Background and Objectives Wnts play an important role in cartilage development, homeostasis and degeneration. Therefore, exploring the therapeutic potential of factors that can modulate Wnts is of great interest. Secreted frizzled related proteins (SFRPs) were identified as secreted Wnt antagonists. Polymorphisms in Frizzled-related protein (FRZB-SFRP3) are associated with osteoarthritis (OA). Here, we study the role of SFRPs and their structural domains in cartilage biology.
Materials and Methods Surgical destabilization of the medial meniscus (DMM) was performed on eight-week-old male Frzb -/-, Sfrp1-/- and wild-type mice. Eight weeks after surgery, mice were sacrificed and histological scores were determined for the femoral and tibial articular surfaces following the OARSI guidelines. Chondrogenesis of ATDC5 micromasses, stably overexpressing pcDNA3.1-FRZB, -FRZB-ΔCRD, -FRZB-ΔNTN, -SFRP1, -SFRP1-ΔCRD, -SFRP1-ΔNTN, pGIPZ-shmiRNA-Frzb or control plasmids, was induced by culturing cells for 14 days in DMEM/F12 + 5%FBS, supplemented with ITS (insulin, transferrin, sodium selenite). On day 14, cells were switched to mineralization medium (αMEM + 5%FBS, supplemented with ITS, β-glycerophosphate and ascorbic acid). mRNA expression of chondrogenic markers (Aggrecan, Col2a1 and Col10a1) were assessed by quantitative RT-PCR. Western blot was used to study canonical (β-catenin) and non-canonical (CamKII) Wnt signalling.
Results The OARSI score showed a significant increase in cartilage damage in DMM-operated Frzb -/-mice compared to wild-type but not in Sfrp1-/- mice. Overexpression of Frzb in ATDC5 micromasses boosted chondrogenesis with up-regulation of Col2a1, whereas Frzb silencing lead to decreased chondrogenesis. These results corresponded with a reduction or increase in the activation of canonical WNT signalling, respectively. Wnt/CamKII signalling was not affected. SFRP1 overexpression led to a decrease in Col2a1 transcription, but to an increase in Col10a1 expression. These results corresponded with increased activation of canonical WNT signalling pathway and decreased phosphorylation of CamKII. Domain studies for Frzb and Sfrp1 showed that overexpression of the NTN domain increased expression of Col2a1 and Aggrecan.
Conclusions Different SFRPs have a strong, but distinct effect on cartilage. Frzb promoted early chondrogenesis through modulation of canonical Wnt signalling. Sfrp1 reduced early chondrogenesis and favoured hypertrophy by affecting both canonical and non-canonical Wnt signalling. Domain studies revealed that the NTN domain of SFRPs is responsible for the positive effect on chondrogenesis.