Reciprocal regulation of Wnt5a and TGF-β1 expression in OA osteoblasts.

<p>Confluent OA osteoblasts were treated with either rhWnt5a or siWnt5a in order to measure the expression of TGF-β1. Conversely, OA osteoblasts were treated with either rhTGF-β1 or siTGF-β1 prior to the measure of Wnt5a expression. Gene expression was measured by RT-qPCR. A) Expression of TGF-β1 in response to siWnt5a treatement (n = 4); B) Expression of TGF-β1 in response to rhWnt5a (n = 3); C) Expression of TGF-β1 in response to siTGF-β1 treatment (n = 6); D) Expression of Wnt5a in response to rhTGF-β1 (n = 3); E) Expression of Wnt5a in response to siTGF-β1 (n = 4).</p

Expression of Wnt5a and LGR4 and LGR5 in normal and OA osteoblasts.

<p>Confluent normal and OA osteoblasts were prepared for RT-qPCR analysis. A) Wnt-5a expression in normal Ob (n = 4) and OA Ob (n = 16); B) LGR4 expression in normal Ob (n = 4) and OA Ob (n = 14); C) LGR5 expression in normal Ob (n = 4) and OA Ob (n = 14); D) Representative western blot of LGR4 production in normal and OA Ob; E) Representative western blot of LGR5 production in normal and OA Ob.</p

Time-dependent effect of siWnt5a treatment on gene expression of targeted genes.

<p>Confluent OA osteoblasts were treated with siWnt5a at time = 0 and every three days until 21 days of treatment. Osteoblasts were lyzed at the indicated time points and prepared for RT-qPCR. A) Expression of Wnt5a during the 21 days of osteoblast differentiation; B) Regulation of LRP5 expression; C) Regulation of LRP6 expression; D) Regulation of CBFA1/Runx2 expression; E) Regulation of Osterix expression. The results are the mean +/- SEM of n = 7 preparations. Statistical significance of siWnt5a treatments vs siSCR are indicated directly on the figure: * p<0.05, ** p<0.01 and *** p<0.005.</p

Effect of Wnt5a and Rspo-2 treatments on non-canonical Wnt pathway signaling in normal and OA osteoblasts.

<p>Confluent normal and OA osteoblasts were treated with rhWnt5a, rhRspo2 or both for 15 minutes. Proteins were prepared from cells for Western blot analysis. A) Representative Western blot analysis of normal and OA osteoblasts; B) Representative Western blot analysis for phospho-PKC and phospho-JNK of OA osteoblasts treated with siWnt5a or a siSCR (control); C) Quantification of the phosphorylation of JNK (<i>p</i>-JNK) in normal and OA osteoblasts relative to actin loading (OA: n = 5, normal: n = 3); D) Quantification of the phosphorylation of PKC (<i>p</i>-PKC) in normal and OA osteoblasts relative to actin loading (OA: n = 4 and N: n = 3); E) NFAT activity level measured with dual-luciferase reporter assay following rhWnt5a treatment in OA osteoblasts (n = 8); F) AP-1 activity level measured with dual-luciferase reporter assay following rhWnt-5a treatment in OA osteoblasts (n = 3); G) TOPflash activity level in response to Wnt3a, Wnt5a or both in OA osteoblasts (n = 4).</p

Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca²⁺ pathways in human osteoarthritis osteoblasts

<div><p>Objective</p><p>Clinical and <i>in vitro</i> studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/β-catenin signaling pathway (cWnt), and a reduced mineralization <i>in vitro</i>. In addition to the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. However, there are no reports of either pathway in OA Ob. Here, we studied the two non-canonical pathways in OA Ob and if they influence their phenotype.</p><p>Methods</p><p>Human primary subchondral Ob were isolated from the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or of normal individuals at autopsy. The expression of genes involved in non-canonical Wnt signaling was evaluated by qRT-PCR and their protein production by Western blot analysis. Alkaline phosphatase activity and osteocalcin secretion (OC) were determined with substrate hydrolysis and EIA, respectively. Mineralization levels were evaluated with Alizarin Red Staining, Wnt/PKC and Wnt/PCP pathways by target gene expression and their respective activity using the NFAT and AP-1 luciferase reporter assays.</p><p>Results</p><p>OA Ob showed an altered phenotype as illustrated by an increased alkaline phosphatase activity and osteocalcin release compared to normal Ob. The expression of the non-canonical Wnt5a ligand was increased in OA Ob compared to normal. Whereas, the expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar. Wnt5a directly stimulated the expression and production of LGR5, contrasting, Wnt5a did not stimulate the expression of LGR4. Wnt5a also stimulated the phosphorylation of both JNK and PKC, as well as the activity of both NFAT and AP-1 transcription factors. The inhibition of Wnt5a expression partially corrects the abnormal mineralization, OC secretion and ALPase activity of OA Ob.</p><p>Conclusion</p><p>These data indicate that the alteration of Wnt5a, a non-canonical Wnt signaling activator, is implicated in the modified signalisation and phenotype observed in OA Ob.</p></div

Regulation of the expression of LGR4 and LGR5 in OA osteoblasts.

<p>Confluent OA osteoblasts were treated with either recombinant human Wnt5a (100 ng/mL) or siWnt5a. LGR4 and LGR5 expression were measured by RT-qPCR. A. Effect of siWnt5a on Wnt5a gene expression (n = 6); B) LGR4 expression in response to siWant5a (n = 6); C) LGR5 expression in response to siWnt5a treatment (n = 6); D) LGR4 expression in response to rhWant5a (n = 7); E) LGR5 expression in response to rhWnt5a in OA Ob (n = 8); F) LGR5 expression in response to rhWnt5a in normal Ob (n = 3). G) Representative western blot of LGR4 production in OA Ob stimulated by rhWnt5a for 48 hours; H) Representative western blot of LGR5 production in OA Ob stimulated by rhWnt5a for 48 hours.</p

Effect of Wnt5a regulation on the phenotype of osteoblasts.

<p>Confluent normal (n = 6) and OA osteoblasts (n = 42) were stimulated with 50 nM 1,25(OH)₂D₃ prior to the determination of alkaline phosphatase activity (A) and osteocalcin secretion (B). OA osteoblasts were also treated with either siWnt5a of rhWnt5a and the activity of alkaline phosphatase or osteocalcin secretion was measured after 1,25(OH)₂D₃ stimulation. C) Effect of siWnt5a treatment on the expression of TNAP (n = 5); D) Effect of rhWnt5a on ALPase activity levels in normal Ob (n = 3); E) Effect of siWnt5a treatment on osteocalcin secretion in OA Ob (n = 8 preparations); F) Effect of rhWnt5a on osteocalcin secretion in normal Ob (n = 3). G) Effect of siWnt5a treatment for 28 days on mineralization levels of OA osteoblasts as measured with Alizarin red staining (n = 5 preparations). Top: representative ARS staining following siSCR or siWnt5a treatment.</p

Effect of diacerein and rhein on the osteoclastic levels of metalloprotease-13 (MMP-13) and cathepsin K

Determination was performed in the conditioned medium for MMP-13 and on cell lysates for cathepsin K. Raw 264.7 cells were incubated for 5 days with RANKL (100 ng/mL), allowing the cells to differentiate into osteoclasts. After this period, the cells were incubated for 2 days together with RANKL in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL) and diacerein or rhein (10 or 20 μg/mL). Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control. RANKL, receptor activator of nuclear factor-κB ligand.<p><b>Copyright information:</b></p><p>Taken from "Diacerein inhibits the synthesis of resorptive enzymes and reduces osteoclastic differentiation/survival in osteoarthritic subchondral bone: a possible mechanism for a protective effect against subchondral bone remodelling"</p><p>http://arthritis-research.com/content/10/3/R71</p><p>Arthritis Research & Therapy 2008;10(3):R71-R71.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2483463.</p><p></p

Effect of diacerein and rhein on subchondral bone osteoblast intracellular mitogen-activated protein (MAP) kinase pathways

Subchondral bone osteoblasts were pre-incubated for 2 hours with diacerein or rhein at 20 μg/mL and incubated for 30 minutes in the presence or absence of interleukin-1-beta (IL-1β) (100 pg/mL). Levels of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2), p38, and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) (p46 and p54) MAP kinases were studied by Western blot and quantified by densitometry as described in Materials and methods. Data are expressed as fold changes compared with IL-1β-treated control, which was assigned a value of 1. Statistical analysis was performed versus IL-1β-treated control.<p><b>Copyright information:</b></p><p>Taken from "Diacerein inhibits the synthesis of resorptive enzymes and reduces osteoclastic differentiation/survival in osteoarthritic subchondral bone: a possible mechanism for a protective effect against subchondral bone remodelling"</p><p>http://arthritis-research.com/content/10/3/R71</p><p>Arthritis Research & Therapy 2008;10(3):R71-R71.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2483463.</p><p></p

Representative immunohistochemical staining section for metalloprotease-13 (MMP-13) and cathepsin K in human osteoarthritis subchondral bone

MMP-13 was detected in the osteoblasts (Ob) as well as in the osteoclasts (Oc). Cathepsin K was detected only in osteoclasts. Original magnification, ×100.<p><b>Copyright information:</b></p><p>Taken from "Diacerein inhibits the synthesis of resorptive enzymes and reduces osteoclastic differentiation/survival in osteoarthritic subchondral bone: a possible mechanism for a protective effect against subchondral bone remodelling"</p><p>http://arthritis-research.com/content/10/3/R71</p><p>Arthritis Research & Therapy 2008;10(3):R71-R71.</p><p>Published online 25 Jun 2008</p><p>PMCID:PMC2483463.</p><p></p