Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence

Abstract

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Keywords: calcification; cartilage; chondrocytes; osteoarthritis; senescence; sortilin.

Figure 1

Sortilin and senescence are increased in OA mouse knees. (A) Safranin Orange staining of young and old mice (left 100× magnification (scale bar 500 µm), right 400× magnification (scale bar 50 µm)). The OARSI Score of young (8-week-old) and old (45-week-old) mice (young: 1.10 ± 0.211; old: 2.70 ± 0.632; p < 0.0001; N = 10). (B) Sortilin-IF staining of young and old mouse knees (400× magnification (scale bar 100 µm).The white square indicates the amplified area in the picture below. Quantification of sortilin-positive chondrocytes (young: 31.02 ± 10.28; old: 67.17 ± 16.81; N = 10). (C) p16-IF staining of young and old mouse knees (400× magnification (scale bar 100 µm). The white square indicates the amplified area in the picture below. Quantification of p16-positive chondrocytes (young: 28.42 ± 14.88, old: 41.04 ± 20.46, p = 0.0006, N < 8). IgG staining was used as a negative control for the specific antibody staining. The statistical evaluation was performed using an unpaired t-test. *** p < 0.001,**** p < 0.0001.

Figure 2

Sortilin and senescence are increased in human OA cartilage. Human cartilage with increasing severity of OA, assessed radiologically by the Kellgren-Lawrence score, was analyzed for sortilin immunostaining and gene expression levels. (A) Representative images of sortilin-IF staining of human cartilage are shown (630× magnification, scale bar 50 µm)). (B) The quantification of sortilin-positive cells shows a significantly increased number in OA cartilage compared with healthy (h) control (one-way ANOVA: F (3, 26) = 5.568; p = 0.004). (C) The analyses of sortilin gene expression using qRT-PCR corroborated this result (one-way ANOVA F (3, 26) = 3.236; p = 0.038). (D) Representative images of p16-IF staining of human cartilage are shown (400× magnification, scale bar, 50 µm). The quantification of p16-positive cells shows a significantly increased number in OA cartilage compared with healthy control (Mann-Whitney test: median: healthy: 2.564; OA: 17.17; p = 0.4). (E) The analyses of p16 gene expression using qRT-PCR corroborated this result (Mann-Whitney test: Median Healthy: 0.004; OA: 0.57; p = 0.04). All pictures were taken at 400× magnification (scale bar 50 µm). IgG staining was used as a negative control for the specific antibody staining. For statistical evaluation, either a one-way ANOVA in the case of comparing more than two groups with Dunett’s post-hoc test or a Mann-Whitney test was performed. The data were analyzed for normal distribution using a Shapiro-Wilk normality test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Sortilin and ALP are upregulated in osteogenically differentiated human chondrocytes. Isolated human chondrocytes were treated with osteogenic medium (OM) for up to 21 days to induce calcification. Chondrocyte medium (CM) was used as a control. (A) Quantification of eluted Alizarin Red S bound to chondrocytes after treatment with CM and OM to measure calcification. Treatment with OM leads to significantly increased calcification at days 7 and 21, compared with control (CM) (two-way ANOVA F (1, 20) = 29.25; p < 0.0001). Furthermore, there is a significant increase in calcification over time (two-way ANOVA F (2, 40) = 22.78; p < 0.0001). (B) Western blot of sortilin and ALP after osteogenic differentiation. (C) Sortilin is significantly increased at day 7 (OM) compared to control (CM). Over time, there continues to be a significant increase in sortilin (two-way ANOVA: F (2, 32) = 13.50; p ≤ 0.0001). (D) ALP shows a significant increase in protein levels at day 21 in the OM group compared to the control. (two-way ANOVA F (2, 28) = 4.206; p = 0.0253). (E) ALP activity assay of CM and OM chondrocytes. At day 21, the ALP activity of OM chondrocytes is significantly increased compared to CM chondrocytes (two-way ANOVA: F (2, 38) = 10.97; p = 0.0002). For statistical evaluation, a two-way ANOVA with Sidak’s post-hoc test was performed. ns: not significant; * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Sortilin is upregulated by senescence. Human chondrocytes were stimulated with mitomycin C for up to 10 days to induce cellular senescence. (A) Gene expression of sortilin after 5 and 10 days of treatment with mitomycin C (RM one-way ANOVA F (1.379, 9.656) = 16.88; p = 0.0013; N = 8). (B) Gene expression of p16 after 5 and 10 days of treatment with mitomycin C (RM one-way ANOVA: F (2, 14) = 5.080; p = 0.0219; N = 8). (C) Gene expression of p21 after 5 and 10 days of stimulation with mitomycin C (RM one-way ANOVA: F (1.367, 9.571) = 49.96; p < 0.0001). (D) Gene expression of ALP after 5 and 10 days of stimulation with mitomycin C (RM one-way ANOVA: F (1.083, 4.331) = 25.42, p = 0.0056). (E) Sortilin immunofluorescence showing percent sortilin positive cells as a function of duration of stimulation and representative images of sortilin immunostaining at days 1, 5, and 10 (RM one-way ANOVA: F (1.028, 2.057) = 1.077; p = 0.4092; N = 3). (F) p16 immunofluorescence with the percentage of p16 pos. cells depending on the duration of stimulation and representative images of p16 staining at days 1, 5, and 10 (RM one-way ANOVA: F (1.263, 2.527) = 1.153; p = 0.399; N = 3). All pictures are taken at 400× magnification (scale bar 50 µm). * p < 0.05, ** p < 0.01, *** p < 0.001.