Browsing by Subject "Phosphat"

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Cellular stress regulates fibroblast growth factor 23 (FGF23) und αklotho
(2023) Münz, Sina; Föller, Michael
Cellular stress is defined as the impairment of regular cell function by internal or external stimuli including critical temperatures, energy deficiency, infections, mechanic injury, or chemical noxae. The present thesis aims to investigate the influence of cellular stress on the expression of FGF23 and αklotho. FGF23 is predominantly produced in bone and regulates the phosphate excretion in the kidney. Thereby, αklotho functions as a co-receptor for FGF23. By binding to the FGF receptor-αklotho complex, FGF23 reduces the reabsorption of phosphate from the tubular lumen by decreasing the abundance of sodium-phosphate co-transporters. Furthermore, FGF23 decreases the synthesis of 1,25(OH)2D3, active vitamin D, and increases its degradation. 1,25(OH)2D3 is a regulator of intestinal phosphate absorption and therefore, FGF23 additionally reduces dietary phosphate uptake. Chronically elevated FGF23 is associated with numerous disorders such as kidney disease or CVD. Beside its function as a co-receptor of FGFR, αklotho has many beneficial FGF23-independent functions. It has originally been identified as an anti-aging hormone, as a loss-of-function mutation in the αklotho gene causes numerous aging-like symptoms such as vascular and tissue calcification, osteoporosis, sterility, and an early death. The present papers investigated the influence of cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inducers PAC-1 and serum depletion on the regulation of FGF23 and αklotho. In UMR106 rat osteoblast-like osteosarcoma cells, a 24 or 48 h-treatment with cisplatin, doxorubicin, PAC-1, or serum reduction and depletion significantly up-regulated Fgf23 expression. Under serum depletion, also FGF23 protein secretion was increased. In addition to FGF23, cisplatin and doxorubicin also increased gene expression of pro-inflammatory cytokine Il6 hinting at the presence of necrotic cell death. By inhibiting Il-6 membrane receptor gp130 it has been shown, that FGF23 stimulation partially depended on IL-6 signaling. The stimulation of FGF23 by inflammatory mediators including IL-6, TNFα, TGF-β, or IL-1β has already been reported by others. Furthermore, inflammatory diseases such as rheumatoid arthritis, CKD, or inflammatory bowel disease are associated with excess FGF23 serum concentrations. In this regard, we investigated gene expression and activation of the transcription factor NFκB, which regulates numerous inflammatory functions. Cisplatin and doxorubicin increased the expression of NFκB subunit Rela and cisplatin also stimulated the phosphorylation of NFκB. Independently, NFκB inhibitors wogonin and withaferin A attenuated cisplatin-mediated stimulation of FGF23 indicating, that FGF23 excess was in part promoted by NFκB signaling. These investigations confirmed a strong impact of cisplatin or doxorubicin-induced inflammation on FGF23 synthesis, whereas PAC-1 and serum depletion have reported to directly induce apoptosis, which is commonly not associated with inflammation. Known factors, induced by all cytotoxic substances used here, are the formation of ROS and activation of HIF1α. Both are positive regulators of FGF23, leading to the conclusion, that cellular stress might regulate FGF23 via HIF1α or oxidative stress. FGF23 excess results in increased bone resorption and suppressed bone formation. Likewise, also chemotherapeutic drugs and serum deficiency reduce bone density. Therefore, the stimulation of FGF23 may cause or further stimulate bone resorption. In paper 2, the influence of the cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inductors PAC-1 or serum depletion on αklotho expression in renal MDCK, NRK-52E, and HK-2 cells has been investigated. In fact, all cytotoxic compounds stimulated gene expression of αklotho while decreasing cell proliferation and viability. By using a combined apoptosis and necrosis assay, we confirmed the induction of apoptosis but also necrosis to a variable extent. Additionally, the transcriptional regulation of apoptotic proteins of the BCL-2 family was assessed and confirmed apoptosis stimulation. Transcription factor PPARγ is a known positive regulator of αklotho. In MDCK cells, we detected a significant influence of cisplatin-mediated stimulation of PPARγ mRNA on the αklotho increase. Furthermore, cisplatin, doxorubicin, PAC-1, and serum deprivation also up-regulated FGFR production in MDCK cells. In cancer cells, overexpression of FGFR is associated with enhanced resistance against chemotherapeutic drugs. Consequently, αklotho and FGFR1 stimulation may be a protective mechanism to prevent hyperphosphatemia during diseases. However, human HK-2 cells treated with cisplatin, paclitaxel, doxorubicin, or serum depletion significantly down-regulated αklotho expression and protein secretion. PAC-1 did not change the expression or production of αklotho in HK-2 cells, which might be explained by the minor effect of PAC-1 on non-carcinogenic cells lacking an overexpression of procaspase-3. The differential regulation of αklotho in MDCK and NRK-52E versus HK-2 cells by cytotoxic stress might have numerous causes. For instance, there is evidence of an increased sensitivity of HK-2 cells to stress stimuli but a better comparability to the animal model. However, immortalized cell lines can not completely reflect the conditions of native tissue especially with regard to cell death. Furthermore, the species, sex or age of the donor organism as well as passage number of the cells and drug transporter expression might impact αklotho regulation. Additionally, the mode of cell death determined by intracellular ATP homeostasis and its regulation of AMPK might play an important role in αklotho regulation. However, all these theories need to be further addressed. In summary, inflammation, ROS formation, or the activation of HIF1α are all reported to correlate in a negative manner with αklotho production or serum levels. αklotho down-regulation may be a tool to increase cell proliferation or prevent hypophosphatemia. In contrast, AMPK activation by intracellular ATP restriction may positively regulate αklotho to promote cell protection and avoid hyperphosphatemia.
Einfluss von Phosphatmangel und erhöhter atmosphärischer CO2-Konzentration auf die Wurzelexsudation und ihre Auswirkungen auf Mobilisierung und Aufnahme von Schwermetallen durch verschiedene Lupinenarten und Tomate
(2011) Kawanishi, Ayumi; Römheld, Volker
There is an increasing awareness of a contamination of the food chain by toxic heavy metals as consequence of anthropogenic induced pollution of the environment since the industrialization in the 18. century. In addition the CO2 concentration might promote the biomass formation of plants and thus, via an increased allocation of photo-assimilates into the roots, chemical changes in the rhizosphere. These changes can promote mobility and uptake of various heavy metals by crop plants, too. Therefore it was the main objective of this Ph.D. research, to study the possible consequences of such observed increase in the atmospheric CO2 concentration on the intensification of the rhizosphere chemistry on the uptake of heavy metals by selected plant species in continuation of the research work by Egle (2003) at the University Göttingen. As plant species various lupinus species and tomato were chosen, which differ in principle in their reaction to a low phosphate nutritional status such as root growth characteristics and secretion of protons and carboxylates. As approach two nutrient solution experiments (Chapter 4 and 5) and a soil experiment with heavy metal polluted soils (Chapter 6) were conducted. In both nutrient solution experiments the well-described root-induced changes such as proton and carboxylate release could be confirmed, which were intensified at higher atmospheric CO2 concentrations (Chapter 4 and 5). Surprisingly the detected increase in proton (tomato) and caboxylate release (particularly by white lupin) with a simultaneously increased mobility of Cu and Cd in the soil did not result in an increased concentration of heavy metals in roots and shoots of the growth experimental plants. The unexpected finding in chapter 6 were discussed in the outlook of chapter 6 (6.7) and a repetition of this experiment with consideration of the discussed aspects is urgently recommended.
Transcriptomics and hormonal regulation of cluster root development in phosphate-deficient white lupin
(2014) Wang, Zhengrui; Neumann, Günter
Among crops, white lupin (Lupinus albus) represents the extraordinary ability to acquire sparingly soluble soil phosphate (Pi) by formation of cluster roots (CRs), mediating intense exudation of phosphorus (P)-mobilising root exudates (citrate, phenolics, protons and acid phosphatase). It is widely used as a model plant for investigations of P acquisition by root-induced chemical modifications of the rhizosphere. During the last two decades, a large pool of information on CR function and physiology was obtained mainly by hypothesis-driven research. Based on these findings, this study was designed to get a more comprehensive picture of the metabolic changes during CR development using a transcriptome sequencing approach. The outcome of the transcriptome analysis was the basis for the formulation of research questions on the regulation of CR formation and function to be investigated more in detail: Chapter I, focuses on transcriptome sequencing used for the first time for a systematic comparison of different stages in CR development. To get insights into the regulatory factors involved in CR formation, special emphasis was placed on hormone-related genes. Initiation of CR primordia in the pre-emergent (PE) zone was reflected by strongest expression of genes involved in transport and biosynthesis of auxins, brassinosteroids (BRs) and cytokinin receptors. Cluster root maturation, involving meristem degeneration and root hair proliferation was associated with strongly increased expression of ethylene-related transcripts and decreased expression of auxin- and BR-related genes. Also transcripts related with abscisic and jasmonic acids and cytokinin degradation were up-regulated in mature (MA) clusters. The primary metabolism, highly expressed in juvenile (JU) clusters, underwent significant modifications during CR maturation with increased contribution of Pi-independent bypass reactions, promoting biosynthesis of organic acids. Citrate catabolism and respiration were down-regulated, triggering citrate accumulation in MA clusters. Up-regulation of phenylpropanoid pathways reflected accumulation of phenolics. Increased expression of transcripts encoding ALMT and MATE transporters may be involved in the exudation of flavonoids and citrate, while up-regulation of transcripts encoding Pi transporters mediates subsequent uptake of mobilised Pi. Predominant expression of nucleotide degradation and secretory acid phosphatase in MA clusters coincides with Pi re-translocation and mobilisation of organic soil P. Up-regulation of the FIT transcription factor, usually mediating the expression of Fe deficiency responses (root hair proliferation, proton extrusion, Fe(III)-reduction, exudation of phenolics) can be observed also in MA clusters of P-deficient Lupinus albus even under Fe-sufficient conditions. This raises the question, whether FIT has a similar function in the regulation of P deficiency responses. Chapter II, addresses the question whether sucrose acts as a shoot-borne signal for CR formation. External sucrose amendments to P-sufficient plants, at concentrations similar to those in PE root zones of P-deficient plants, induced CR formation comparable to P-deficient plants. Palatinose (25 mM), and combined application of glucose/fructose (both at 12.5 mM) failed to induce CR formation under P-sufficient conditions, demonstrating a specific signal function of sucrose and excluding osmotic and carbon source effects. However, CRs induced by sucrose were not functional with respect to citrate exudation, acid phosphatase and phosphoenolpyruvate carboxylase (PEPC) activities and expression of related genes (LaMATE, LaSAP and LaPEPC) quantified by RT-qPCR. In Chapter III, the interactions of different phytohormones and sucrose on CR formation were investigated more in detail by an integrated approach of RT-qPCR, hormone translocation analyses and exogenous application of hormones or hormone antagonists. Shoot-to-root translocation of auxin was unaffected by P limitation, supporting the hypothesis that sucrose, rather than auxins, acts as major shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose or P limitation. As reported in other plant species, moderately increased production of ethylene and brassinosteroids (BRs) may induce biosynthesis and transport of root-borne auxins, indicated by increased expression of respective genes (YUCCA, PIN1, AUX1, BR, ACC_oxidase) in pre-emergent clusters. A role of BR in CR formation is further underlined by inhibitory effects of BR antagonists. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the inhibition of lateral rootlets growth during CR maturation, indicated by a massive increase of gene expression involved in ethylene production, associated with decline of transcripts with stimulatory effects (BR- and auxin-related genes). Based on these findings, models for the regulatory networks involved in CR formation (Chapter III) and function (Chapter I) have been developed.