Prokineticin 2 is a catabolic regulator of osteoarthritic cartilage destruction in mouse

Abstract

Background: Our preliminary study indicates that the multi-functional protein, prokineticin 2 (Prok2), is upregulated in osteoarthritic (OA) chondrocytes as a target of the hypoxia-inducible factor (HIF)-2α. This study aims to elucidate the potential roles of Prok2 in OA.

Methods: Prok2 expression was assessed through microarray analysis in chondrocytes and confirmed via immunostaining in OA cartilage. Experimental OA was induced through destabilization of the medial meniscus (DMM). Functions of Prok2 were assessed by adenoviral overexpression, intra-articular (IA) injection of recombinant Prok2 (rProk2), and knockdown of Prok2 in joint tissues. We also explored the potential utility of Prok2 as an OA biomarker using enzyme-linked immunosorbent assay (ELISA).

Results: HIF-2α upregulated Prok2, one of the prokineticin signaling components, in OA chondrocytes of mice and humans. Adenoviral overexpression of Prok2 in chondrocytes and cartilage explants, as well as the application of rProk2, led to an upregulation of matrix metalloproteinase (MMP)3 and MMP13. Consistently, the overexpression of Prok2 in joint tissues or IA injection of rProk2 exacerbated cartilage destruction and hindpaw mechanical allodynia induced by DMM. However, the knockdown of Prok2 in joint tissues did not significantly affect DMM-induced cartilage destruction. Additionally, despite being a secreted protein, the serum levels of Prok2 in OA mice and human OA patients were found to be below the range detected by ELISA.

Conclusion: The upregulation of Prok2 exacerbates OA cartilage destruction and hindpaw mechanical allodynia. However, its knockdown is not sufficient to inhibit experimental OA and Prok2 is not a potential candidate serum biomarker of OA.

Keywords: Biomarker; Chondrocytes; Matrix-degrading enzymes; Mechanical allodynia; Osteoarthritis; Prokineticin.

Fig. 1

Prok2 is a target of HIF-2α in chondrocytes and exhibits upregulation in OA cartilage of mice and humans. A Primary-culture mouse chondrocytes were treated with vehicle alone or IL-1β (1 ng/ml, 36 h). Alternatively, chondrocytes were infected with 800 MOI of Ad-C (control), Ad-HIF-2α, or Ad-ZIP8 for 36 h. Presented is a heatmap of microarray analysis data for prokineticins (Prok) and their receptors (ProkR, n=3). B, C RT-PCR (B) and qRT-PCR (C) analysis of the indicated molecules in chondrocytes infected with 800 MOI of Ad-C (control) or the indicated MOI of Ad-HIF-2α for 36 h (n=7). D Positioning of HRE sequences in Prok2 promoter. E Representative immunostaining images of Prok2 in cartilage from sham- or DMM-operated mice and semi-quantification of Prok2 positive pixels (n=6 mice per group). F Immunostaining images and semi-quantification of Prok2 positive pixels in damaged and undamaged human OA cartilage (n=5 patients). Values are presented as mean ± 95% CI, and significance was evaluated by paired t-test. Scale bar: 50 μm

Fig. 2

Prok2 stimulates expression of MMP3 and MMP13 in chondrocytes. A, B RT-PCR (A) and qRT-PCR (B) of the indicated molecules in chondrocytes infected with 800 MOI of Ad-C or the indicated MOI of Ad-Prok2 for 36 h (n=7 cell cultures). C, D RT-PCR (C) and qRT-PCR (D) of the indicated molecules in chondrocytes treated with vehicle alone or the indicated amount of recombinant Prok2 (rProk2) for 36 h (n=8 cell cultures). Values are presented as mean ± 95% CI, and significance was evaluated by paired t-test

Fig. 3

Prok2 modulates protein levels of MMP13 and type II collagen in cartilage explants. A Mouse cartilage explants, sourced from 4-day-old ICR mice, were treated with 1 × 10⁹ pfu of Ad-C, Ad-HIF-2α, or Ad-Prok2 for 24 h. Presented are representative immunofluorescence images of type II collagen, MMP13, and DAPI in sections of cartilage explants (n=10 explants). B Mouse cartilage explants were treated with PBS as a control, 100 ng/ml of IL-6 as a positive control, and 100 ng/ml of rProk2 for 24 h. Representative immunofluorescence images of type II collagen, MMP13, and DAPI in sections of cartilage explants are presented (n=10 explants)

Fig. 4

Adenoviral overexpression of Prok2 in joint tissues exacerbates post-traumatic OA cartilage destruction and hindpaw mechanical allodynia in mice. A, B Mice were IA injected with 1 × 10⁹ pfu of Ad-C (control), Ad-Prok2 (Ad-P), or Ad-HIF-2α (Ad-H) once a week for 3 weeks (n=10 mice per group). OARSI grade and osteophyte maturity were determined at 9 weeks after the first IA injection (A). Representative safranin-O staining images of joint sections at 6 weeks and 9 weeks after the first IA injection (B). C, D Sham- or DMM-operated mice were IA injected with 1 × 10⁹ pfu of Ad-C (control) or Ad-Prok2 (Ad-P) once a week for 3 weeks (n=13 mice per group). OARSI grade and osteophyte maturity were determined at 6 weeks after surgery (C). Representative safranin-O staining images of joint sections at 6 weeks after surgery (D). E von Frey assays were performed at the indicated weeks after DMM operation (n=22 mice per group). Data for OARSI grade and osteophyte maturity are presented as median ± interquartile range (IQR) and paw withdrawal threshold is presented as mean with s.e.m. Significance was evaluated by Kruskal-Wallis with post hoc Bonferroni (A), Mann-Whitney U test (B), and Student’s t test (E), respectively. Scale bar: 50μm

Fig. 5

IA injection of recombinant Prok2 (rProk2) exacerbates post-traumatic OA cartilage destruction. A, B Sham- or DMM-operated mice were IA injected with BSA or rProk2 (10 μg/10 μ1/joint) once a week for 3 weeks (n=8 mice per group). Representative safranin-O staining images of joint sections at 6 weeks after surgery (A). Quantification of OARSI grade and osteophyte maturity at 6 weeks after surgery (B). Values are presented as median ± interquartile range (IQR) and significance was evaluated by Mann-Whitney U test. Scale bar: 50 μm

Fig. 6

Knockdown of Prok2 in joint tissues alone is insufficient to inhibit post-traumatic OA cartilage destruction. A Representative immunostaining images of Prok2 (left panel) and semi-quantification of Prok2-positeive pixels (right panel) in cartilage sections of mice received IA injection of 1 × 10⁹ pfu of Ad-shC (control) or Ad-shProk2 (n=6 mice per group). B, C Sham and DMM-operated mice were subject to IA injections of 1 × 10⁹ pfu of Ad-shC or Ad-shProk2 once a week for 3 weeks. Representative safranin-O staining images (B) and scoring of OARSI grade and osteophyte maturity (C, n=14 mice per group). D qRT-PCR analysis of the indicated molecules in primary-culture chondrocytes treated with 400 MOI of Ad-C and Ad-HIF-2α, with or without the specified MOI of Ad-sh-C and Ad-shProk2 (n=9). Values are presented as mean ± 95% CI, and significance was evaluated by paired t-test

Fig. 7

Prok2 is not detected in the sera of OA model mice or human OA patients. A Primary-culture mouse chondrocytes were infected with 800 MOI of Ad-C, Ad-HIF-2α, or Ad-Prok2 for 36 h. Five milliliters of cell culture supernatant from five 35-mm dishes was concentrated to 100 μl an ELISA was used to detect secreted Prok2 (n=8 independent cultures). B Sera were collected from mice IA injected in both knees with 1 × 10⁹ pfu of Ad-C, Ad-HIF-2α, or Ad-Prok2 once a week for 3 weeks. Prok2 levels in 100 μl of collected sera were determined by ELISA (n=8 mice per group). C Sera was collected from mice that were sham- or DMM-operated in both knees and sampled at 8 weeks after the operation. Prok2 levels were determined by ELISA from 100 μl of collected sera (n=18 mice per group). D Prok2 levels were determined from 100 μl of sera collected from KL grade 0 non-OA individuals and KL grade 3~4 OA patients (n=18 females per group). Because the Prok2 levels in all samples (except the Ad-Prok2-infected chondrocytes in A) were below the range of detection, the detected values are presented as optical density. Statistical values are presented as means ± s.e.m, and were assessed with paired t-test for ELISA of cell culture media, ANOVA with Bonferroni’s post hoc test for ELISA of mouse serum, and Student’s t test for ELISA of human serum