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. 2018 Jul 1;27(13):2276-2289.
doi: 10.1093/hmg/ddy133.

Noonan Syndrome-Causing SHP2 Mutants Impair ERK-dependent Chondrocyte Differentiation During Endochondral Bone Growth

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Free PMC article

Noonan Syndrome-Causing SHP2 Mutants Impair ERK-dependent Chondrocyte Differentiation During Endochondral Bone Growth

Mylène Tajan et al. Hum Mol Genet. .
Free PMC article

Abstract

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins. In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Figures

Figure 1.
Figure 1.
 Homogeneous postnatal growth retardation in NS mice. (A) Growth curves of male Ptpn11+/+ (WT) (n=12) and Ptpn11D61G/+ (NS) (n=9) mice are shown. Compared with WT mice, NS mice were significantly shorter by 2 weeks of age (2-way repeated-measures ANOVA plus Bonferroni post-test; ***P<0.001, **P<0.01). (B and C) Representative gross images (B) and Faxitron X-ray composites images of entire skeleton, lower legs, and skull (C) of 4-week-old WT and NS mice. NS mice displayed shortened axial and appendicular skeletons, mid-facial hypoplasia, and rounded cranium, without obvious bone deformity.
Figure 2.
Figure 2.
 Decreased growth plate width in NS mice. Histological sections from tibiae of 4-week-old WT (n=12) and NS (n=9) mice. (A and B) Alcian blue (A) and COL10 (B) staining of growth plate. (C) Measurements of different chondrocyte zones, showing a decrease of the widths of the hypertrophic zone, and to a lesser extent of the proliferating zone, in NS mice. (D) PCNA immunostaining to evaluate chondrocyte proliferation. (E) Quantification of the number of cells positive for PCNA per surface in the proliferating zone of the growth plate. There was no difference between WT and NS mice. (F) Colorimetric TUNEL assay to evaluate chondrocyte apoptosis. Values are means ± SEM. Significant statistical differences between groups: **P<0.01, *P<0.05 (two-tailed Student’s t-test).
Figure 3.
Figure 3.
 NS-causing PTPN11 mutants impair chondrocyte differentiation. Expression of chondrocyte differentiation markers in WT (n=7) and NS (n=7) primary murine chondrocytes, as determined by quantitative RT-PCR at baseline (T0) and after 7 days (T7) of differentiation with ascorbic acid. (A–K) Markers of the proliferating (A–C), pre- (D–F), early- (G and H), and late- (I–K) hypertrophic stages. In NS primary chondrocytes, transcripts associated with pre- and early-hypertrophic stages were very low and did not change during differentiation, suggesting impaired differentiation. Results are expressed as a fold change in mRNA expression, compared with WT mice at baseline. Values are means±SEM. The statistical significance of fold-changes was determined by using a two-way ANOVA for comparison between T0 and T7, and a two-tailed Student’s t-test for comparison between WT and NS primary chondrocytes. Significant statistical differences between groups: ***P<0.001, **P<0.01, *P<0.05.
Figure 4.
Figure 4.
 Alkaline phosphatase staining and activity are decreased in NS primary murine chondrocytes. (A and B) Proliferation of WT (n=7) and NS (n=7) primary murine chondrocytes cultured with 10 ng/ml FGF2. (A) Counting of the total cell number at days 1, 2 and 3 showing that the cell number doubled the first 2 days and then reached a plateau without difference between NS and WT chondrocytes. (B) Incorporation of EdU, a cell proliferation marker incorporated in cells during the S-phase of the cell cycle, into DNA between days 1 and 2 and days 2 and 3. The incorporated EdU in DNA coupled to Oregon Green-azide was assessed by the fluorescence detected at an excitation/emission wavelength of 490/585 nm; results were expressed as percentage of WT fluorescence. The proliferation rate was similar between WT and NS chondrocytes. (C and D) Alkaline phosphatase staining and activity in WT (n=7) and NS (n=7) primary murine chondrocytes at baseline (T0) and after 7, 14, and 21 days of differentiation with ascorbic acid. (C) Representative images showing ALP staining. Note significant decreased in NS chondrocytes, compared with WT chondrocytes. (D) ALP activity was determined by fluorometric method using p-nitrophenol-phosphate as a substrate, and normalized to the total DNA content of each sample. ALP activity was significantly decreased in NS chondrocytes compared to WT chondrocytes. Values are means ± SEM. The statistical significance of fold changes was determined using a two-way repeated-measures ANOVA for comparison between T0 and T7 and a two-tailed Student’s t-test for comparison between WT and NS primary chondrocytes. Significant statistical differences between groups: ***P<0.001, **P<0.01, *P<0.05.
Figure 5.
Figure 5.
 NS mutants increase RAS/ERK activation in chondrocytes in vitro and in vivo. (A) Histological sections from tibiae of 4-week-old WT (n=8) and NS mice (n=8) processed for phosphoERK1/2 immunostaining. (B) Quantification of phosphoERK1/2-positive cells per surface in the proliferating and hypertrophic zones of the growth plate. Data are expressed as means ± SEM for two sections per mouse. Significant statistical differences between groups: ***P<0.001, **P<0.01, *P<0.05 (two-tailed Student’s t-test). (C) ATDC5 cells were transduced with adenovectors encoding SHP2-WT and D61del. These cells were either left unstimulated or stimulated with 400 ng/ml GH or 10 ng/ml FGF2 for the indicated times, lysed, and probed by western-blot analysis to determine the level of ERK1/2 phosphorylation with an anti-phospho-ERK1/2 antibody. Note the exposure time was much more important for GH than for FGF2, explaining the detection of bands under unstimulated conditions. Membranes were reprobed with anti-SHP2 and anti-tubulin antibodies to verify SHP2 expression and loading homogeneity. Results from three independent experiments were quantified using ImageJ software and expressed as means ± SEM. Only significant differences versus WT cells for the corresponding time are indicated: ***P<0.001, **P<0.01, *P<0.05 (two-tailed Student’s t-test).
Figure 6.
Figure 6.
 RAS/ERK inhibition partially restores growth and growth plate abnormalities in NS mice. (A) Body length of 4-week-old WT and NS mice, treated i.p. from 1 to 4 weeks of age with vehicle (n WT/NS=11/10), IGF1 (4 mg/kg twice daily; n WT/NS=10/7) or U0126 (5 mg/kg daily; n WT/NS=11/6), are shown. IGF1 and U0126 treatments partially restored growth retardation in NS mice. (B) Measurement of growth plate zones widths after Alcian blue staining. IGF1 led to an increase of the length of the proliferating zone without correcting the hypertrophic zone shortening, while U0126 treatment restored the length of the growth plate, by simultaneously increasing the length of the proliferating and hypertrophic zones. (C and D) ALP expression determined by quantitative RT-PCR (C) and activity determined by fluorometric method using p-nitrophenol-phosphate used as a substrate (D), in WT (n=7) and NS (n=7) primary murine chondrocytes at baseline (T0) and after 7 days of differentiation with ascorbic acid, in the presence or absence of U0126 (10 μM) added to the culture medium. U0126 treatment restored ALP expression and activity in NS chondrocytes. Results of Alp expression are expressed as a fold change in mRNA expression compared with WT mice at baseline. ALP activity was normalized to the total DNA content of each sample. Values are means ± SEM. The statistical significance of fold changes was determined using a two-way repeated-measures ANOVA for comparison between T0 and T7 and a two-tailed Student’s t-test for comparison between WT and NS primary chondrocytes. Significant statistical differences between groups: ***P<0.001, **P<0.01, *P<0.05.
Figure 7.
Figure 7.
 Inhibition of RAS/ERK activation by U0126 and statin treatments correct impaired chondrocyte differentiation in vitro. (A) Body length of 4-week-old WT and NS mice, treated i.p. from 1 to 4 weeks of age with vehicle (n WT/NS=12/9) or rosuvastatin (1 mg/kg daily; n WT/NS=12/8), are shown. Statin treatment partially restored growth retardation in NS mice. (B) Measurement of growth plate zone widths after Alcian blue staining. Rosuvastatin treatment restored the length of the growth plate, mostly by increasing the length of the hypertrophic zone. (C and D) ALP expression determined by quantitative RT-PCR (C) and activity determined by fluorometric method using p-nitrophenol-phosphate used as a substrate (D), in WT (n=7) and NS (n=7) primary murine chondrocytes at baseline (T0) and after 7 days of differentiation with ascorbic acid, in the presence or absence of lovastatin (1 μM) added to the culture medium. Lovastatin treatment restored ALP expression and activity in NS chondrocytes. Results of Alp expression are expressed as a fold change in mRNA expression compared with WT mice at baseline. ALP activity was normalized to the total DNA content of each sample. Values are means ± SEM. The statistical significance of fold changes was determined using a two-way repeated-measures ANOVA for comparison between T0 and T7 and a two-tailed Student’s t-test for comparison between WT and NS primary chondrocytes. Significant statistical differences between groups: ***P<0.001, **P<0.01, *P<0.05.

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