STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2
Malika Sahni1,
Regina Raz2,
J. Douglas Coffin3,
David Levy2 and
Claudio Basilico1,*
1 Department of Microbiology, NYU School of Medicine, New York, NY 10016, USA
2 Department of Pathology, NYU School of Medicine, New York, NY 10016, USA
3 Department of Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA
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Fig. 1. Loss of STAT1 function leads to correction of the long bone phenotype observed in TgFGF mice. Radiography of the long bones of wild-type (WT), Stat1-/-, TgFGF and TgFGF/Stat1-/- mice at 3 months of age (A). Femurs and tibia of TgFGF are shorter and thicker than in the WT and Stat1-/- mice. TgFGF/Stat1-/- bones are similar to the WT and Stat1-/-. (B) Histogram representation of the ratio between bone length (femurs) and bodyweight. Bodyweight was similar in all the groups analyzed. TgFGF femurs are significantly shorter than WT, Stat1-/- and TgFGF/Stat1-/-. Each histogram represents the average of six independent measurements performed on four mice in each group. Bars correspond to s.d.
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Fig. 2. BrdU incorporation in metatarsal chondrocytes. E15.5 metatarsals were maintained in organ culture for 48 hours and were labeled with 50 µg BrdU/ml for the last 6 hours of the culture. Histological sections (5 µm) were stained with specific anti-BrdU antibody. The positive cells were visualized by DAB staining. Sections were counterstained with Alcian Blue. (A) Metatarsals from TgFGF are shorter and wider than those of wild type (WT) and Stat1-/-. Loss of the Stat1 gene in TgFGF (TgFGF/Stat1-/-) increases the length of the metatarsals in TgFGF/Stat1-/- mice. (B) Chondrocytes in the proliferating zone labeled with BrdU. The rate of DNA synthesis is higher in WT (62±1%) and Stat1-/- (66±0.8%) versus TgFGF (34±0.6%). Absence of Stat1 in TgFGF attenuates the inhibitory effect of TgFGF on chondrocyte proliferation (TgFGF/Stat1-/-, 44±0.5%).
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Fig. 3. TUNEL staining of growth plate chondrocytes at P1 and P10. Longitudinal femur sections of wild-type (WT), TgFGF, Stat1-/- and TgFGF/Stat1-/- mice at P1 (A) and P10 (B) were analyzed for the presence of apoptotic chondrocytes in the epiphyseal region as well as in the metaphyseal growth plates. Apoptotic chondrocytes were present at high frequency around the secondary centers of ossification in TgFGF at P10 as well as in the proliferating zone. WT femurs show low frequency of chondrocyte DNA fragmentation in the epiphyseal region and in the proliferating zone. In Stat1-/- mice, the levels of apoptosis is near absent along the femur, except in the hypertrophic zone.
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Fig. 4. TUNEL staining of cultured primary chondrocytes. Primary chondrocytes from wild type (WT), Stat1-/-, TgFGF and TgFGF/Stat1-/- mice were isolated from P10 growth plate and plated at similar density. After 24 hours in the culture, the cells were fixed and stained for TUNEL. (A) Hoechst staining visualizing the nuclei of the cells; (B) TUNEL-positive cells in the same field as A.
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Fig. 5. TUNEL staining of osteoblastic cells undergoing apoptosis in the post-frontal suture of calvaria. Cross sections of calvarial bone at the post-frontal suture (PF) were stained with TUNEL to visualize the apoptotic cells (A) or with Toluidine Blue (B). Wild type (WT) and Stat1-/- calvarial sutures show no signs of apoptosis (A), while TgFGF and TgFGF/Stat1-/- show numerous apoptotic cells around the PF suture and osteogenic front (indicated by arrows in B). The osteogenic front is more advanced in WT and Stat1-/- mice (B), while in TgFGF and TgFGF/Stat1-/- the fronts remain far apart from each other.
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Fig. 6. Histological sections of P5 and P15 femur growth plates stained with Hematoxylin and Eosin. (A) Longitudinal section of femurs from P5 mice. h, hypertrophic zone; ph, prehypertrophic zone; pr, proliferating zone; rz, reserve zone. (B) Longitudinal sections of femurs from P15 mice. The asterisks indicate hypertrophic chondrocytes detected in the reserve zone, while the arrows indicate the trabecular bone fronts. SCO, secondary center of ossification.
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Fig. 7. Measurements of the height of the proliferating zones and of the distance between opposing growth plates during femur development. Each bone section was stained with anti-ColX antibody to distinguish the proliferating (ColX negative) from the hypertrophic (ColX positive) zone. Measurements of the height of the proliferative and hypertrophic zone were performed along the central axis of a longitudinal section of the growth plate. (A) Height of the proliferating zone; (B) distance between opposing growth plates measured from the inner borders of the collagen X positive hypertrophic zones. Values (±s.d.) represent the mean of measurements from four different sections performed using a measuring microscope eyepiece and are expressed in arbitrary units. Statistical analysis of the differences between wild-type (WT) and Stat1-/- mice, or WT and TgFGF values, respectively, shows significant differences (P<0.01-0.001) for the values indicated with an asterisk.
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© The Company of Biologists Ltd 2001
