Background/Purpose: CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. Excessive mechanical stress loading is a risk factor for OA, but the molecular mechanism underlying the relationship is unclear. We hypothesized that mechanical stress-induced enhancement of CD44-ICD production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression.

Methods: Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading using a stretching system (STB-140) at various intensities. An ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD(pCMV/myc/cyto-CD44ICD).

Results: CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2) in an intensity-dependent manner. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading in a dose-dependent manner. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading.
Induction of chondrocyte de-differentiation by CD44-ICD overexpression
The effect of CD44-ICD overexpression on the expression of chondrogenic differentiation-related genes was also assessed. As shown in Fig. A, two CD44-ICD bands of about 15 kD (a strong band and weak band) were observed in lysates of BACs transfected with the CD44-ICD plasmid by Western blot, while corresponding bands were absent in BACs that were not transfected or were transfected with control plasmid. SOX9 protein expression was reduced in BACs transfected with CD44-ICD, but not in controls (Fig. A). CD44-ICD significantly decreased the mRNA expression of SOX9, aggrecan, and COL2, while increasing the expression of COL1 mRNA (Fig. B). This suggests that the ability to maintain the chondrocyte phenotype was disrupted by CD44-ICD overexpression. These results suggest that CD44-ICD overexpression or CD44-ICD production upon excess mechanical stress loading can promote the de-differentiation of articular chondrocytes.

Conclusion: Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-cd44-intracellular-domain-production-suppresses-bovine-articular-chondrocyte-de-differentiation-induced-by-excessive-mechanical-stress-loading/