DYRK1A negatively correlated with the pathological progression of OA in adult mice and humans
To explore the role of DYRK1A in OA, DMM surgery was performed on the knee joints of 2-mouth-old WT mice to simulate OA model and mice received sham surgery as control (Sham)[17]. After DMM operation, paraffin sections were performed on the knee joints of mice, and then HE and Fast green/Safranin O staining were performed which were evaluated by the modified makin score and OARSI score respectively[18]. The results showed that compared with the sham operation group, the damage degree of the DMM group was significantly increased, indicating that the successful establishment of OA model. (Supplemental Fig. 5A-D). The expression changes of DYRK1A in the knee joints of WT mice at 1 month, 2months and 3months after DMM surgery were detected by immunohistochemistry staining. At 2 mouths and 3 mouths after DMM surgery, the expression of DYRK1A in the OA cartilage was significantly down-regulated compared with sham surgery group. These differences show statistical significance. The expression of DYRK1A also showed differences with the progression of OA. Compared with the knee joint of mice at 1 mouth after operation, the expression of DYRK1A in the knee joints of mice at 2 mouths and 3 mouths after surgery was further reduced and had a statistical difference (Fig. 1A, B). We collected the articular cartilage of patients requiring joint replacement surgery due to OA (Most involved) and segment of relatively complete OA cartilage as control group (Least involved)[19]. The results of hematoxylin and eosin (HE) (Supplemental Fig. 5E, F), Fast green/Safranin O staining, the modified makin score and OARSI score confirmed more severe cartilage degradation than that of the Least involved group (Fig. 1C, D). We found that DYRK1A was significantly down-regulated in the Most involved group compared to the Least involved group (Fig. 1E, F).
Our data showed that DYRK1A was downregulated during the pathogenesis of OA and may be negatively correlated with disease progression.
Accelerated Articular Cartilage Destruction In Adult Mice With Chondrocyte-specific Deletion Of Dyrk1a
Five days after tamoxifen injection, the expression of DYRK1A protein in articular chondrocytes specific deletion of DYRK1A (DYRK1A-cKO) mice was significantly reduced (Fig. 1G-I), indicating that the DYRK1A gene was effectively deleted. In adult DYRK1A-cKO mice, µCT scans were performed 3 months after DMM surgery (Fig. 1J). From the reconstructed 3D images, it was observed that OA was significantly aggravated in adult mice with DYRK1A-cKO compared to Cre-negative mice. However, the subchondral bone plate Bone Volume/Tissue Volume (BV/TV); Trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th) were statistically analyzed, and there was no significant difference (Supplemental Fig. 5G-J). The effect of DYRK1A deficiency on the development of osteoarthritis was subsequently assessed. One month after DMM, DYRK1A-cKO mice had early symptoms of OA, such as unevenness, small pits on the joint surface and loss of cartilage and proteoglycan, which could be detected by HE staining or by Fast green/Safranin O staining. These phenotypes were more severe than that in Cre-negative mice. Two months after DMM surgery, compared with Cre-negative mice, DYRK1A-cKO mice showed more extensive destruction of most areas of articular cartilage and partial exposure of subchondral bone. Three months after DMM surgery, compared with Cre-negative mice, DYRK1A-cKO mice had further aggravated articular cartilage destruction and extensive exposure of subchondral bone (Fig. 2A). Histological analysis showed that the thickness of superficial cartilage in the articular cartilage of DYRK1A-cKO mice was less than that of Cre-negative mice at 1 month, 2 months, and 3 months after surgery, and the number of hypertrophic chondrocytes was higher than that of Cre-negative mice (Fig. 2C). At 2 and 3 months after DMM surgery, using modified makin score and OARSI score, the degree of joint damage in DYRK1A-cKO mice was more severe than that in Cre-negative mice, and DYRK1A-cKO mice after DMM surgery arthritis progression worsened over time (Fig. 2B,D).
These results suggest that DYRK1A-cKO mice mice exacerbates the DMM surgery-induced osteoarthritis phenotype.
Chondrocyte-specific Deletion Of Dyrk1a Disrupts The Balance Of Anabolism And Catabolism In Mouse Articular Cartilage
The first line of defense against OA initiation is the superficial layer. It produces lubricant proteins, harbors cartilage precursor cells, resists shear stress, acts as a sliding surface, and plays an important role in protecting chondrocytes. In OA, degenerative changes begin with decreased cellular anabolism and increased catabolism[5]. DYRK1A has the effect on promoting cell proliferation and cell anabolism. DYRK1A is abundantly expressed in superficial chondrocytes, and deletion of DYRK1A aggravates the progression of OA[20].
To elucidate the mechanism of accelerated OA in DYRK1A-cKO mice, we detected the protein levels related to anabolism and catabolism in chondrocytes by immunohistochemistry. Collagen (Col II) and aggrecan are important anabolic markers in the extracellular matrix of cartilage[21]. Compared with Cre-negative mice, DYRK1A-cKO mice showed lower levels of immunoreactivity for Col II in articular cartilage and aggrecan (Fig. 3A, B; Fig. 4C, D). Collagen (Col X) is a marker of chondrocyte hypertrophy and promotes catabolism[22]. Compared with Cre-negative mice, the immunoreactivity of Col X (Fig. 3C, D) in articular cartilage of DYRK1A-cKO mice was increased. This was associated with reduced Fast green/Safranin O staining of articular cartilage in mutant mice. These results suggest that DYRK1A deficiency may directly promote catabolic activity and inhibit anabolic activity of articular chondrocytes. This disrupts the homeostasis of articular cartilage in adult mice.
Decreased Egfr-erk Signaling And Aggrecan Expression Mediate The Development Of Dyrk1a-deficient Osteoarthritis
EGFR-ERK signaling is important for maintaining the normal function of superficial cartilage during skeletal development. Furthermore, EGFR-ERK signaling is thought to be an anabolic mediator in articular cartilage destruction[5]. Pozo et al observed that DYRK1A can prevent the endocytic degradation of EGFR to maintain the activity of EGFR and downstream signaling[7]. DYRK1A inhibits the endocytic degradation of EGFR and maintains the phosphorylation level of downstream ERK by EGFR to promote the expression of p-ERK, aggrecan, Col II and other chondrocyte anabolic factors [23, 24]. Using immunohistochemistry to detect EGFR, EGFR was down-regulated in knee cartilage of DYRK1A-cKO mice compared with Cre-negative mice at 3 months after DMM modeling (Fig. 3E, F). The expression of EGFR in the knee joints of WT mice at 3 months after DMM modeling was significantly lower than that of mice in the Sham group (Fig. 4A, B). We found that EGFR was down-regulated in Most involved group compared to Least involved group in human cartilage (Fig. 4E, F). The cartilage of DYRK1A-cKO mice 3 months after DMM, were selected for Western Blot detection of EGFR expression. We found that the expression of EGFR in DYRK1A-cKO mice was decreased compared with that of Cre-negative mice, but there was no statistical significance. This may be related to the fact that other tissues besides cartilage, such as synovium and meniscus, which also express EGFR (Fig. 4K, L). Using GEO2R analysis, it was found that EGFR in cartilage of osteoarthritis patients was significantly down-regulated compared with normal human cartilage (Fig. 4I, J). Therefore, we investigated whether EGFR-ERK signaling mediates the aggravation of OA caused by DYRK1A deficiency. We found that p-ERK levels were significantly down-regulated in knee cartilage of DYRK1A-cKO mice compared with Cre-negative mice at 3 months after DMM surgery (Fig. 4G, H), and aggrecan expression was also significantly decreased. (Fig. 4C, D). This suggests that DYRK1A deletion may attenuate the expression of cellular anabolic markers such as aggrecan and Col II through EGFR-ERK signaling. Therefore, we propose a possible mechanism for this phenomenon (Fig. 4M).