Decreased proliferation and altered differentiation in osteoblasts from genetically and clinically distinct craniosynostotic disorders

Abstract

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Calpha and protein kinase Cepsilon isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Czeta levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype.

Figure 1.

Phase contrast microphotographs representing comparative morphology of primary osteoblasts cultured for 2 weeks (second passage). A: Control osteoblasts; B: NCM osteoblasts; C: P253R osteoblasts; D: C342R osteoblasts. Original magnification, ×120.

Figure 2.

In vitro cell proliferation comparison between control osteoblasts and osteoblasts isolated from distinct craniosynostotic patients. Data represent pool of three independent experiments performed in triplicate, and are expressed as means ± SE. *P <0.001 versus control cells.

Figure 3.

Comparative histochemical analysis of ALP activity in control osteoblasts and in osteoblasts isolated from craniosynostotic patients. Cells were cultured for one week in standard conditions before analysis. A: Control osteoblasts; B: NCM osteoblasts; C: P253R osteoblasts; D: C342R osteoblasts. Microphotographs are representative of at least four experiments performed between the second and third passages. Original magnification, ×200.

Figure 4.

Biochemical analysis of ALP activity expressed as mean of Sigma units/mg total proteins, assayed in control osteoblasts and in osteoblasts isolated from craniosynostotic patients. Data represent pool of three independent experiments performed in triplicate, and are expressed as means ± SE. *P <0.001 versus control cells.

Figure 5.

Comparison of in vitro mineral deposition profile in osteoblast cultures isolated from control individual and patients with different craniosynostotic disorders. Osteoblasts were cultured in 24 wells/plate for 6 weeks in presence of dexamethasone. After 2 (A, C, E, and G) and 6 (B, D, F, and H) weeks of culture, plates were fixed and mineralization was detected by von Kossa’s staining. Control osteoblasts produced appreciable mineralization only after 6 weeks (A and B); NCM (C and D) and P253R (E and F) osteoblasts showed positive mineralization profiles after 2 weeks that sensibly increased after 6 weeks of culture. In C342R osteoblast cultures no mineralized nodules were detected during 6 weeks of culture (G and H). Photographs are representative of three distinct experiments.

Figure 6.

Changes in noncollagenous matrix protein expression in osteoblast cultures obtained from control individual and patients with different craniosynostotic disorders by Western blotting analysis. A: Composite autoradiograms from representative experiments. Western blotting analysis was performed using anti-OPN, anti-BSP, anti-ONC, and anti-β-actin (Actin) antibodies in osteoblasts cultured with (+) or without (−) 10% FBS for 72 hours. B: Bar graphs represent quantification of protein levels by densitometric scanning of autoradiograms obtained from multiple experiments. Striped bars and white bars refer to cultures with and without 10% FBS, respectively. Values are means ± SE and are represented as ratios of the specific protein to β-actin to compensate for any loading differences. *P <0.05 versus control cells.

Figure 7.

Differential expression and activation of PKCα, PKCε, and PKCζ isoenzymes in control osteoblasts and in osteoblasts isolated from patients with different craniosynostotic disorders by Western blotting analysis. Cytosol (c) and membrane (m) fractions were separated from subconfluent cultures. A: Composite autoradiograms from a single representative experiment. B: Bar graphs of PKCα, PKCε, and PKCζ isoenzymes represent quantification of protein levels by densitometric scanning of autoradiograms obtained from a single representative experiment of three distinct experiments performed. White bars and striped bars refer to the cytosol fraction and membrane fraction, respectively. Values are represented as ratios of PKC isoenzyme to β-actin to compensate for any loading differences.

Figure 8.

Effect of treatment with hr-FGF2 (20 ng/ml) and heparin (50 μg/ml) on cell proliferation of osteoblasts isolated from control individual and patients with different craniosynostotic conditions. Cells were serum-free cultured before treatment for 24 hours. FGF2 was added to the medium culture for 24 hours. Data are representative of three distinct experiments performed in quadruplicate and are expressed as cpm/well means ± SE. *P <0.001 versus untreated cells.

Figure 9.

Effect of treatment with hr-FGF2 (20 ng/ml) and heparin (50 μg/ml) on in vitro mineralization of control (A, E, and I), NCM (B, F, and J), P253R (C, G, and K), and C342R (D, H, and L) osteoblast cultures. At the end of cultures, plates were fixed and mineralization was detected by von Kossa’s staining. Cultures were serum-deprived for 24 hours before treatment. During the 2 weeks of culture, FGF2 and heparin were continuously added in the mineralization medium supplemented with 0.1% FBS (I, J, K, and L). FGF2 treatment inhibited mineralization in all samples. Osteoblasts were also cultured in mineralization medium without growth factor as a control, supplemented with 0.1% FBS (E, F, G, and H) and 10% FBS (A, B, C, and D). Note the increased mineralization in P253R in 0.1% FBS cultures (G). Microphotographs were representative of three distinct experiments performed in triplicate. Original magnification, ×50.