Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Serum levels of ( A ) total FGF23 (cFGF23), ( B ) intact FGF23 (iFGF23), ( C ) intact to total FGF23 ratio (i/c FGF23), ( D ) parathyroid hormone (PTH), ( E ) 1,25-dihydroxyvitamin D [1,25(OH) 2 D], ( F ) calcium (Ca 2+ ), and ( G ) phosphate (Pi); ( H ) fractional excretion of Pi (FePi); ( I ) body weight, ( J ) tail length, and ( K ) femur length; 3D-μCT scan reconstruction of ( L ) distal femur trabecular metaphysis (scale bar = 200 μm); ( M ) midshaft femur cortical diaphysis (scale bar = 500 μm); ( N ) 2D μCT analysis of cortical bone porosity (scale bar = 100 μm); ( O ) red fluorescence microscopy imaging of alizarin red S–stained (ARS-stained) mineralization fronts; ( P ) bright-field microscopy imaging of modified trichrome Goldner staining; and ( Q ) tartrate-resistant acidic phosphatase (TRAcP) staining of longitudinal histology sections of distal femur (scale bar = 100 μm for ARS, 500 μm for Goldner and TRAcP). All analyses were performed in 12-week-old WT ( n ≥ 5) and Dmp1 KO ( n ≥ 5) mice fed a diet containing 0.7% Pi (normal Pi, NP) or 2% Pi (high Pi, HP) from 6 to 12 weeks of age. Values are expressed as mean ± SEM; P < 0.05 vs. a NP-WT, b HP-WT, c NP- Dmp1 KO ; P < 0.1 vs. d NP-WT, e HP-WT. Statistical tests were ANOVA test followed by post hoc t tests and multiple-testing correction using Holm-Bonferroni method.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: Fluorescence, Microscopy, Imaging, Staining, Modification

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Serum levels of ( A ) total FGF23 (cFGF23), ( B ) intact FGF23 (iFGF23), ( C ) intact to total FGF23 ratio (i/c FGF23), ( D ) parathyroid hormone (PTH), ( E ) 1,25-dihydroxyvitamin D [1,25(OH) 2 D], ( F ) calcium (Ca 2+ ), and ( G ) phosphate (Pi); ( H ) fractional excretion of Pi (FePi); and kidney mRNA expression of ( I ) NaPi2a , ( J ) Cyp27b1 , and ( K ) Cyp24a1 in 12-week-old WT ( n ≥ 5), Fgf23 Dmp1-cKO ( n ≥ 5), Dmp1 KO ( n ≥ 3), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 5) mice. Values are expressed as mean ± SEM; P < 0.05 vs. a WT, b Fgf23 cKO , c Dmp1 KO . Statistical tests were ANOVA test followed by post hoc t tests and multiple-testing correction using Holm-Bonferroni method.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: Expressing

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: ( A ) Mouse gross appearance, ( B ) body weight, ( C ) tail length, ( D ) femur length, ( E ) femur gross appearance, and ( F ) 3D μCT representation of total femur in sagittal plane (scale bar = 1 mm) in 12-week-old WT ( n ≥ 6), Fgf23 Dmp1-cKO ( n ≥ 5), Dmp1 KO ( n ≥ 5), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 4) mice. Values are expressed as mean ± SEM; P < 0.05 vs. a WT, b Fgf23 cKO , c Dmp1 KO . Statistical tests were performed using ANOVA test followed by post hoc t tests and multiple-testing correction using Holm-Bonferroni method.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques:

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: 3D μCT ( A ) scan reconstruction of distal femur trabecular metaphysis (scale bar = 500 μm) and ( B – G ) parameters of trabecular bone microarchitecture; ( H ) scan reconstruction of midshaft femur cortical diaphysis (scale bar = 500 μm) and ( I – O ) parameters of cortical bone microarchitecture. All analyses were performed in 12-week-old WT ( n ≥ 5), Fgf23 Dmp1-cKO ( n ≥ 5), Dmp1 KO ( n ≥ 5), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 5) mice. Values are expressed as mean ± SEM; P < 0.05 vs. a WT, b Fgf23 cKO , c Dmp1 KO . Statistical tests were ANOVA test followed by post hoc t tests and multiple-testing correction using Holm-Bonferroni method. BV/TV, bone volume to tissue volume ratio; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn.Dens, connectivity density; SMI, structural model index; mat BMD, material bone mineral density; Ma.Ar, marrow area; CSA, cross-sectional area; Ct.Ar, cortical area; Ct.Th, cortical thickness; Ct.Po, cortical porosity.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques:

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: ( A and B ) High-resolution μCT analysis of cortical bone porosity (scale bar = 100 μm), ( C ) acid-etched scanning electron microscopy of femur cortical bone (scale bar = 20 μm), ( D ) red fluorescence microscopy imaging of ARS-stained mineralization fronts (top), and bright-field microscopy imaging of modified trichrome Goldner staining (middle) and tartrate-resistant acidic phosphatase (TRAcP) staining (bottom) of longitudinal histology sections of distal femur (scale bar = 100 μm for ARS, 250 μm for Goldner and TRAcP). For each staining, top (×3.5 original magnification) and bottom (×1.8 original magnification) zoom-in panels represent regions of interest in trabecular and in cortical bone, respectively. All analyses were performed in 12-week-old WT ( n ≥ 5), Fgf23 Dmp1-cKO ( n ≥ 5), Dmp1 KO ( n ≥ 5), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 5) mice.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: Electron Microscopy, Fluorescence, Microscopy, Imaging, Staining, Modification

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Bone marrow stromal cells were isolated from 12-week-old WT ( n ≥ 3), Fgf23 Dmp1-cKO ( n ≥ 3), Dmp1 KO ( n ≥ 3), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 4) mice, then cultured for 14 (D14) and 21 (D21) days in osteoblast differentiation medium containing 3, 7, or 10 mM of beta-glycerophosphate (bGP). Levels of ( A ) total DMP1 (cDMP1) and ( B ) total FGF23 (cFGF23) in conditioned media collected at D21. ( C and D ) Alkaline phosphatase (ALP) staining and quantification and ( E and F ) alizarin red S (ARS) staining and quantification. Values are expressed as mean ± SEM; P < 0.05 vs. bGP treatment–matched a WT, b Fgf23 cKO , c Dmp1 KO , and genotype-matched *3 mM and **7 mM within each time point. d P = 0.1 vs. 10 mM Dmp1 KO . Statistical tests were ANOVA test followed by Bonferroni post hoc tests.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: Isolation, Cell Culture, Staining

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Bulk RNA-sequencing analysis was performed on bone marrow stromal cells isolated from 12-week-old WT ( n = 3), Fgf23 Dmp1-cKO ( n = 3), Dmp1 KO ( n = 3), and Dmp1 KO Fgf23 Dmp1-cKO ( n = 3) mice and cultured for 21 days in osteoblast differentiation medium containing 10 mM of beta-glycerophosphate. ( A ) Venn diagram identifies genes showing altered expression in Dmp1 KO but not in Dmp1 KO Fgf23 cKO osteoblasts ( tan area ). Heatmaps represent the expression of ( B ) Fgf23 and ( C ) genes identified in A and used in Ingenuity Pathway Analysis (IPA; QIAGEN) to define the most represented canonical pathways regulated by FGF23. ( D ) Venn diagram identifies genes showing altered expression in Dmp1 KO and in Dmp1 KO Fgf23 cKO osteoblasts ( burgundy area ). Heatmaps represent the expression of ( E ) Dmp1 and ( F ) genes identified in D and used in IPA to define the most represented canonical pathways regulated by DMP1. Statistical tests were unpaired t test and corrected by the false discovery rate ( P < 0.1).

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: RNA Sequencing Assay, Isolation, Cell Culture, Expressing

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Single-cell RNA-sequencing analysis was performed on bone marrow stromal cells isolated from 12-week-old WT ( n = 3), Fgf23 Dmp1-cKO ( n = 3), Dmp1 KO ( n = 3), and Dmp1 KO Fgf23 Dmp1-cKO ( n = 3) mice and cultured for 21 days in osteoblast differentiation medium containing 10 mM of beta-glycerophosphate. Uniform manifold approximation and projection (UMAP) plot of ( A ) total isolated cells and ( B ) osteoblast lineage cells only. ( C ) Dot plot of cluster-enriched markers in osteoblastic cells segregated by clusters of differentiation. Pseudotime analysis of osteoblastic cluster from ( D ) all groups combined and ( E ) segregated by group. ( F ) Percentage of osteoblast-like cells in the total number of isolated cells segregated by cluster of differentiation and by group.

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: RNA Sequencing Assay, Isolation, Cell Culture

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: Single-cell RNA-sequencing analysis was performed on bone marrow stromal cells isolated from 12-week-old WT ( n = 3), Fgf23 Dmp1-cKO ( n = 3), Dmp1 KO ( n = 3), and Dmp1 KO Fgf23 Dmp1-cKO ( n = 3) mice and cultured for 21 days in osteoblast differentiation medium containing 10 mM of beta-glycerophosphate. ( A ) Venn diagram identifies genes showing altered expression in Dmp1 KO but not in Dmp1 KO Fgf23 cKO osteoblasts ( colored area ) in each cluster of differentiation. ( B ) Heatmaps represent the expression of genes identified in A in the osteoprogenitor cluster ( green dot ) and used in Ingenuity Pathway Analysis (IPA) to define the most represented canonical pathways regulated by FGF23. ( C – J ) Violin plots representing the expression of most regulated target genes in Dmp1 KO and corrected in Dmp1 KO Fgf23 cKO osteoprogenitors. ( K ) IPA gene network analysis showing most connected gene targets in the osteoprogenitor cluster and identifying FGF receptor 1 (FGFR1), ERK1/2, and PI3K/AKT as common regulators of these targets. Statistical tests were Mann-Whitney’s U test and corrected by the false discovery rate ( P < 0.1).

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: RNA Sequencing Assay, Isolation, Cell Culture, Expressing

Journal: JCI Insight

Article Title: FGF23 directly inhibits osteoprogenitor differentiation in Dmp1 -knockout mice

doi: 10.1172/jci.insight.156850

Figure Lengend Snippet: ( A – C ) mRNA expression of markers of osteoblast differentiation in MC3T3-E1 osteoblasts cultured for 21 days and treated with recombinant FGF23 (0, 25, and 50 ng/mL) for the last 6 and 48 hours of culture ( n ≥ 4). Levels of ( D ) total DMP1 (cDMP1) and ( E ) total FGF23 (cFGF23) measured by ELISA in conditioned culture media from bone marrow stromal cells (BMSCs) isolated from 12-week-old WT ( n ≥ 4), Fgf23 Dmp1-cKO ( n ≥ 5), Dmp1 KO ( n ≥ 5), and Dmp1 KO Fgf23 Dmp1-cKO ( n ≥ 5) mice, then cocultured for 21 days with BMSCs isolated from the same group (isogenic), or immortalized BMSCs displaying genetic overexpression of Fgf23 ( Fgf23 TG ) or Dmp1 ( Dmp1 TG ). ( F and G ) Alkaline phosphatase (ALP) staining and quantification and ( H and I ) alizarin red S (ARS) staining and quantification. Values are expressed as mean ± SEM; P < 0.05 vs. ( A – C ) time point–matched a Ctr, b FGF23 25 ng/mL, ( D – I ) culture-matched a WT, b Fgf23 cKO , c Dmp1 KO , and *genotype-matched isogenic. Statistical tests were ANOVA test followed by post hoc t tests and multiple-testing correction using Holm-Bonferroni method ( A – C ) and by Bonferroni’s post hoc tests ( D – I ).

Article Snippet: In all mice, we measured serum iFGF23 levels using a murine iFGF23 ELISA that measures the intact bioactive protein exclusively and total FGF23 (cFGF23) using the cFGF23 ELISA that recognizes the full-length protein and its C-terminal cleavage fragments (both from Quidel).

Techniques: Expressing, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Over Expression, Staining

Journal: Pflugers Archiv : European journal of physiology

Article Title: Controlled dietary phosphate loading in healthy young men elevates plasma phosphate and FGF23 levels.

doi: 10.1007/s00424-024-03046-4

Figure Lengend Snippet: Fig. 3 Phosphatropic hormones after 5 days of low- or high- phosphate (Pi) diet. Levels of A serum parathyroid hor- mone (PTH), B plasma intact fibroblast growth factor 23 (iFGF23), C plasma C-terminal FGF23 (cFGF23), D the ratio of iFGF23 (ng/l)/cFGF23 (ng/l) as well as serum E calcidiol, and F calcitriol, and G plasma soluble Klotho, and H Fetuin-A on the final day of both the 5-day low and high Pi diet. One reference unit (RU)/ml corre- sponds to 2 ng/l cFGF23. Blue dots represent differences (Δ) between high Pi diet and low Pi diet expressed as mean differ- ence ± 95% confidence interval and blue dashed line represents the zero line of the right y-axis. Data was analyzed by the paired t-test (A–E) or Wilcoxon test (F–H), n = 10, *p < 0.05

Article Snippet: Daily fractional excretion of Pi (FEPi) was calculated by the following equation using the absolute 24-h urine Pi and creatinine: Daily renal filtered Pi load was calculated with the following equation: Plasma iFGF23, cFGF23, Fetuin-A, and urinary metanephrine were measured with the human iFGF23 and cFGF23 enzyme-linked immunosorbent assay (ELISA) (iFGF23 and cFGF23, Quidel, 60–6600 and 60–6100, respectively), Fetuin-A ELISA (R&D Systems, DFTA00, Lot P413286), and the Metanephrine Urine ELISA (Demeditec Diagnostics GmbH, DEE8400) according to manufacturers’ protocols.

Techniques: Clinical Proteomics