Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

<div><p>Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.</p></div

FGFR3 signal transduction in chondrocytes and mechanisms of FGFR3 inhibitors.

<p>Activations of MAPK (mitogen-activated protein kinase) and STAT (signal transducers and activators of transcription) negatively regulate chondrocyte proliferation and differentiation. MAPK signaling includes sequential stimulation of a signaling cascade involving RAS, RAF, MEK, and ERK. CNP binding to natriuretic peptide receptor-B induces the generation of the second messenger cGMP, which activates PKG and leads to attenuation of the MAPK pathway by inhibiting RAF activation. NF449 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081569#pone.0081569-Krejci3" target="_blank">[14]</a>, A31 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081569#pone.0081569-Jonquoy1" target="_blank">[16]</a>, and P3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081569#pone.0081569-Jin1" target="_blank">[15]</a> are recently identified FGFR3 inhibitors. NF449 and A31 have inhibitory effects on the kinase activity of FGFR3. P3 has an affinity for extracellular domain of FGFR3. Meclozine attenuates ERK phosphorylation.</p

Meclozine promotes chondrocyte proliferation and ameliorates loss of extracellular matrix in FGF2-treated RCS cells.

<p>(A, B) RCS cells were treated with 5 ng/ml FGF2 and the indicated concentrations of meclozine for 48 hours. Cell growth was quantified using the MTS assay (A) or by counting cells (B). Data are normalized to that without meclozine and indicated by the mean and SD (<i>n</i> = 8 for A and 6 for B). Meclozine rescued the FGF2-mediated growth arrest of RCS cells. (C) Meclozine (10 µM) ameliorated FGF2-mediated alteration of cellular shape and loss of extracellular matrix. RCS cells were treated with 5 ng/ml FGF2 with and without 0.2 µM CNP or 20 µM meclozine for 72 hours, and cartilage-like sulfated proteoglycan matrix was stained by Alcian blue. Growing RCS cells were round-shaped and produced abundant cartilage-like sulfated proteoglycan matrix in the absence of FGF2. FGF2 treatment transformed some cells to fibroblast-like shapes and prominently suppressed expression of sulfated proteoglycan matrix. In the RCS cells treated with CNP or meclozine, the cellular shape remained round and the intensity of Alcian blue staining approximated that of FGF2-negative cells. Representative images of triplicated experiments are shown. Magnified images of the middle panels are shown in the rightmost column. Bars in the left, middle, and right panels are 750, 150, and 30 µm, respectively. (D) Meclozine (20 µM) inhibited mRNA expression of matrix metalloproteinases in FGF2-treated RCS cells. Cells were treated with FGF2 and either CNP or meclozine for four hours and mRNAs were quantified by real-time RT-PCR. Expression levels of <i>Mmp10</i>, <i>Mmp13</i>, and <i>Adamts1</i> are presented as the mean and SD normalized to that of FGF2-negative cells (<i>n</i> = 3). FGF2-mediated increases of <i>Mmp10</i>, <i>Mmp13</i>, and <i>Adamts1</i> mRNA were antagonized by CNP and meclozine. Statistical significance is estimated by Student's t-test.</p

Meclozine attenuates FGFR3-mediated ERK phosphorylation in FGF2-treated RCS cells.

<p>(<b>A</b>) RCS cells were pretreated with 20 µM meclozine for 30 minutes before adding 5 ng/ml FGF2 and the levels of ERK and MEK phosphorylation were determined by Western blotting. As a loading control, the membranes were reprobed with antibodies against MEK and ERK. Meclozine suppressed FGF2-mediated ERK phosphorylation but not MEK phosphorylation after adding FGF2. (<b>B</b>) RCS cells were infected by lentivirus expressing constitutively active (ca) ERK, MEK, and RAF mutants. Cells were treated with 20 µM meclozine and their proliferation potencies were quantified using the MTS assay. The 490-nm absorbance was normalized to that without meclozine and the mean and SD are presented (<i>n</i> = 3). Meclozine rescued caMEK- and caRAF-mediated growth arrest, but had no effect on caERK-mediated growth arrest.</p

Meclozine facilitates chondrocyte differentiation of ATDC5 cells expressing mutant FGFR3 in micromass culture.

<p>ATDC5 cells were infected with lentivirus expressing FGFR3-WT (wild-type), FGFR3-G380R causing ACH, or FGFR3-K650E causing TDII. Similar expressions of the transgenes are shown by immunoblotting in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081569#pone.0081569.s003" target="_blank">Figure S3</a>. ATDC5 cells in micromass culture were treated with CNP (0.2 µM) or meclozine (20 µM) for six days and were stained with Alcian blue. Experiments were repeated six times and representative images are shown. After staining, matrix proteoglycans in the cell lysate were quantified by measuring absorbance at 610 nm. The data are presented as the mean and SD (<i>n</i> = 6). CNP and meclozine increased the Alcian blue staining of ATDC5 cells expressing FGFR3-G380R and –K650E in micromass culture. Statistical significance is estimated by Student's t-test.</p

Meclozine increases the longitudinal length of embryonic tibiae with or without FGF2 treatment in bone explant culture.

<p>Unstained bilateral tibiae of the same individual were photographed side by side on day six of explant culture. Longitudinal bone lengths were normalized to that of untreated contralateral tibia, and the mean and SD are indicated. FGF2 causes inhibition of longitudinal bone growth. In the presence of FGF2, CNP (0.2 µM) and meclozine (20 µM) significantly increased the longitudinal length of embryonic tibiae. Even without FGF2, meclozine (20 µM) facilitated the growth of embryonic tibiae but without statistical significance (Student's t-test).</p

Design of additional oligoprobes based on clinical samples.

<p>Design of additional oligoprobes based on clinical samples.</p

Comparison of the results between sequencing and PCR-SSOP-Luminex assay.

<p>Comparison of the results between sequencing and PCR-SSOP-Luminex assay.</p

Improvement of the detection by the additional probes at K65 locus.

<p>The ordinate shows the percentage of the genotype at K65 locus determined by the PCR-SSOP-Luminex DR assay. One hundred % is the sample number (74) successfully amplified by PCR.</p

Validation of PCR-SSOP-Luminex assay using plasmids as templates.

<p>(A) Agarose gel electrophoresis of amplified fragments. Lanes 1–6: Fragment a (547 bp). Lanes 7–14: Fragment c (512 bp). 1, wild type; 2, M41L-TTG; 3, M41L-CTG; 4, K65R-AGA; 5, 70R-AGA; 6, 70R-AGG; 7, wild type; 8, 103N-AAC; 9, K103N-AAT; 10, M184V-GTG; 11, T215Y-TAC; 12, T215F-TTC. (B) Median fluorescence intensities (MFIs). The plasmid in the test sample is indicated on the top of each panel. Oligoprobes used for detection are indicated at the bottom. Matched results are shown as black bars, mismatched results as white bars. Assays were performed in triplicate. The mean ± standard deviation is shown at the top of each bar.</p