Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

<div><p>Objectives</p><p>Fibroblast Growth Factor 23 (FGF23) is well documented as a crucial player in the systemic regulation of phosphate homeostasis. Moreover, loss-of-function experiments have revealed that FGF23 also has a phosphate-independent and local impact on skeletogenesis. Here, we used ATDC5 cell line to investigate the expression of FGF23 and the role it may play locally during the differentiation of these cells.</p><p>Methods</p><p>ATDC5 cells were differentiated in the presence of insulin, and treated with recombinant FGF23 (rFGF23), inorganic phosphate (Pi) and/or PD173074, an inhibitor of FGF receptors (FGFRs). The mRNA expressions of <i>FGF23</i>, <i>FGFRs</i> and markers of hypertophy, <i>Col X and MMP13</i>, were determined by qPCR analysis and FGF23 production was assessed by ELISA. FGFR activation was determined by immunoprecipitation and immunoblotting.</p><p>Results</p><p>FGF23 mRNA expression and production were increased during ATDC5 differentiation. At D28 in particular, rFGF23 stimulation increased hypertrophic markers expression, as <i>Col X</i> and <i>MMP13</i>, and mineralization. A synergic effect of Pi and rFGF23 stimulation was observed on these markers and on the mineralization process. The use of PD173074, a pan-FGFR inhibitor, decreased terminal differentiation of ATDC5 by preventing rFGF23 pro-hypertrophic effects.</p><p>Conclusions</p><p>Altogether, our results provide evidence that FGF23 plays an important role during differentiation of ATDC5 cell line, by promoting both hypertrophy and mineralization.</p></div

Influence of FGF23 on chondrogenic differentiation.

<p>Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 or 28 days and stimulated or not with 100ng/mL mouse rFGF23 for 24h, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance at D14 and D28 of FGF23 (A,B), COLX (C,D) and MMP13 (E,F) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p<0.05 vs ITS. Data are expressed as mean ± SD, n = 3.</p

FGF23 expression and production in ATDC5 differentiation.

<p>Total RNA was extracted from ATDC5 cultured in ITS conditions for 0, 7, 14, 21 and 28 days, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, **: p< 0,01 vs D0. Data are expressed as mean ± SD, n = 4. FGF23 production in media was quantified by ELISA (B), **: p< 0,01 vs D0, ***: p< 0,001, ND: Non Detectable. Total proteins were extracted from ATDC5 (0, 7, 14, 21 or 28 days post-insulin) and 10 μg was subjected to SDS-PAGE with FGF23 (1/200) antibody; ß-actin was used as loading control (C).</p

Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 28).

<p>Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 28).</p

Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 14).

<p>Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 14).</p

Effect of specific inhibition of FGFR activation during ATDC5 differentiation process and after FGF23 stimulation.

<p>Total RNA was extracted from ATDC5 cultured in ITS conditions for 28 days pre-treated or not with 1 μM PD173074 one hour before stimulation with 100 ng/ml of rFGF23 (24h for RNA or 48h for proteins), then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A), MMP13 (B) and COL X (C) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p< 0.05 vs ITS, £: p< 0.01 vs rFGF23. Data are expressed as mean ± SD, n = 3.</p

Influence of FGF23 on Pi-induced mineralisation process.

<p>ATDC5 were fixed with 4% PFA, and then stained by Alizarin red for 45min after 21 or 28 days of insulin stimulation with or without 100 ng/mL of mouse rFGF23. Images presented are representative of 5 independent experiments (A). ATDC5 micromasses were cultured for 14 days with insulin stimulation in the presence of 3 mM Pi or 100 ng/mL rFGF23 or both. Then they were fixed with 4% PFA and stained by Alizarin red for 45min. Images presented are representative of 3 independent experiments (B). Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 days, then reverse transcribed into cDNA and analysed by real-time PCR and compared to ITS. The relative abundance of COL X, MMP13 and FGF23 were normalized to RPS29 mRNA (C, D, E). Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to ITS. *: p < 0.05 vs ITS, #: p < 0.05 vs ITS + Pi and £: p < 0.05 vs ITS + rFGF23. Data are expressed as mean ± SD, n = 3.</p

Expression of FGFRs during ATDC5 differentiation.

<p>Total RNA was extracted from ATDC5 cultured in ITS conditions for 0, 7, 14, 21 and 28 days, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGFR1 (A), FGFR3 (B), FGFR4 (C) and Klotho (D) were normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, **: p< 0,01 vs D0. Data are expressed as mean ± SD, n = 4. Total proteins were extracted from ATDC5 (7, 14, 21 or 28 days post-insulin) and 10 μg was subjected to SDS-PAGE with anti-FGFR1, FGFR3 or FGFR4 (1/200) antibodies; ß-actin was used as loading control (E).</p

Respective contributions of and to transforming growth factor-beta-1 (TGF-β1)-induced increase in extracellular inorganic pyrophosphate (ePPi) production

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Effect of small interfering RNA (siRNA) on Ank and PC-1 mRNA levels. Rat chondrocytes were transfected with siRNA 24 hours before TGF-β1 stimulation. Total RNA was extracted from rat chondrocytes exposed to 10 ng/mL of TGF-β1 for 12 hours (Ank) or 24 hours (PC-1) and then subjected to real-time polymerase chain reaction analysis. The level of Ank, PC-1, and L27 mRNAs was normalized to that of S29 mRNA and expressed as mean percentages (± SD) over control values. Effect of Ank or PC-1 siRNA on ePPi levels. Shown are levels of ePPi in culture supernatant of rat chondrocytes transfected with siRNA and then stimulated for 12 or 24 hours with 10 ng/mL of TGF-β1. ePPi levels were normalized to the amount of total cell proteins (= 6) and are expressed as mean (± SD) in picomoles per microgram of protein. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05

Effect of Smad 7 overexpression on transforming growth factor-beta-1 (TGF-β1)-induced responses in rat chondrocytes

<p><b>Copyright information:</b></p><p>Taken from "Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes"</p><p>http://arthritis-research.com/content/9/6/R122</p><p>Arthritis Research & Therapy 2007;9(6):R122-R122.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2246241.</p><p></p> Rat chondrocytes were electroporated with either empty vector or wild-type Smad 7 overexpressing plasmid (2 μg/well of six-well plate) and then treated for 12 hours with 10 ng/mL of TGF-β1. Total RNA was extracted and subjected to real-time polymerase chain reaction analysis. The mRNA level of aggrecan and Ank was normalized to that of S29 mRNA and is expressed as mean percentages (± standard deviation) over control values from three independent experiments. Statistically significant differences from the control are indicated as *< 0.05 and from TGF-β1-treated cells as #< 0.05