Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
Syndecan-4 (Sdc4), family member of type I transmembrane heparan sulfate proteoglycans (HSPGs), is a regulator of various cartilage-related processes including osteoarthritis (OA). Blockade of Sdc4 signaling protects mice from cartilage degradation in experimentally induced OA. OA is characterized by hypertrophic differentiation of chondrocytes and matrix remodeling. Various signaling pathways including the WNT signaling pathway may trigger this induction of chondrocyte differentiation. Experiments investigating the effect of different WNT3a concentrations on WT and Sdc4 deficient chondrocytes have emphasized a complex dialogue between canonical and non-canonical WNT pathways. We hypothesize that Sdc4 controls the chondrocyte phenotype by specific modulation of WNT signaling pathways.
Methods
In vitro analyses were performed using neonatal wild type (wt) and Sdc4-/- chondrocytes, or blocking antibodies against Sdc4. The influence of WNT3a on glycosaminoglycan (GAG) production was analyzed using alcian blue staining of micromass cultures. Expression of marker genes (e.g. aggrecan, collagen2, MMP13) was measured by quantitative RT-PCR. Effects of WNT3a on canonical and noncanonical WNT signaling were analyzed using Western Blot and luciferase reporter assay (AP-1, NFAT, TCF/Lef). The influence of WNT3a on the remodeling of the ECM was investigated by electron microscopy. Basal calcium concentrations without and upon WNT3a stimulation were examined using the Fura-2 method. In vivo relevance was investigated upon induction of OA using the DMM model.
Results
Micromass cultures revealed a higher basal GAG production by Sdc4-/- chondrocytes. WNT3a stimulation led to a decrease in GAG production in wt cells, which was absent in Sdc4-/- chondrocytes. qRT-PCR showed a 10x higher basal production of aggrecan and collagen2 in Sdc4-/- chondrocytes. WNT3a increased the expression of both genes in Sdc-4 -/-, whereas it decreased the expression in wt chondrocytes. MMP13 was significantly less expressed in Sdc4-/- chondrocytes and, unlike in wt cells, was not upregulated upon WNT3a stimulation. Western blot showed that ß-catenin is strongly reduced and not upregulated upon stimulation with WNT3a in Sdc4-/- chondrocytes. LRP6 was less phosphorylated and TCF/Lef promotor was less activated upon WNT3a stimulation in Sdc4-/- chondrocytes. pCamKII was increased under basal conditions, but decreased upon WNT3a stimulation in Sdc4-/-. The same effects on canonical and noncanonical WNT signaling upon WNT stimulation were obtained by using a blocking anti-Sdc-4 antibody. Upon WNT3a stimulation, Sdc4-/- cells displayed a finer, more condensed and disorganized ECM structure compared to wt. Sdc4-/- chondrocytes had increased intracellular Ca2+ levels, which were reduced after 24h incubation with WNT3a. In vivo stainings confirmed in vitro results.
Conclusion
Sdc4 is a major regulator of the chondrocyte cellular response to WNT signalling through facilitating the induction of the canonical WNT signaling pathway. The blockade of Sdc4 protects from OA induced changes in chondrocyte phenotype by inhibiting WNT induced differentiation of chondrocytes.
Disclosure:
C. K. Clarke,
None;
A. Held,
None;
R. Stange,
None;
U. Hansen,
None;
L. Godmann,
None;
J. Bertrand,
None;
T. Pap,
None;
G. Nalesso,
None;
F. Echtermeyer,
None;
F. Dell’Accio,
None;
J. Sherwood,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/syndecan-4-regulates-chondrocyte-phenotype-and-cartilage-homeostasis-via-the-wnt-signaling-pathway/