Session Title: Biology and Pathology of Bone and Joint
Session Type: Abstract Submissions (ACR)
Background/Purpose: The main cellular events of articular chondrocytes during osteoarthritis are a loss of the traits as permanent cartilage, and a transition to hypertrophic chondrocytes responding to excessive stress. Persistant stress in an articular cartilage make the hypertrophied chondrocytes to be dead through apoptosis or form osteophytes through endochondral ossification. The research about molecular mechanisms controlling the endochondral ossification in the growth plate of bone can give us an important clue to understand the molecular mechanisms of osteoarthritis. Stromal cell-derived factor-1 alpha (SDF-1α) is elevated in joint fluid of osteoarthritis and is implicated in osteoarthritis, but it’s exact function in chondrocyte biology or molecular mechanisms by which SDF-1α acts still remains unclear. In this study, we investigated the roles of SDF-1α on the endochondral ossification.
Methods: Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with PBS vehicle or recombinant mouse SDF-1α. Real-time PCR analysis was performed using Applied Biosystems 7900 HT Real-Time PCR System and TaqMan® Gene Expression Assays for Sox9, Col2a1, Acan, MMP13, Col10a1, Runx2. Western blot was performed with MMP13, Runx2, type 10 collagen, proliferating chondrocyte nuclear antigen (PCNA), SOX9, p-Smad1/5/8, p-ERK and p-p38. Organ culture tissues were stained with safranin O/fast green and alcian blue/alizarin red. Immunohistochemical analysis was also performed on tissue sections with Caspase3, MMP13, Runx2, type 10 collagen, PCNA and SOX9. For quantification of chondrocyte apoptosis and necrosis, cells were stained with FITC-conjugated annexin V and propidium iodide, and analyzed on a FACS Aria II.
Results: Primary chondrocyte cultures revealed that SDF-1α significantly increased the expression of Acan, and Col10a1 (p<0.05). The master regulator gene of chondrocyte hypertrophy, Runx2, was also up-regulated in messenger RNA level by SDF-1α (p<0.05). SDF-1α increased the protein expression of Sox9, PCNA, Runx2, type 10 collagen. In addition, SDF-1α increased the phosphorylation of Smad1/5/8, Erk and p38 MAP kinase. The proportion of apoptotic cells which has Annexin V-FITC positive staining was 9.35 and 14.77% in untreated and SDF-1α treated cells, respectively. To gain further insights into the role of SDF-1α in endochondral ossification, we examined the effects of SDF-1α in tibia organ cultures. The length of tibias, compared with the controls, was significantly increased in SDF-1α treatment group (p<0.05). Immunohistochemical staining of organ cultures showed the expression of PCNA, the marker for chondrocyte proliferation and Sox9 markedly increased in chondrocytes of proliferating zone. In addition to proliferation marker, type 10 collagen, Runx2 and caspase3, were up-regulated in hypertrophic zone by SDF-1α.
Conclusion: Our findings reveal that SDF-1α has an effect on chondrocyte proliferation, hypertrophy and apoptosis by up-regulation of Sox9 and Runx2 through Smad and MAPK pathway during endochondral ossification.
S. E. Usmani,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-stromal-cell-derived-factor-1-alpha-through-smad-and-mapk-pathway-on-endochondral-ossification/