Pip5k1c Loss in Chondrocytes Causes Spontaneous Osteoarthritic Lesions in Aged Mice

Abstract

Osteoarthritis (OA) is the most common degenerative joint disease affecting the older populations globally. Phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c), a lipid kinase catalyzing the synthesis of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), is involved in various cellular processes, such as focal adhesion (FA) formation, cell migration, and cellular signal transduction. However, whether Pip5k1c plays a role in the pathogenesis of OA remains unclear. Here we show that inducible deletion of Pip5k1c in aggrecan-expressing chondrocytes (cKO) causes multiple spontaneous OA-like lesions, including cartilage degradation, surface fissures, subchondral sclerosis, meniscus deformation, synovial hyperplasia, and osteophyte formation in aged (15-month-old) mice, but not in adult (7-month-old) mice. Pip5k1c loss promotes extracellular matrix (ECM) degradation, chondrocyte hypertrophy and apoptosis, and inhibits chondrocyte proliferation in the articular cartilage of aged mice. Pip5k1c loss dramatically downregulates the expressions of several key FA proteins, including activated integrin β1, talin, and vinculin, and thus impairs the chondrocyte adhesion and spreading on ECM. Collectively, these findings suggest that Pip5k1c expression in chondrocytes plays a critical role in maintaining articular cartilage homeostasis and protecting against age-related OA.

Keywords: Pip5k1c; aging; articular chondrocytes; osteoarthritis.

Figure 1.

Genetic deletion of Pip5k1c in aggrecan-expressing chondrocytes in adult mice. (A) PCR genotyping using tail DNA. Pip5k1c flox KO band, ~380bp; Pip5k1c flox wildtype (WT) band, ~250bp; Aggrecan^CreERT2, ~650bp. (B) A schematic diagram illustrating the breeding strategy and experimental design. (C) Immunofluorescent (IF) staining of knee joint sections showing the reduced expression of Pip5k1c in articular cartilage (AC) and growth plate (GP) after TAM injections. White dashed boxes indicate the higher magnification images in the right panels. Scale Bar: 50 mm. (D) Percentages of Pip5k1c-positive cells in AC and GP. N = 6 mice per group. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.

Figure 2.

Pip5k1c loss induces subchondral bone sclerosis and osteophyte formation in aged mice. (A) In vivo μCT scans of knee joints from control and cKO mice at 5- and 13-months post-TAM injections. Scale bar, 1 mm. Red arrowheads indicate the formation of osteophytes. Yellow arrowheads indicate subchondral bone sclerosis. (B, C) Quantitative analyses of bone mineral density (BMD) (B) and the volume of calcified meniscus and synovial tissue (C). N = 6 mice per group. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.

Figure 3.

Loss of Pip5k1c in chondrocytes promotes OA-like lesions in aged mice. (A) Representative images of safranin O & fast green (SO&FG)-stained knee joint sections from control and cKO mice at 13 months after TAM injections. Black dashed boxes indicate the higher magnification images of AC, GP, meniscus, and synovium in lower panels. Black arrowheads indicate the degradation of AC. Blue arrowheads indicate the loss of integrity of GP. Red arrowheads indicate the formation of osteophytes. Scale bar, 50 μm. (B) The severity of OA-like lesions was analyzed using the Osteoarthritis Research Society International (OARSI) scoring system. (C, D) Quantitative analyses of safranin O-positive areas in the AC (c) and GP (d). (E, F) Osteophyte score (E) and synovitis score (F) were performed using histological sections. N = 6 mice per group. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.

Figure 4.

Pip5k1c loss causes ECM degradation and chondrocyte hypertrophic differentiation in aged mice. (A) IF staining for expressions of aggrecan, Col2a1, Mmp13, Adamts5, Col10a1, and Runx2 using knee joint sections from control or cKO mice at 13 months after TAM injections. White dashed boxes indicate the higher magnification images in the right panels. White dashed lines indicate the cartilage surfaces. Scale bar: 50 µm. (B-G) Quantitative analyses of the percentages of aggrecan-, Col2a1-, Mmp13-, Adamts5-, Col10a1-, and Runx2-positive cells in AC. N = 6 mice per group. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.

Figure 5.

Pip5k1c loss inhibits chondrocyte proliferation and induces chondrocyte apoptosis in aged mice. (A) IF staining for expression of Ki67 using knee joint sections from control or cKO mice at 13 months after TAM injections. White dashed boxes indicate the higher magnification images in the right panels. White dashed lines indicate the cartilage surfaces. Scale bar: 50 µm. (B) Quantitative data of (A). (C) Fluorescent TUNEL staining. Scale bar: 50 µm. (D) Quantitative data of (d). (E) Western blotting. Protein extracts were isolated from cultured ATDC5 cells transfected with negative control (NC) siRNA or Pip5k1c siRNA and subjected to western blot analyses with indicated antibodies. t: total; p: phosphorylated. (F) Relative protein levels normalized to the NC siRNA group. In vitro siRNA knockdown experiments were independently repeated four times. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.

Figure 6.

Pip5k1c loss reduces expression of FA proteins and impairs chondrocyte-ECM adhesion. (A) IF staining for expressions of activated integrin β1 (9EG7), talin, and vinculin using knee joint sections from control or cKO mice at 13 months after TAM injections. White dashed boxes indicate the higher magnification images in the right panels. White dashed lines indicate the cartilage surfaces. Scale bar: 50 µm. (B-D) Quantitative data of (a). (E) Western blotting. Protein extracts were isolated from cultured ATDC5 cells which had been transfected with NC siRNA or Pip5k1c siRNA. (F) Relative protein levels of talin, vinculin, integrin β1, and integrin β3 in ATDC5 cells transfected with NC siRNA or Pip5k1c siRNA. (g) Representative images of attachment and spreading of ATDC5 cells on type II collagen-coated surfaces after transfection of NC siRNA or Pip5k1c siRNA. (H) Percentages of attached cells. In vitro experiments were independently repeated at least three times. Results are expressed as mean ± standard deviation (s.d.). The exact P values are shown in the figures.