RESUMO
One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis are unclear. This study used MC3T3-E1 FGF23-producing cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 were only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in cells treated with organic phosphate while cells treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of Fgf23 to organic phosphate, suggesting it activates FGF23. TGF-ß signaling inhibition increased Fgf23 expression after the addition of organic phosphate, while the negative TGF-ß regulator Skil decreased this response. In summary, the observed differential response of FGF23-producing to phosphate types may have consequences for phosphate homeostasis.
RESUMO
Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with ß-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders.
Assuntos
Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos , Glicerofosfatos , Espécies Reativas de Oxigênio , Sirtuína 1 , Animais , Sirtuína 1/metabolismo , Sirtuína 1/genética , Glicerofosfatos/farmacologia , Glicerofosfatos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , Fator de Crescimento de Fibroblastos 23/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição HES-1/metabolismo , Fatores de Transcrição HES-1/genética , Linhagem Celular , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Carbazóis/farmacologiaRESUMO
Fibroblast growth factor 23 (FGF23) is produced and secreted by osteocytes and is essential for maintaining phosphate homeostasis. One of the main regulators of FGF23, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is primarily synthesized in the kidney from 25-hydroxyvitamin D (25(OH)D) by 1α-hydroxylase (encoded by CYP27B1). Hitherto, it is unclear whether osteocytes can convert 25(OH)D and thereby allow for 1,25(OH)2D3 to induce FGF23 production and secretion locally. Here, we differentiated MC3T3-E1 cells toward osteocyte-like cells expressing and secreting FGF23. Treatment with 10-6â M 25(OH)D resulted in conversion of 25(OH)D to 150â pmol/L 1,25(OH)2D3 and increased FGF23 expression and secretion, but the converted amount of 1,25(OH)2D3 was insufficient to trigger an FGF23 response, so the effect on FGF23 was most likely directly caused by 25(OH)D. Interestingly, combining phosphate with 25(OH)D resulted in a synergistic increase in FGF23 expression and secretion, likely due to activation of additional signaling pathways by phosphate. Blockage of the vitamin D receptor (VDR) only partially abolished the effects of 25(OH)D or 25(OH)D combined with phosphate on Fgf23, while completely inhibiting the upregulation of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1), encoding for 24-hydroxylase. RNA sequencing and in silico analyses showed that this could potentially be mediated by the nuclear receptors Retinoic Acid Receptor ß (RARB) and Estrogen Receptor 2 (ESR2). Taken together, we demonstrate that osteocytes are able to convert 25(OH)D to 1,25(OH)2D3, but this is insufficient for FGF23 activation, implicating a direct effect of 25(OH)D in the regulation of FGF23, which occurs at least partially independent from its cognate VDR. Moreover, phosphate and 25(OH)D synergistically increase expression and secretion of FGF23, which warrants investigating consequences in patients receiving a combination of vitamin D analogues and phosphate supplements. These observations help us to further understand the complex relations between phosphate, vitamin D, and FGF23.
Assuntos
Calcitriol , Osteócitos , Humanos , Calcifediol , Calcitriol/farmacologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Oxigenases de Função Mista , Osteócitos/metabolismo , Fosfatos , Receptores de Calcitriol/genética , Vitamina D/farmacologia , Animais , CamundongosRESUMO
Fibroblast growth factor 23 (FGF23) has been described as an important regulator of mineral homeostasis, but has lately also been linked to iron deficiency, inflammation, and erythropoiesis. FGF23 is essential for the maintenance of phosphate homeostasis in the body and activating mutations in the gene itself or inactivating mutations in its upstream regulators can result in severe chronic hypophosphatemia, where an unbalanced mineral homeostasis often leads to rickets in children and osteomalacia in adults. FGF23 can be regulated by changes in transcriptional activity or by changes at the post-translational level. The balance between O-glycosylation and phosphorylation is an important determinant of how much active intact or inactive cleaved FGF23 will be released in the circulation. In the past years, it has become evident that iron deficiency and inflammation regulate FGF23 in a way that is not associated with its classical role in mineral metabolism. These conditions will not only result in an upregulation of FGF23 transcription, but also in increased cleavage, leaving the levels of active intact FGF23 unchanged. The exact mechanisms behind and function of this process are still unclear. However, a deeper understanding of FGF23 regulation in both the classical and non-classical way is important to develop better treatment options for diseases associated with disturbed FGF23 biology. In this review, we describe how the currently known upstream regulators of FGF23 change FGF23 transcription and affect its post-translational modifications at the molecular level.