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Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

Matsushita M, Kitoh H, Ohkawara B, Mishima K, Kaneko H, Ito M, Masuda A, Ishiguro N, Ohno K - PLoS ONE (2013)

Bottom Line: We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan ; Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.

ABSTRACT
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.

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Meclozine attenuates FGFR3-mediated ERK phosphorylation in FGF2-treated RCS cells.(A) 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) 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 (n = 3). Meclozine rescued caMEK- and caRAF-mediated growth arrest, but had no effect on caERK-mediated growth arrest.
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pone-0081569-g005: Meclozine attenuates FGFR3-mediated ERK phosphorylation in FGF2-treated RCS cells.(A) 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) 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 (n = 3). Meclozine rescued caMEK- and caRAF-mediated growth arrest, but had no effect on caERK-mediated growth arrest.

Mentions: We next scrutinized the effects of meclozine on the downstream signaling pathways of FGFR3 in FGF2-treated RCS cells. RCS cells were pretreated with meclozine for 30 minutes before adding FGF2, and the phosphorylation levels of ERK and MEK were determined by Western blotting. The FGF2-mediated ERK1/2 phosphorylation was attenuated by meclozine, while MEK1/2 phosphorylation remained unchanged (Figure 5A). We next introduced constitutively active (ca) mutants of ERK, MEK, and RAF into RCS cells using lentivirus and quantified cell growth with the MTS assay. As predicted, meclozine ameliorated caMEK- and caRAF-mediated growth inhibition, whereas meclozine had no effect on caERK-mediated growth inhibition (Figure 5B). We observed the similar effects by counting cells (Figures S5). Both data point to a notion that meclozine is likely to inhibit MEK1/2-mediated ERK1/2 phosphorylation or activate phosphatase(s) for phosphorylated ERK1/2 (Figure 6).


Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

Matsushita M, Kitoh H, Ohkawara B, Mishima K, Kaneko H, Ito M, Masuda A, Ishiguro N, Ohno K - PLoS ONE (2013)

Meclozine attenuates FGFR3-mediated ERK phosphorylation in FGF2-treated RCS cells.(A) 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) 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 (n = 3). Meclozine rescued caMEK- and caRAF-mediated growth arrest, but had no effect on caERK-mediated growth arrest.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852501&req=5

pone-0081569-g005: Meclozine attenuates FGFR3-mediated ERK phosphorylation in FGF2-treated RCS cells.(A) 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) 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 (n = 3). Meclozine rescued caMEK- and caRAF-mediated growth arrest, but had no effect on caERK-mediated growth arrest.
Mentions: We next scrutinized the effects of meclozine on the downstream signaling pathways of FGFR3 in FGF2-treated RCS cells. RCS cells were pretreated with meclozine for 30 minutes before adding FGF2, and the phosphorylation levels of ERK and MEK were determined by Western blotting. The FGF2-mediated ERK1/2 phosphorylation was attenuated by meclozine, while MEK1/2 phosphorylation remained unchanged (Figure 5A). We next introduced constitutively active (ca) mutants of ERK, MEK, and RAF into RCS cells using lentivirus and quantified cell growth with the MTS assay. As predicted, meclozine ameliorated caMEK- and caRAF-mediated growth inhibition, whereas meclozine had no effect on caERK-mediated growth inhibition (Figure 5B). We observed the similar effects by counting cells (Figures S5). Both data point to a notion that meclozine is likely to inhibit MEK1/2-mediated ERK1/2 phosphorylation or activate phosphatase(s) for phosphorylated ERK1/2 (Figure 6).

Bottom Line: We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan ; Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus