HtrA1, Ddr-2, and Mmp-13 are reliable biomarkers for osteoarthritis (OA), yet the exact mechanism for the upregulation of HtrA-1 is unknown. Some have shown that chondrocyte hypertrophy is associated with early indicators of inflammation including TGF-β and the Receptor for Advanced Glycation End-products (RAGE). To examine the correlation of inflammation with the expression of biomarkers in OA, we performed right knee destabilization surgery on 4-week-old-wild type and RAGE knock-out (KO) mice. We assayed for HtrA-1, TGF-β1, Mmp-13, and Ddr-2 in articular cartilage at 3, 7, 14, and 28 days post-surgery by immunohistochemistry on left and right knee joints. RAGE KO and wild type mice both showed staining for key OA biomarkers. However, RAGE KO mice were significantly protected against OA compared to controls. We observed a difference in the total number of chondrocytes and percentage of chondrocytes staining positive for OA biomarkers between RAGE KO and control mice. The percentage of cells staining for OA biomarkers correlated with severity of cartilage degradation. Our results indicate that the absence of RAGE did protect against the development of advanced OA. We conclude that HtrA-1 plays a role in lowering TGF-β1 expression in the process of making articular cartilage vulnerable to damage associated with OA progression.
The proximal and distal femur epiphysis of mice are both weight bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH) in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in Thyroid stimulating hormone receptor (Tshr-/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo, and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Ibsp, Bglap2, Dmp1, Spp1, and Alpl). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.
The critical importance of hypoxia-inducible factor (HIF)s in the regulation of endochondral bone formation is now well established. HIF protein levels are closely regulated by the prolyl hydroxylase domain-containing protein (PHD) mediated ubiquitin-proteasomal degradation pathway. Of the three PHD family members expressed in bone, we previously showed that mice with conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in the trabecular bone mass of the long bones. By contrast, loss of Phd3 expression in chondrocytes had no skeletal effects. To investigate the role of Phd1 expressed in chondrocytes on skeletal development, we conditionally disrupted the Phd1 gene in chondrocytes by crossing Phd1 floxed mice with Collagen 2α1-Cre mice for evaluation of a skeletal phenotype. At 12 weeks of age, neither body weight nor body length was significantly different in the Cre+; Phd1flox/flox conditional knockout (cKO) mice compared to Cre−; Phd1flox/flox wild-type (WT) control mice. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype differences were found for any of the trabecular bone measurements of either femur or tibia. Similarly, cortical bone parameters were not affected in the Phd1 cKO mice compared to control mice. Histomorphometric analyses revealed no significant differences in bone area, bone formation rate or mineral apposition rate in the secondary spongiosa of femurs between cKO and WT control mice. Loss of Phd1 expression in chondrocytes did not affect the expression of markers of chondrocytes (collage 2, collagen 10) or osteoblasts (alkaline phosphatase, bone sialoprotein) in the bones of cKO mice. Based on these and our published data, we conclude that of the three PHD family members, only Phd2 expressed in chondrocytes regulates endochondral bone formation and development of peak bone mass in mice.
HtrA1 and Mmp‐13 are reliable biomarkers of osteoarthritis (OA) and are upregulated at days post knee destabilization surgery (Polur et al., 2010). While expression of RAGE and its ligands are suggested, the exact mechanism for HtrA1 upregulation is not known (Cecil et al., 2005; Loeser et al., 2005). Our objective was to examine biomarkers of OA in the presence (control mice) and absence (RAGE knock‐out) of RAGE. We performed right knee destabilization and sham surgery on 8 week old control and RAGE knock‐out (KO) mice. We assayed for HtrA1, Mmp‐13, RAGE and S‐100 at 3, 7, 14 and 28 days post‐surgery by immunohistochemistry on both knee joints. We did not detect biomarkers of OA including Mmp‐13 in any sham surgery mice. In RAGE KO mice, we observed negligible staining of HtrA1 and faint staining of S‐100 at 28 days post‐surgery but not Mmp‐13. In contrast, we detected the presence of S‐100, HtrA1 and Mmp‐13 at focal points 3 days post‐surgery in control mice. By 28 days post‐surgery HtrA1 and Mmp‐13 expression is uniformly expressed in articular cartilage in control mice. RAGE KO does not entirely prevent HtrA1 upregulation but does protect against Mmp‐13 activation. Since the expression of Mmp‐13 is a terminal event resulting in OA, we conclude that RAGE KO protects against the most vital component of articular cartilage degradation and subsequently, OA.
The proximal and distal femur epiphysis of mice are both weight bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH) in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in Thyroid stimulating hormone receptor (Tshr -/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo, and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Bsp, Ocn, Dmp1, Opn, and Alp). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.
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