FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence

Abstract

Oncogenic activation of the RAS-ERK MAP kinase signaling pathway can lead to uncontrolled proliferation but can also result in apoptosis or premature cellular senescence, both regarded as natural protective barriers to cell immortalization and transformation. In FGFR3-related skeletal dyplasias, oncogenic mutations in the FGFR3 receptor tyrosine kinase cause profound inhibition of cartilage growth resulting in severe dwarfism, although many of the precise mechanisms of FGFR3 action remain unclear. Mutated FGFR3 induces constitutive activation of the ERK pathway in chondrocytes and, remarkably, can also cause both increased proliferation and apoptosis in growing cartilage, depending on the gestational age. Here, we demonstrate that FGFR3 signaling is also capable of inducing premature senescence in chondrocytes, manifested as reversible, ERK-dependent growth arrest accompanied by alteration of cellular shape, loss of the extracellular matrix, upregulation of senescence markers (alpha-GLUCOSIDASE, FIBRONECTIN, CAVEOLIN 1, LAMIN A, SM22alpha and TIMP 1), and induction of senescence-associated beta-GALACTOSIDASE activity. Our data support a model whereby FGFR3 signaling inhibits cartilage growth via exploiting cellular responses originally designed to eliminate cells harboring activated oncogenes.

FIGURE 1

FGFR3 effects on proliferation, extracellular matrix production and cellular shape in RCS chondrocytes. (A) Cells were treated with FGF2 for 72 hours and counted. Note the potent FGF2-mediated inhibition of RCS growth. Data represent an average from four wells with the indicated standard deviation. (B) RCS chondrocytes were treated with FGF2 for 72 hours and stained with Alcian blue to visualize the cartilaginous extracellular matrix (upper panel). The corresponding bright field with contrast photograph is also shown (lower panel, 100×). Note the significant loss of extracellular matrix in FGF2-treated cells. (C) RCS chondrocytes were treated with FGF2 for 72 hours in the presence of [³⁵S]sulfate and the amount of incorporated radioactivity was determined by liquid scintillation. Note the potent downregulation of [³⁵S]sulfate incorporation by FGF2. Data represent an average from four wells with the indicated standard deviation. (D) RCS chondrocytes were treated with FGF2 for 72 hours and photographed. The left panel shows the round to cubical shape of control cells. Note the cellular shape alteration that accompanies the FGF2-mediated growth arrest (right panel, 200×). (E) RCS chondrocytes were treated with FGF2 (10 ng/ml) for 72 hours, co-stained for polymerized actin (Alexa Fluor 594-phalloidin) and focal adhesions (vincullin-FITC) and photographed. Note the significant cytoskeleton remodelling accompanied by increased substrate-adhesion that is induced by FGFR3 signaling. Scale bar: 20 μm.

FIGURE 2

FGFR3-mediated senescence in RCS chondrocytes. (A) Cells were treated with FGF2 for 72 hours and the activity of SA-β-GALACTOSIDASE was determined as described in the Materials and Methods. Note a potent, FGF2-mediated induction of SA-β-GALACTOSIDASE activity that appears as blue cytoplasmic staining (upper panels). The corresponding bright field with contrast photograph is also shown (lower panels, 100×). (B) The experiment presented in (A) was quantified by counting the SA-β-GALACTOSIDASE positive cells under the light microscope. The data represent an average from 10 counts (50 cells each) with the indicated standard deviation. The results are representative for three experiments. (C) Cells were treated with FGF2 (20 ng/ml) for the indicated times and expression of a panel of senescence markers was determined by real-time RT-PCR. Quantities of the transcript are relative to untreated cells. The data represent an average from technical duplicates with the indicated range. Results are representative for five experiments. (D) RCS chondrocytes were treated with FGF2 (20 ng/ml) for the indicated times and the quantities of given proteins were determined by WB. The α-GLUCOSIDASE signal was quantified by densitometry and graphed. ACTIN serves as a loading control. Data are representative for four experiments.

FIGURE 3

Reversal of FGF2-mediated senescence by chemical inhibition of the ERK pathway. (A) Cells were treated with FGF2 for 24 hours in the presence of the MEK inhibitor U0126, and analyzed for expression of a panel of senescence markers by real-time RT-PCR. Quantities of the transcript are relative to untreated cells. Note that U0126 partially antagonizes FGF2-mediated induction of all tested markers. The data represent an average from technical duplicates with the indicated range. Results are representative for three experiments. A similar experiment was performed in (B), with the quantities of the given proteins determined by WB. Note the FGF2-mediated induction or downregulation of senescence markers, which was rescued by U0126 in all but one case (α-GLUCOSIDASE, evidenced by densitometry). ACTIN serves as a loading control. Results are representative for four experiments. SM22α was not analyzed by WB since its protein induction by FGF2 required at least 48 hours of treatment (Fig. 2D).

FIGURE 4

FGFR3-mediated senescence is a reversible phenotype. (A) RCS chondrocytes were seeded and treated with FGF2 for four days as indicated. In some samples, the length of FGF2 treatment was restricted by washing away after one, two and three days of cultivation. Cell lysates were analyzed by WB for CAVEOLIN 1 and LAMIN A/E expression at the indicated times. ACTIN serves as a loading control. Note the FGF2-mediated induction of CAVEOLIN 1 and LAMIN A/E which was reversed when FGF2/FGFR3 stimulus diminished as a result of FGF2 removal. (B) The experiment was carried-out similarly to (A) except that the all cell cultures were treated with FGF2 for the whole duration of the experiment, and FGFR3 signaling was inhibited by addition of the FGFR inhibitor SU5402. Note the reversal of the FGF2/FGFR3-mediated effect on senescence markers by SU5402. The results are representative for three experiments. (C) RCS chondrocytes were grown for up to five days, treated with FGF2 (10 ng/ml) for a variable length of time and counted at the indicated times. Note that despite the complete growth arrest caused by the initial FGF2 treatment, the cells resumed normal growth upon FGF2 removal. The results are representative for three experiments.

FIGURE 5

Apoptosis induced by FGFR3 overexpression. (A) RCS chondrocytes were transfected with wild-type FGFR3 as well as its activating mutants associated with hypochondroplasia (N540K), ACH (G380R), and TD (R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 24 hours later. Note the typical migration pattern of FGFR3 in both K650 mutants that form only an immature, intracellular variant of FGFR3 (lower band). Also note the different levels of ERK activation (P-ERK blot) that correspond to the severity of the disease. KD is an inactive, kinase-dead FGFR3 mutant (K508M), whereas cells transfected with a GFP or empty plasmid serve as transfection controls. ACTIN serves as loading control. (B) Cells were co-transfected with indicated FGFR3 mutants together with a GFP plasmid. 48 hours later, GFP-positive cells were isolated by cell-sorting as described in Material and Methods, plated and grown for additional 48 or 72 hours, and photographed (left panel, bright field with contrast, 100×), or counted (right graph). Note the poor attachment of cells transfected with FGFR3 carrying highly activating K650M and K650E mutants, with many small, dying (white arrows) or dead (black arrows) cells. Also note the poor growth in GFP-positive cells expressing activating FGFR3 mutants when compared to GFP-positive cells expressing KD-FGFR3 or an empty plasmid. Also note the growth of GFP-negative cells (right graph, open bars), which was normal in all the transfections. Data represent an average from three wells with the indicated standard deviation. (C) Cells were transfected as indicated, grown for 48 hours, stained with propidium iodide (PI) and annexin-FITC, and analyzed by flow-cytometry to visualize the apoptotic (annexin positive) and necrotic (PI/annexin positive) cells. The data represent an average from three independent experiments with the indicated standard deviation. Cells transfected with an empty plasmid serve as a transfection control. Control – untransfected cells, FGF2 – untransfected cells treated with FGF2 (25 ng/ml) for 24 hours.