Asporin expression is highly regulated in human chondrocytes

A significant association between a polymorphism in the D repeat of the gene encoding asporin and osteoarthritis, the most frequent of articular diseases, has been recently reported. The goal of the present study was to investigate the expression of this new class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes. First, we studied the modulation of asporin (ASPN) expression by cytokines by Western blot and reverse transcription-polymerase chain reaction. Interleukin-1$\beta$ and tumor necrosis factor-$\alpha$ downregulated ASPN, whereas transforming growth factor-$\beta$1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. Finally, we found an important role of the transcription factor Sp1 in the regulation of ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity. In addition, using gene reporter assay and electrophoretic mobility shift assay, we showed that Sp1 mediated its effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene. In conclusion, this report is the first study on the regulation of asporin expression by different cytokines in human articular chondrocytes. Our data indicate that the expression of this gene is finely regulated in cartilage and suggest a major role of Sp1.

💡 Research Summary

This study investigates how the expression of asporin (ASPN), a newly identified class I small leucine‑rich proteoglycan (SLRP), is regulated in human articular chondrocytes. The authors first examined the impact of pro‑inflammatory cytokines on ASPN levels. Both interleukin‑1β (IL‑1β) and tumor necrosis factor‑α (TNF‑α) markedly reduced ASPN mRNA and protein, as demonstrated by RT‑PCR and Western blot analyses, indicating that inflammatory environments typical of osteoarthritis (OA) suppress this matrix component. In contrast, transforming growth factor‑β1 (TGF‑β1) up‑regulated ASPN when chondrocytes were cultured in serum‑free medium; the effect was blunted in the presence of serum, suggesting that serum factors can interfere with TGF‑β1 signaling.

The second major focus was the relationship between chondrocyte differentiation status and ASPN expression. Serial passaging, which drives dedifferentiation, caused a progressive decline in ASPN levels across passages 1 to 5. Conversely, three‑dimensional alginate culture, which promotes redifferentiation, restored ASPN expression to levels comparable with early‑passage cells. These findings underscore that ASPN is a differentiation‑dependent gene, tightly linked to the chondrocytic phenotype and extracellular matrix (ECM) homeostasis.

The most novel aspect of the work concerns transcriptional regulation by the zinc‑finger transcription factor Sp1. Ectopic Sp1 expression in HEK293 cells increased ASPN mRNA 2‑ to 3‑fold and strongly activated a luciferase reporter containing the ASPN promoter (−2000 bp to +100 bp). Systematic 5′‑deletion mapping identified a critical Sp1‑responsive region between –473 and –140 bp upstream of the transcription start site. Electrophoretic mobility shift assays (EMSA) confirmed direct binding of Sp1 to this segment, and supershift experiments with anti‑Sp1 antibodies validated the specificity of the interaction. Pharmacological inhibition of Sp1 with mithramycin A reduced ASPN transcription, further establishing Sp1 as a key positive regulator.

Collectively, the data reveal a finely tuned regulatory network: inflammatory cytokines suppress ASPN, anabolic TGF‑β1 promotes it, cellular dedifferentiation diminishes it, and redifferentiation restores it, all converging on Sp1‑mediated transcriptional activation. The authors conclude that ASPN expression is highly dynamic in cartilage and that Sp1 plays a central role in maintaining its levels. Given the association of ASPN polymorphisms with OA susceptibility, these mechanistic insights suggest that modulating Sp1 activity or ASPN expression could become a therapeutic avenue for preserving cartilage integrity in degenerative joint disease.