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Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations

Orit Rozenblatt-Rosen1, Efrat Mosonego-Ornan1,2, Einat Sadot1, Liora Madar-Shapiro2, Yuri Sheinin2, Doron Ginsberg1 and Avner Yayon*1,2

1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
2 ProChon Biotech Ltd, Kiryat Weizmann, Rehovot 76114, Israel



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  		Fig. 1. Effects of FGF9 on RCS cell proliferation, FGFR3 activation and gene expression. RCS cells were harvested at the indicated time points following stimulation with 20 ng/ml of human recombinant FGF9 and 1µg/ml of heparin. (A) Equal amounts of cell lysates were analyzed by SDS-PAGE and western blotting with anti-FGFR3 antibodies (upper panel), anti-pMAPK antibodies (middle panel) and anti-FRS2 antibodies (lower panel). (B) Cells were harvested, and RNA was prepared and subjected to northern blot analysis using human FGFR3 cDNA as a probe. (C) RCS cells (60,000 cells/well) were incubated with FGF9 and heparin for 0 (untreated control), 8, 16, 24 and 72 hours as indicated. Then, FGF9 was washed out and the cells were cultured further under normal conditions for 72 hours, at which point the cell number was determined. The initial cell number seeded in each system was 0.6x105. The respective cell numbers obtained at the end of the 72-hour incubation were 7.2x105, 6.8x105, 3.5x105, 2.9x105 and 1.1x105. Values are averages of triplicates, with standard deviation <10% in all cases.

 


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  		Fig. 2. FGF9 inhibits RCS cells proliferation via a G1 cell cycle arrest. RCS cells were incubated with FGF9 and heparin for 16 hours (A) or for the indicated times (B) and subjected to cell cycle analysis using the FACSORT. Controls include untreated cells or cells that were incubated in the presence of heparin. The percentage of cells accumulated at the G1, S and G2/M stages of the cell cycle is indicated.

 


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  		Fig. 3. Modulation of gene expression by FGF9. (A) Differential hybridization to Atlas rat gene array. Total RNA prepared from either RCS cells incubated with FGF9 and heparin for three hours or untreated cells was used to generate two radiolabeled cDNA probes. The probes were hybridized to the Clontech Atlas membranes (7738-1) as described in Materials and Methods. Each cDNA is present on the filter in two adjacent spots. The frames indicate several genes regulated by FGFR3. The previously unreported, upregulated genes were (fold-induction is given in brackets) c-Jun (x2.5), Jun D (x15.1), Fra 2 (x5.6), cyclin D1 (x2.3), NF-{kappa}B1(p50/p105) (x3.6), STAT3 (x3.1) and Ezrin (x4.0), whereas ID1 was downregulated three-fold. (B) Kinetics of protein expression following FGF9-induced growth arrest. RCS cells were exposed to FGF9 and heparin for the indicated time intervals, lysed and analyzed by SDS-PAGE and western blotting with anti c-Jun, Jun-D, p21, Id1 and Ezrin antibodies.

(C) Immunohistochemical analysis of epiphyseal growth plates from normal and transgenic G380R mutant h-FGFR3 expressing mice. Immunostaining for Rel A (NF-{kappa}B p65) was performed on sections of proximal tibia growth plates from normal and transgenic littermates harboring the G380R mutated FGFR3. The different zones of the growth plate (PZ-proliferation zone, MZ-maturation zone, HZ-hypertrophic zone) are noted.

 


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  		Fig. 4. FGF9 induces changes in RCS cell morphology and the subcellular localization of FGFR3. (A) RCS cells were stimulated with FGF9, heparin or FGF9 with heparin for 48 hours and visualized by phase microscopy. (B) Cells were subjected to double-labeled immunofluoresent staining with anti-FGFR3 and anti-vinculin antibodies followed by reaction with a secondary anti-rabbit antibody conjugated to Cy3 and anti-mouse conjugated to Alexa 488 antibodies, respectively. Coimmunoflorescence of green and red signals identifies the sites where the two proteins colocalize. A hypass filter was used in order to emphasize staining of the focal adhesions.

 


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  		Fig. 5. Effects of FGF9 on RCS cell morphology, cytoskeletal organization and FGFR3 localization. RCS cells were incubated with FGF9 and heparin for 10 minutes, 1 hour or 6 hours, fixed and double stained with antibodies to FGFR3 and anti-pTYR antibodies (A) or anti-vinculin antibodies (B) or phalloidin (C).

 


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  		Fig. 6. A schematic model suggesting how NF{kappa}B, Id1 and possibly Twist may interact to control FGFR gene expression. In this hypothetical scheme, it is proposed that the feedback regulation of FGFR expression in RCS cells involves principally NF{kappa}B and Id, acting via an unknown intermediary factor, which may be a b-HLH protein such as (Twist). Activation of FGFR induces upregulation of NF-{kappa}B subunits NF-{kappa}B1 (p50/p105) and RelA(p65) (Ghosh et al., 1998Go) and downregulation of Id1, a general inhibitor of terminal differentiation, which was shown to inhibit Twist by direct interaction with the protein. Several studies have demonstrated that activated NF-{kappa}B can upregulate Twist protein either directly or through inhibition of BMP4 signaling, which can directly regulate the expression of Id1. Since the involvement of Twist in this cell system is unknown and hypothetical, it has been placed in parentheses. FGFR may also downregulate the expression of Id1 via a NF-{kappa}B-independent signaling pathway. Both downregulation and signaling shut-off of FGFR3 are tightly regulated during chondrocyte maturation and terminal differentiation. `FGFR3' refers to the total signal-transduction activity mediated via FGFR3, determined by the sum of activated FGFR3 molecules in the cell. The left side of the figure denotes alterations in chondrocyte morphology and their correlation with FGFR3 activity. Initially, FGFR3 signalling level is high and is associated with a transition in chondrocyte morphology from a polygonal to a rounded shape. In the last stage of the process (bottom cell), FGFR3 is downregulated and its signaling activity ceases as the cells attain a fully rounded shape.

 

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