CD38 Drives Progress of Osteoarthritis by Affecting Cartilage Homeostasis

Abstract

Objective: To observe expression of CD38, a key modulator of nicotinamide dinucleotide (NAD+) metabolism in mice with knee osteoarthritis, and protective effect of CD38 inhibition during the osteoarthritis (OA) development.

Method: The destabilization of the medial meniscus (DMM) model was performed in mice to mimic the process of OA. Immunofluorescence of CD38 was performed to evaluate its response during the OA process. Limb bud-derived mesenchymal cells were isolated for micromass culture. 100 nM or 1 μM CD38 inhibitor (78c) treatment for 14 days and CD38 sgRNA infection were then used to explore the effects of chondrogenic differentiation via Alcian blue staining. The expressions of chondrogenic markers were detected using RT-PCR and Western blot. To explore the protective effect of CD38 inhibitor on cartilage degradation during OA in vivo, a CD38 inhibitor was injected into the knee joint after DMM operations. Micro-CT analysis and Safranin O-fast green staining were used to evaluate subchondral bone micro-architecture changes and cartilage degeneration.

Results: Compared to the control group, the CD38 expression in superficial cartilage was obviously increased in DMM group (P < 0.05). During the normal chondrogenic differentiation, the extracellular matrix formed and expression of Sox9, Col2, aggrecan increased apparently while CD38 expression decreased, which could be reversed with ablation of CD38 in limb bud-derived mesenchymal cells. Consistent with findings in vitro, CD38 blockage via CD38 inhibitor injection protected against osteosclerosis in medial subchondral bone and cartilage degeneration in DMM-induced experimental mice. Compared to the Sham group, DMM mice showed significantly increased values of BV and BV/TV in subchondral bone (P < 0.05) and Mankin score, which could be rescued by 78c treatment (P < 0.05). Also the CD38 inhibitor contributed to homeostasis of anabolism and catabolism by upregulating Sox9, Col2, aggrecan and downregulating Runx2, Col10 and Mmp13.

Conclusion: This study primarily implicates CD38 as an important regulator of chondrogenic differentiation. Inhibition of CD38 demonstrated protection against cartilage degeneration, which suggests that CD38 could be a potential therapeutic target for OA.

Keywords: CD38; Cartilage; Chondrocyte; NAD; Osteoarthritis.

Fig. 1

CD38 mRNA (A) and protein expression (B) were detected by RT‐PCR and immunofluorescence in control and DMM groups. CD38 expression was upregulated during the development of osteoarthritis. Quantitative analysis of the relative density of CD38 by densitometric analysis. Scale bar = 100 μm; Data are presented as mean ± SD. *P < 0.05, n = 6.

Fig. 2

CD38 and chondrogenic marker expression during differentiation. (A) Cartilaginous nodules and extracellular matrix formed after 6 days of chondrogenic differentiation (n = 3). Alcian blue staining intensity was measured using Image J software. Scale bar = 1 mm; (B) Western blot was performed to detect the protein level of Col‐2 and Aggrecan in progenitor chondrogenic cells (n = 3). Quantification of the protein expressions was obtained using Image J software. (C) There is no obvious changes in cell quantity during the first three days of cell differentiation (n = 6). (D) CD38 gene expression decreased and Sox9, Aggrecan and Col2 increased during chondrogenic differentiation (n = 3). Data are presented as mean ± SD. *P < 0.05.

Fig. 3

Ablation of CD38 promote chondrogenic differentiation. (A) 100 nM and 1 μM 78c treatment increases extracellular matrix formation in micromass cultures (n = 3). Alcian blue staining intensity was measured. Scale bar = 1 mm; (B) Compared with 100 nM 78c treatment, 1 μM significantly inhibit CD38mRNA expression and upregulate Col2 and aggrecan mRNA expression (P < 0.05), but has no effect on Sox9 mRNA expression (n = 3). Data are presented as mean ± SD. * P < 0.05.

Fig. 4

CRISPR/Cas9 Lentivirus knockout of CD38 promote chondrogenic differentiation. A. CRISPR/Cas9 Lentivirus transfection effectively decreased CD38 expression in primary micromass cells (n = 3). Quantification of CD38 protein expression was analyzed. B. Alcian‐Blue positive nodules are increased with CD38 knockout (n = 3). Scale bar = 1 mm; C. CD38 knockout significantly increased Sox9, Col2 and aggrecan gene expression (n = 3). Data are presented as mean ± SD. * P < 0.05

Fig. 5

78c protected against cartilage degeneration in DMM‐induced mice. Micro‐CT analysis andSafranin O‐fast green staining were performed to evaluate the degree of joint degeneration. Scale bar = 100 μm; Data are presented as mean ± SD. * P < 0.05, n = 12.

Fig. 6

(A) 78c stimulates upregulation of anabolic genes Sox9, Col2 and aggrecan, and downregulates hypertrophic gene Col10 and catabolic markers Runx2 and Mmp13 in DMM‐induced mice (P < 0.05). (B) Col2 and MMP13 protein expression were assessed by immunohistochemistry and its quantitative analysis. Red arrows: MMP13‐positive cells. Scale bar = 500 μm. Data are presented as mean ± SD. * P < 0.05, n = 6.