Abstract
ObjectiveClinical and in vitro 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 in vitro. 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.MethodsHuman 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.ResultsOA 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.ConclusionThese 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.
Highlights
Osteoarthritis (OA) is the most common form of arthritis, and knee OA is amongst the most frequent type along with hip OA
The expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar
We previously reported that OA Ob demonstrate better cell proliferation [3] and an elevation in markers of differentiation such as alkaline phosphatase (ALPase), osteocalcin (OC) secretion, type 1 collagen production [4, 5] and growth factors such as transforming growth factor β1 (TGF-β1) [4, 6] compared to normal Ob
Summary
Osteoarthritis (OA) is the most common form of arthritis, and knee OA is amongst the most frequent type along with hip OA. Femoral heads of patients with OA obtained at autopsy showed a low mineralization pattern compared to normal tissues [9, 10] This may explain the apparent bone mineral density increase in OA as representing an increase in material density and not mineral density [11]. The production of collagen type I and the ratio of collagen type I α1 to α2 chains, which is increased in OA tissue compared to normal, is the cause for an increase in undermineralized osteoid matrix [12] This situation was observed in OA osteoblasts in vitro and linked to elevated levels of TGF-β1 [4]
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