Klf10 is involved in extracellular matrix calcification of chondrocytes alleviating chondrocyte senescence

Abstract

Osteoarthritis (OA) is a chronic degenerative disease resulting joint disability and pain. Accumulating evidences suggest that chondrocyte extracellular matrix calcification plays an important role in the development of OA. Here, we showed that Krüppel-like factor 10 (Klf10) was involved in the regulation of chondrocyte extracellular matrix calcification by regulating the expression of Frizzled9. Knockdown of Klf10 attenuated TBHP induced calcification and reduced calcium content in chondrocytes. Restoring extracellular matrix calcification of chondrocytes could aggravate chondrocyte senescence. Destabilization of a medial meniscus (DMM) mouse model of OA, in vivo experiments revealed that knockdown Klf10 improved the calcification of articular cartilage and ameliorated articular cartilage degeneration. These findings suggested that knockdown Klf10 inhibited extracellular matrix calcification-related changes in chondrocytes and alleviated chondrocyte senescence.

Fig. 1

Extracellular matrix calcification occurs in senescence chondrocytes and OA cartilage. A, B Mouse articular chondrocytes were treated with different concentrations of TBHP, alizarin red dye was used to show the size of calcium nodules (n = 3). Scale bar: 100 μm. C The alkaline phosphatase kit was used to measure the alkaline phosphatase activity of chondrocytes treated with TBHP at different concentrations (n = 3). D–G Micro-CT was used to detect the knee joints of mice after DMM 2 and 8 weeks, and SKYscan software was used to analyze osteophyte volume, bone density and bone volume fraction (n = 5). H, I Alizarin red was used to stain the knee sections without decalcification. The black line area represented the calcified cartilage area (n = 5). Scale bar: 20 μm

Fig. 2

Klf10 knockdown attenuates TBHP induced chondrocytes extracellular matrix calcification. A, B Chondrocytes were treated with TBHP and siRNA (Klf10) and its negative control, and alizarin red staining was used to detect the formation of calcium nodules (n = 3). Scale bar: 100 μm. C The alkaline phosphatase kit was used to measure the alkaline phosphatase activity of chondrocytes (n = 3). D, E Relative calcification related gene mRNA expressions of murine chondrocytes were detected using RT-qPCR (n = 3)

Fig. 3

Klf10 downregulation affects calcium ion content but not phosphate ion. A Q-PCR was used to detect the expression of related genes (n = 3). B The ATP test kit is used to detect ATP produced by articular cartilage cells (n = 3). C–G Confocal microscopy of cells treated with Fluo-8 dye. The red arrows are positive cells. Data analysis using Zeiss software ZEN (n = 3). Scale bar:100 μm. H, I Relative gene mRNA expressions of murine chondrocytes were detected using RT-qPCR (n = 3)

Fig. 4

Klf10 regulates Fzd9 to improve chondrocyte extracellular matrix calcification induced by TBHP. A–C Q-PCR was used to detect the expression of Fzd9 (n = 3). D Mouse articular chondrocytes were treated with siRNA (Fzd9) for 48 h. The expressions of Fzd9 was detected by western blot. E, F Alizarin red dye is used to stain calcium nodules formed by treated chondrocytes (n = 3). Scale bar: 100 μm. G The alkaline phosphatase kit was used to measure the alkaline phosphatase activity of chondrocytes (n = 3). H, I Relative calcification related gene mRNA expressions of murine chondrocytes were detected using RT-qPCR (n = 3). J Serially truncated and mutated Fzd9 promoter constructs were cloned and transfected into cells. The relative luciferase activities were determined after Klf10 overexpression. K Selective mutation (left panel) analyses identified Klf10-responsive regions in the Fzd9 promoter (right panel)

Fig. 5

Restoration of FZD9 expression aggravates chondrocyte extracellular matrix calcification, and recovery of calcification aggravates chondrocyte senescence. A, B Alizarin red dye shows the ability of overexpression treated chondrocytes to form calcified nodules (n = 3). Scale bar: 100 μm. C The alkaline phosphatase kit was used to measure the alkaline phosphatase activity of chondrocytes (n = 3). D, E Relative calcification related gene mRNA expressions of murine chondrocytes were detected using RT-qPCR (n = 3). F Murine articular chondrocytes were treated with lentivirus. The expressions of P16, P21 and Mmp13 were detected by western blot

Fig. 6

Restoration of Fzd9 expression aggravates chondrocyte extracellular matrix calcification, and recovery of calcification aggravates chondrocyte senescence. A, B Murine articular chondrocytes were treated with CPPD. The expressions of P16, P21, Mmp13 and Col2a1 were detected by western blot (n = 3). C, D, G, H β-Galactosidase staining was used to show senescent chondrocytes, and image J was used to calculate proportions (n = 3). Scale bar: 100 μm. E, F Murine articular chondrocytes were treated with BCP. The expressions of P16, P21, Mmp13 and Col2a1 were detected by western blot (n = 3)

Fig. 7

Klf10 downregulation inhibits chondrocyte extracellular matrix calcification and senescence, attenuating articular cartilage degeneration in DMM mice. A Schematic of mouse DMM model construction and lentivirus intra-articular injection. B, C Representative images of Safranin O/Fast Green staining of mouse knee cartilage. The degeneration of articular cartilage in different treatment groups was evaluated using the OARSI score. Scale bar: 100 μm, 50 μm. D–G Micro-CT was used to detect the knee joints of mice after DMM or sham, and SKY Scan software was used to analyze osteophyte volume, bone density and bone volume fraction (n = 5). H, I Alizarin red was used to stain the knee sections without decalcification (n = 5)

Fig. 8

Klf10 downregulation inhibits chondrocyte extracellular matrix calcification and senescence, attenuating articular cartilage degeneration in DMM mice. A–G Immunohistochemical analysis of the expression of Klf10, Fzd9, Runx2, Mmp13, p21, and p16 in different treatment groups. Scale bar = 20 μm, n = 5