Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Abstract

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1(-/-) chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.

Keywords: Akt PKB; FOXO; cartilage; fibroblast growth factor (FGF); fibroblast growth factor receptor (FGFR); osteoarthritis.

FIGURE 1.

Phlpp1 regulates bone mass. A, femur lengths from 4-week-old animals. B, percent change in bone volume per total volume (BV/TV); (C) trabecular number (Tb. N); (D) trabecular thickness (Tb. Th); (E) trabecular spacing (Tb. Sp); and (F) connective density (Conn. D) of distal femurs from 8-week-old WT (n = 3) and Phlpp1^−/− (n = 4) mice were determined by micro-computed tomography. *, p < 0.05 compared with control. G and H, microCT reconstructions are shown of cancellous bone and secondary spongiosa from distal femurs of 8-week-old WT and Phlpp1^−/− mice.

FIGURE 2.

Phlpp1 deficiency increases the number of cells in the proliferative zone. A, sections from paraffin embedded tibias of 4-week-old WT (n = 3) or Phlpp1^−/− (n = 4) mice were stained with Alcian blue. Proliferative (P) and hypertrophic (H) zones are noted. B, average number of cells/tissue area within the proliferative zone (P) was determined. C, BrdU staining of tibias from P5 mice, n = 5 WT, n = 5 Phlpp1^−/−.

FIGURE 3.

Phlpp1 deficiency enhances matrix production and Mek/Erk activation in chondrocytes. Chondrocyte micromass cultures from WT or Phlpp1^−/− mice were cultured for 3 days. A, cultures were fixed and stained with Alcian blue. Relative expression levels of type 2a1 collagen (B) aggrecan (C), PTHR (D), PRTrP (E), and Ihh (F) were assayed by real time PCR. *, p < 0.05 compared with control. G, Western blotting was performed as indicated. This experiment was repeated three times with data from a representative experiment shown.

FIGURE 4.

Phlpp1 deficiency increases Fgf18 expression and Erk1/2 signaling. A–C, Chondrocyte micromass cultures from WT or Phlpp1^−/− mice were cultured for 3 days. A, expression levels of indicated morphogens in Phlpp1^−/− cells relative to control WT cells were assayed by real-time PCR. The control value was set to 1. *, p < 0.1; **, p < 0.05 compared with control. B, immature chondrocytes from WT or Phlpp1^−/− mice were cultured in the presence of 100 nm PD173074. Western blotting was performed with the indicated antibodies and (C) the average gray value of the normalized phospho-Erk1/2 signal was determined (n = 3, normalization against total Erk1/2). D and E, immature chondrocytes from WT or Phlpp1^−/− mice were cultured in monolayer in the presence of the indicated concentrations of the Fgfr inhibitor (PD173074), the Mek1/2 inhibitor (U0126) or vehicle control (DMSO) for 4 days. MTS assays were performed. These experiments were repeated three times. Data from a representative experiment are shown. *, p < 0.05 compared with WT vehicle-treated cells; ^#, p < 0.05 compared with Phlpp1^−/− vehicle-treated cells.

FIGURE 5.

FoxO1 represses Fgf18 expression downstream of Phlpp1. A, ATDC5 cells were transfected with the indicated plasmids and relative expression of Fgf18 was assayed by real time PCR. *, p < 0.05 compared with vector-transfected cells. B, Western blotting was performed to verify expression of each construct. This experiment was repeated three times and a representative experiment is shown.

FIGURE 6.

A Phlpp inhibitor increases Fgf18 expression and matrix production in chondrocytes. A, ATDC5 cells were treated with okadaic acid (Oka), increasing concentrations of the Phlpp inhibitor NCS 117079, or vehicle (DMSO) for 24 h. Western blotting was performed as indicated. B, ATDC5 micromass cultures were cultured in the presence of 25 μm of the Phlpp inhibitor NCS 117079 or vehicle (DMSO). Cultures were fixed and stained with Alcian blue. C, ATDC5 cells were treated with 5 μm NCS 117079 or vehicle (DMSO) for 24 h. Relative expression levels of Fgf18 were assayed by real time PCR. *, p < 0.05 compared with control. These experiments were performed three times with data from a representative experiment shown.

FIGURE 7.

Working Model. Phlpp1 represses Akt2 activity in chondrocytes leading to increased FoxO1-mediated repression of Fgf18 expression. In the absence of Phlpp1, increased Akt activity diminishes FoxO1 levels and increases Fgf18 expression, which stimulates Erk1/2 phosphorylation and chondrocyte proliferation.