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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 mitigates abnormally suppressed proliferation of HCS-2/8 chondrocytes infected with lentivirus expressing mutant FGFR3.(A) HCS-2/8 cells expressing either FGFR3-K650E causing TDII, -K650M causing SADDAN, or G380R causing ACH were cultured with 20 µM meclozine for 48 hours and the proliferation was quantified by the MTS assay. Values are normalized to that without meclozine and the mean and SD are presented (n = 12). Meclozine ameliorated growth arrest driven by the mutations. (B) CNP (0.2 µM) and meclozine (20 µM) also alleviated K650E- and G380R-mediated growth inhibition visualized by Venus fluorescence expressed by the infected lentivirus. Fluorescence microscopy shows that the transfection efficiency is more than 90%. Both CNP and meclozine increased the Venus signal areas. Data are presented as the mean and SE that are normalized to that of vehicle (n = 3). Statistical significance is estimated by Student's t-test.
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pone-0081569-g002: Meclozine mitigates abnormally suppressed proliferation of HCS-2/8 chondrocytes infected with lentivirus expressing mutant FGFR3.(A) HCS-2/8 cells expressing either FGFR3-K650E causing TDII, -K650M causing SADDAN, or G380R causing ACH were cultured with 20 µM meclozine for 48 hours and the proliferation was quantified by the MTS assay. Values are normalized to that without meclozine and the mean and SD are presented (n = 12). Meclozine ameliorated growth arrest driven by the mutations. (B) CNP (0.2 µM) and meclozine (20 µM) also alleviated K650E- and G380R-mediated growth inhibition visualized by Venus fluorescence expressed by the infected lentivirus. Fluorescence microscopy shows that the transfection efficiency is more than 90%. Both CNP and meclozine increased the Venus signal areas. Data are presented as the mean and SE that are normalized to that of vehicle (n = 3). Statistical significance is estimated by Student's t-test.

Mentions: We examined the effects of meclozine on chondrocyte proliferation under the influence of FGFR3 mutants. As RCS cells express high levels of wild-type FGFR3, we used human chondrosarcoma (HCS-2/8) cells to observe unequivocal effects of the transduced FGFR3 mutants. We first introduced lentivirus carrying active mutants of FGFR3 (K650E in TDII, K650M in SADDAN, and G380R in ACH) into HCS-2/8 cells. The lentivirus carried IRES2-driven Venus cDNA downstream of FGFR3 cDNA. The MTS assay demonstrated that K650E-expressing HCS-2/8 cells showed significantly suppressed cellular proliferation (Figure S2). Meclozine partially rescued the growth arrest without apparent cellular toxicity in HCS-2/8 cells expressing the three FGFR3 mutants (Figure 2A). We also observed that the areas of Venus signals, which should be proportional to the number of Venus-positive cells, were increased by meclozine as well as by CNP in K650E- and G380R-expressing HCS-2/8 cells (Figure 2B).


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 mitigates abnormally suppressed proliferation of HCS-2/8 chondrocytes infected with lentivirus expressing mutant FGFR3.(A) HCS-2/8 cells expressing either FGFR3-K650E causing TDII, -K650M causing SADDAN, or G380R causing ACH were cultured with 20 µM meclozine for 48 hours and the proliferation was quantified by the MTS assay. Values are normalized to that without meclozine and the mean and SD are presented (n = 12). Meclozine ameliorated growth arrest driven by the mutations. (B) CNP (0.2 µM) and meclozine (20 µM) also alleviated K650E- and G380R-mediated growth inhibition visualized by Venus fluorescence expressed by the infected lentivirus. Fluorescence microscopy shows that the transfection efficiency is more than 90%. Both CNP and meclozine increased the Venus signal areas. Data are presented as the mean and SE that are normalized to that of vehicle (n = 3). Statistical significance is estimated by Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0081569-g002: Meclozine mitigates abnormally suppressed proliferation of HCS-2/8 chondrocytes infected with lentivirus expressing mutant FGFR3.(A) HCS-2/8 cells expressing either FGFR3-K650E causing TDII, -K650M causing SADDAN, or G380R causing ACH were cultured with 20 µM meclozine for 48 hours and the proliferation was quantified by the MTS assay. Values are normalized to that without meclozine and the mean and SD are presented (n = 12). Meclozine ameliorated growth arrest driven by the mutations. (B) CNP (0.2 µM) and meclozine (20 µM) also alleviated K650E- and G380R-mediated growth inhibition visualized by Venus fluorescence expressed by the infected lentivirus. Fluorescence microscopy shows that the transfection efficiency is more than 90%. Both CNP and meclozine increased the Venus signal areas. Data are presented as the mean and SE that are normalized to that of vehicle (n = 3). Statistical significance is estimated by Student's t-test.
Mentions: We examined the effects of meclozine on chondrocyte proliferation under the influence of FGFR3 mutants. As RCS cells express high levels of wild-type FGFR3, we used human chondrosarcoma (HCS-2/8) cells to observe unequivocal effects of the transduced FGFR3 mutants. We first introduced lentivirus carrying active mutants of FGFR3 (K650E in TDII, K650M in SADDAN, and G380R in ACH) into HCS-2/8 cells. The lentivirus carried IRES2-driven Venus cDNA downstream of FGFR3 cDNA. The MTS assay demonstrated that K650E-expressing HCS-2/8 cells showed significantly suppressed cellular proliferation (Figure S2). Meclozine partially rescued the growth arrest without apparent cellular toxicity in HCS-2/8 cells expressing the three FGFR3 mutants (Figure 2A). We also observed that the areas of Venus signals, which should be proportional to the number of Venus-positive cells, were increased by meclozine as well as by CNP in K650E- and G380R-expressing HCS-2/8 cells (Figure 2B).

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