesis. To this end, UMR106 osteoblast-like cells were cultured, Fgf23 transcript levels determined by qRT-PCR, FGF23 protein measured by ELISA, and SOCE analyzed by fluorescence optics. UMR106 cells expressed TGF-β receptors 1 and 2. TGF-β2 enhanced SOCE and potently stimulated the production of FGF23, an effect significantly attenuated by SB431542, an inhibitor of the transforming growth factor-β (TGF-β) type I receptor activin receptor-like kinases ALK5, ALK4, and ALK7. Furthermore, the TGF-β2 effect on FGF23 production was blunted by SOCE inhibitor 2-APB. We conclude that TGF-β2 induces FGF23 production, an effect involving up-regulation of SOCE.

factor NFκB up-regulates the calcium release-activated calcium (CRAC) channel Orai1/STIM1 which mediates store-operated calcium entry (SOCE) 18 . Orai1/STIM1-mediated SOCE then triggers FGF23 production. Of note, TGF-β has also been shown to up-regulate Orai1/STIM1 in endometrial tumor cells 19 . Moreover, the production of TGF-β is also enhanced in some of the aforementioned disorders associated with high serum FGF23 levels (e.g. chronic kidney disease 3 ).
The present study addressed the question whether the TGF-β-isoform TGF-β2 is a regulator of FGF23 formation.

TGF-β2 induced Fgf23 transcription in UMR106 cells. Our first series of experiments aimed to
explore whether TGF-β2 is capable of influencing the production of FGF23. To this end, we treated UMR106 osteoblast-like cells with and without TGF-β2 for 24 h and determined Fgf23 transcripts by qRT-PCR to estimate FGF23 synthesis. Figure 1A illustrates that the presence of TGF-β2 significantly up-regulated Fgf23 transcript levels, pointing to enhanced FGF23 production. Next, we sought to investigate whether the increase in Fgf23 transcript levels upon incubation with TGF-β2 indeed translated into enhanced FGF23 protein formation. Thus, we determined C-terminal FGF23 protein in the supernatant retrieved from cells incubated with and without TGF-β2 for 24 h using ELISA. As shown in Fig. 1B, TGF-β2 significantly stimulated FGF23 production. Figure 1C depicts the dose response effect of TGF-β2 on Fgf23 transcripts in UMR106 cells.
The TGF-β2 effect on Fgf23 expression was mediated by downstream TGF-β receptor signaling. The cellular effects of TGF-β2 are typically mediated by the joint action of TGF-β receptors 1 and 2.
Employing RT-PCR, we therefore studied the expression of these receptors in UMR106 cells. It is demonstrated in Fig. 2 that UMR106 cells expressed TGF-β receptors 1 and 2. Moreover, cells retrieved from mouse bone also expressed both receptors (Fig. 2). As a control, expression of TGF-β receptors 1 and 2 in rat NRK-52E cells is provided (Fig. 2). protein concentration (pg/ml) in the cell culture medium of UMR106 cells incubated without (ctrl, white bars) or with TGF-β2 (black bars, 10 ng/ml, 24 h). **p < 0.01, ***p < 0.001 indicate significant difference compared to UMR106 cells treated with vehicle only (paired t-test). (C) Arithmetic mean ± SEM (n = 5) of Fgf23 mRNA levels (relative to Tbp mRNA) in UMR106 cells after 24-hour treatment without (ctrl, white bar) or with TGF-β2 (0.3-10 ng/ml, black bars). ***p < 0.001 indicates significant difference compared to UMR106 cells treated with vehicle only (ANOVA).
As a next step, we explored whether the stimulating effect of TGF-β2 on the formation of FGF23 was dependent on downstream TGF-β receptor signaling which involves TGF-β receptor-related kinases. Hence, we determined the TGF-β2 effect on Fgf23 transcripts in the presence and absence of SB431542, an antagonist of TGF-β receptor-related kinases. As shown in Fig. 3, the effect of TGF-β2 on Fgf23 mRNA was significantly attenuated in the presence of SB431542. This result demonstrates that TGF-β receptor-related kinases were required for TGF-β2 to enhance FGF23 generation.

Store-operated calcium entry (SOCE) was needed for the TGF-β2 effect on Fgf23 expression.
Store-operated calcium entry through Orai1/STIM1 has been shown to induce FGF23 formation in UMR106 cells 18 . Moreover, TGF-β stimulates Orai1/STIM1 expression and thus SOCE in other cell types 19 . We therefore sought to clarify whether SOCE participates in TGF-β2-induced FGF23 release. To this end, we utilized Fura-2-dependent Ca 2+ imaging in UMR106 cells after depletion of sarcoplasmic Ca 2+ stores with thapsigargin to assess SOCE. As illustrated in Fig. 4, treatment with TGF-β2 enhanced the thapsigargin-induced increase of cellular Ca 2+ and SOCE in UMR106 cells.
Given the stimulating effect of TGF-β2 on SOCE in UMR106 cells, we addressed the question, whether SOCE is required for the induction of Fgf23 transcription by TGF-β2. In order to test this, we carried out experiments with SOCE inhibitor 2-APB. According to Fig. 5, the induction of FGF23 synthesis by TGF-β2 was significantly blunted by 2-APB. This result suggests that SOCE contributes to the TGF-β2 effect on FGF23 production.
Finally we sought to explore whether the extracellular Ca 2+ concentration influenced Fgf23 transcription in UMR106 cells. To this end, we incubated the cells at different Ca 2+ concentrations (adjusted by the addition of Ca 2+ chelator EGTA or CaCl 2 , resp.) and determined Fgf23 transcripts after 24 h. As a result, in the absence of  Effect of TGF-β2 on Fgf23 transcript levels in UMR106 cells is blunted by TGF-β receptor-related kinase inhibitor SB431542. Arithmetic means ± SEM (n = 9) of Fgf23 mRNA abundance (relative to Tbp mRNA) in UMR106 cells incubated without (white bars) or with (black bars) TGF-β2 (10 ng/ml) in the absence or presence of downstream TGF-β receptor-related kinase inhibitor SB431542 (10 μM) for 24 h. ***p < 0.001 indicates significant difference between cells treated with or without TGF-β2. ## p < 0.01 indicates significant difference between cells treated with TGF-β2 alone (2 nd bar) and cells treated with TGF-β2 and SB431542 (4 th bar) (ANOVA).

Discussion
According to our observations, transforming growth factor-β2 (TGF-β2) up-regulated the expression of fibroblast growth factor 23 (FGF23) in UMR106 osteoblast-like cells.
As a versatile cytokine, TGF-β is involved in a myriad of cellular effects in most tissues and organs including bone, the main site of FGF23 formation. TGF-β is required for both, the formation of bone during development as well as the maintenance and continuous remodeling throughout life 20 . Bone structures such as perichondrium, periosteum, and the epiphyseal growth plate express all TGF-β isoforms 20 . The analysis of transgenic mouse models has, however, revealed that only Tgfb2-deficient mice have an abnormal skeleton whereas Tgfb1-or Tgfb3-deficient mice do not exhibit a bone-related phenotype 20 . Moreover, it is TGF-β2 that is essential for the  development of the skeleton during embryonic development 20 . Therefore, we used TGF-β2 in this study. A putative role of the other TGF-β isoforms in the regulation of FGF23 synthesis must be addressed in a future study. In later life, TGF-β is involved in bone resorption and formation and has several effects on osteoblast differentiation 20 .
Fibroblast growth factors (FGF) have already been demonstrated to be part of downstream TGF-β signaling 21 , and FGF2, FGF4, and FGF6 share the ability with TGF-ß to induce osteoblast proliferation 22 . Our study adds to these results demonstrating that bone-derived FGF23 is directly controlled by TGF-β2.
An important regulator of FGF23 formation is PTH which increases FGF23 expression 15 . Notably, TGF-β receptor 2 phosphorylates the PTH type I receptor thereby attenuating PTH signaling resulting in more trabecular and less cortical bone 23 . Thus, TGF-β is an important modulator of PTH action in bone 24 . Our present results demonstrate that the expression of FGF23, an important target of PTH, is also modulated by TGF-β.
Similar to what has been demonstrated in other cells types 19 , TGF-β2 induced SOCE, but also the thapsigargin-induced elevation in cytosolic Ca 2+ in UMR106 cells. In addition, our study shows that TGF-β2-induced gene expression of Fgf23 was sensitive to 2-APB, an inhibitor of SOCE. These results suggest that the full effect of TGF-β2 required SOCE. In line with this, UMR106 cells have been demonstrated to express Orai1/STIM 18 , and Fgf23 gene expression is enhanced upon Orai1-mediated calcium entry 18 . Moreover, TGF-β up-regulates Orai1 19 and induces cellular calcium influx 28 .
Our results suggest that in the absence of TGF-β2 the extracellular Ca 2+ concentration does not per se influence Fgf23 transcription in UMR106 cells. In contrast, Orai1-mediated Ca 2+ influx from the extracellular space is required for the full TGF-β2 effect on FGF23.
Notably, the stimulating effect of advanced glycation end products (AGEs) 29 and aldosterone 30 on Fgf23 transcription was also significantly blunted by SOCE inhibitor 2-APB, and NFκB has similarly been shown to induce Fgf23 gene expression by enhancing SOCE 18 . Therefore, it appears to be possible that other regulators of SOCE similarly influence Fgf23 transcription.
An elevated FGF23 plasma level is observed in many acute and chronic disorders 14 . Among them, the role of FGF23 has most extensively been studied in chronic kidney disease (CKD) where the FGF23 plasma concentration rises even before the plasma phosphate concentration increases 31 . Notably, CKD and CKD-associated renal fibrosis are associated with enhanced TGF-β formation 32 . Given our present results, it appears likely that enhanced TGF-β production in CKD and other disorders contributes to the high FGF23 plasma concentration common in those diseases. According to a recent study, FGF23 also influences TGF-β expression 33 . Together with our findings, this paper suggests that FGF23 and TGF-β signaling are closely intertwined.
Taken together, TGF-β2 enhanced gene expression and protein synthesis of FGF23 in osteoblast-like cells. The effect was paralleled by enhanced SOCE which was required for the full effect on Fgf23 gene expression. TGF-β2-induced FGF23 formation presumably contributes to high plasma FGF23 levels in acute and chronic diseases.

Methods
Cell culture. Cell culture experiments were conducted as previously described 34 . Briefly, UMR106 rat osteoblast-like cells and NRK-52E rat tubular epithelial kidney cells were cultured in DMEM high glucose medium (Gibco, Life Technologies, Darmstadt, Germany) containing 1.8 mM Ca 2+ supplemented with 10% FBS (Gibco, Life Technologies) (or 5% BCS (Sigma, Schnelldorf, Germany) for NRK-52E cells, resp.) and 100 U/mL penicillin/100 μg/ml streptomycin (Gibco, Life Technologies) under standard culture conditions. UMR106 cells do not have appreciable amounts of Fgf23 mRNA per se. Fgf23 expression was therefore induced by pre-treatment with 100 nM 1,25(OH) 2 D 3 (Tocris, Bristol, UK) 35 . After 24 h, cells were additionally treated with TGF-β2 with or without 25 μM 2-APB or 10 µM SB431542 (all from Sigma) for another 24 h or treated with vehicle only. For the calcium measurements, cells were treated with or without 10 ng/ml TGF-β2 for 1 h.
Calcium concentration in the medium of pretreated UMR106 cells was modified by adding EGTA (0.6-1.8 mM) or CaCl 2 (0.6 and 1.2 mM) for another 24 h.

ELISA.
To assess FGF23 release into the cell culture medium, UMR106 cells were treated for 24 h without or with TGF-β2 (10 ng/ml). Cell culture media were collected for subsequent FGF23 (C-terminal) measurements using an ELISA kit (Immutopics, San Clemente, CA, USA) according to the manufacturer's protocol. Qualitative expression analysis. Total RNA was extracted from UMR106 cells treated for 48 h with 100 nM 1,25(OH) 2 D 3 only or from NRK-52E cells. For analysis of TGF-β receptor type 1 and 2 expression in murine bone tissue, femur and tibia were scrubbed removing residual soft tissue. Both epiphyses from femur and tibia were removed and the bone marrow was flushed out of the bone with 0.9% NaCl. Bone tissue was homogenized in liquid nitrogen using a mortar and pestle. Total RNA from bone was extracted with Tri-Fast (Peqlab). Next, the RNeasy Mini Kit (QIAGEN) was employed according to the manufacturer's protocol.
The following primers were used:  Statistics. Data are provided as means ± SEM, n represents the number of independent experiments. All data were tested for significance using paired or unpaired Student's t-test or one-way ANOVA. Only results with p < 0.05 were considered statistically significant.