27 research outputs found
Verapamil suppresses Wnt-mediated protein expression and nuclear translocation of β-catenin in human osteoarthritic chondrocytes (OAC) cells, and verapamil rescues Wnt-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells.
<p>(A) Immunoblotting of β-catenin in OAC cells treated with Wnt3A-CM and verapamil for 72 hrs. (B) Immunofluorescence staining with anti-β-catenin antibody in OAC cells. Untreated cells (upper panels), cells treated with Wnt3A-CM alone (middle panels), and cells treated with Wnt3A-CM and 25 μM verapamil (lower panels) for 72 hours are stained with anti-β-catenin antibody (green, left panels) and DAPI (blue, middle panels). Wnt3A-induced nuclear localization of β-catenin is blocked by verapamil. Scale bar = 20 μm. (C) Alcian blue staining of ATDC5 cells that are differentiated to chondrocytes with ITS for two weeks. The cells are subsequently treated with Wnt3A-CM and verapamil for 72 hrs. Proteoglycans are quantified by measuring the optical density at 630 nm of the cell lysates. The mean and SEM (<i>n</i>  = 3) are indicated. *<i>p</i><0.05 versus control by one-way ANOVA with Tukey's test.</p
Verapamil activates <i>FRZB</i> promoter and reduces Wnt/β-catenin signaling.
<p>Luciferase activities in human chondrosarcoma (HCS) cells treated with the indicated concentrations of verapamil for 24 hrs. Firefly luciferase activity for <i>FRZB</i> promoter (A) or TOPFlash reporter activity (B) are normalized by the TK promoter-driven Renilla luciferase activity and expressed as relative luciferase units. The mean and SEM (<i>n</i> = 12) are indicated. ** <i>p</i><0.01 and *<i>p</i><0.05 versus control by one-way ANOVA with Tukey's test.</p
Verapamil prevents OA progression and β-catenin accumulation in rat DMM model.
<p><b>(A, B)</b> DMM surgery induces mild OA phenotype in Wistar/ST rat and verapamil prevents OA progression. (A) Representative staining of knee joints with Safranin O and fast green. Scale bars = 200 μm. (B) Verapamil suppresses OA progressions evaluated by modified Mankin score at four and eight weeks after the surgery. Three rats in each group had DMM and sham surgeries in the right and left knees, respectively. Mean and SEM (<i>n</i> = 3) are indicated. (C) Immunofluorescence staining with anti-β-catenin antibody in rat articular cartilage. Articular chondrocytes at the weight-bearing sites are stained with anti-β-catenin antibody and DAPI. Nuclear translocation of β-catenin in DMM is blocked by verapamil. Scale bar = 20 μm.</p
Verapamil Protects against Cartilage Degradation in Osteoarthritis by Inhibiting Wnt/β-Catenin Signaling
<div><p>In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce <i>FRZB</i> expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of <i>ACAN</i> encoding aggrecan, <i>COL2A1</i> encoding collagen type II α1, and <i>SOX9</i>, and suppressed Wnt-responsive <i>AXIN2</i> and <i>MMP3</i> in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.</p></div
Verapamil upregulates chondrogenic makers (<i>ACAN</i> encoding aggrecan, <i>COL2A1</i>, and <i>SOX9</i>) and downregulates Wnt-responsive <i>AXIN2</i> and <i>MMP3</i> in human osteoarthritic chondrocytes (OAC) cells.
<p>Expression levels of each mRNA are normalized to that without treatment. (A) Verapamil upregulates the native <i>FRZB</i> at the mRNA and protein levels in OAC cells (<i>n</i> = 3). (B) Gene expressions of <i>ACAN</i>, <i>COL2A1</i>, and <i>SOX9</i> are increased by verapamil in 24 hrs. Purified Wnt3A protein increases <i>AXIN2</i> and <i>MMP3</i> mRNA expressions in 24 hrs (C), and verapamil inhibits the Wnt3A-induced gene expressions (D). (E) Verapamil does not suppress LiCl-induced <i>AXIN2</i> and <i>MMP3</i> expressions. (F) <i>FRZB</i> siRNAs (siFRZB-1 and siFRZB-2) cancel the effects of verapamil in Wnt3A-treated OAC cells. The mean and SEM (<i>n</i> = 3) are indicated. *<i>p</i><0.05 versus control by one-way ANOVA with Tukey's test.</p
Verapamil suppresses hypertrophic differentiation of chondrocytes and β-catenin staining in growth plates in explanted mouse fetal tibiae on embryonic day 17.5.
<p>(A) Tibiae are cultured with LiCl or verapamil for 10 days, and coronal slices of paraffin sections are stained with Alcian blue combined with hematoxylin and eosin staining. Three layers of proliferative (green), prehypertrophic (yellow), and hypertrophic (red) zones are indicated by bars. Scale bar = 200 μm. (B) Verapamil suppresses the number of chondrocytes in the hypertrophic zone in (A). (C) Immunofluorescence with antibody against β-catenin in proximal tibiae of mouse embryo (E17.5). Color bars indicate layers as indicated in (A). Scale bar = 200 μm. (D) Verapamil suppresses the number of β-catenin-positive cells in the proliferative zone. The mean and SEM (<i>n</i> = 3) are indicated. *<i>p</i><0.05 versus control by one-way ANOVA with Tukey's test.</p
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
Staining for β-catenin proteins is increased adjacent to the injured site in the rat Achilles tendon.
<p><b>(A, B)</b> The Achilles tendon (AT) between calcaneum (C) and gastrocnemius (G) was punctuated with a 14-gauge needle (N) at the injured site (IS). Scale bar = 2 mm. <b>(C, D)</b> Hematoxylin-eosin staining of sagittal sections of injured <b>(C)</b> and sham-operated tendons <b>(D)</b> on postoperative day 14. Position of IS with abundant inflammatory cells is indicated by a double-headed arrow in <b>C</b>. Tendons are placed with the distal side on the left and the proximal side on the right. Scale bar = 200 μm. <b>(E-G)</b> High magnification of the areas indicated in <b>C</b> and <b>D</b>. <b>(E)</b> An image distant from IS and close to the calcaneum. <b>(F</b>) An image adjacent to IS and near the center of the tendon. <b>(G)</b> An image in sham-operated tendon. Scale bar = 200 μm. <b>(H)</b> Immunostaining for β-catenin (green) with DAPI (blue). Scale bar = 5 μm. <b>(I)</b> The ratio of β-catenin-positive cells in the field of ~36,000 μm<sup>2</sup> is indicated by mean and SD (<i>n</i> = 3 rats each). The numbers of cells counted in the ~36,000 μm<sup>2</sup>-image field for Distal to IS, Proximal to IS, and Sham tendon are 21–56 cells, 29–72 cells, and 28–55 cells, respectively. The number of β-catenin positive cells is divided by the number of DAPI-positive cells in the field. <b>(J)</b> Mean and SD (<i>n</i> = 3 rats each) of intensities of total cellular and nuclear β-catenin signals of the tendon cells indicated in <b>(I)</b>. Each intensity is normalized by the number of DAPI-positive cells. <i>p</i> < 0.05 by one-way ANOVA. *<i>p</i> < 0.05, **<i>p</i> < 0.01 by Tukey-Kramer post-hoc test.</p