Dysregulated FOXO transcription factors in articular cartilage in aging and osteoarthritis

Abstract

Objective: Aging is a major risk factor for osteoarthritis (OA). Forkhead-box class O (FoxO) transcription factors regulate mechanisms of cellular aging, including protein quality control, autophagy and defenses against oxidative stress. The objective of this study was to analyze FoxO transcription factors in normal, aging and OA cartilage.

Design: Knee joints from humans ages 23-90 and from mice at the age of 4-24 months and following surgically induced OA were analyzed for expression of FoxO proteins. Regulation of FoxO protein expression and activation was analyzed in cultured chondrocytes.

Results: Human cartilage expressed FOXO1 and FOXO3 but not FOXO4 proteins. FOXO1 and FOXO3 were more strongly expressed the superficial and mid zone as compared to the deep zone and were mainly localized in nuclei. During human joint aging, expression of FOXO1 and FOXO3 was markedly reduced in the superficial zone of cartilage regions exposed to maximal weight bearing. In OA cartilage, chondrocyte clusters showed strong FOXO phosphorylation and cytoplasmic localization. Similar patterns of FOXO expression in normal joints and changes in aging and OA were observed in mouse models. In cultured chondrocytes, IL-1β and TNF-α suppressed FOXO1, while TGF-β and PDGF increased FOXO1 and FOXO3 expression. FOXO1 and FOXO3 phosphorylation was increased by IL-1β, PDGF, bFGF, IGF-1, and the oxidant t-BHP.

Conclusions: Normal articular cartilage has a tissue specific signature of FoxO expression and activation and this is profoundly altered in aging and OA in humans and mice. Changes in FoxO expression and activation may be involved in cartilage aging and OA.

Keywords: Aging; Cartilage; FoxO; Osteoarthritis.

Fig. 1

FOXO protein expression in human articular cartilage. A. Protein extracts from articular cartilage were analyzed by western blotting using antibodies to FOXOs as indicated. Graph shows the results of a total of five normal and five OA donors. B. Photograph of osteochondral slab from the central region of the medial femoral condyle showing regions that were used to cut sections from the most proximal (1) to the most distal (6) location. C. Immunohistochemistry was performed for FOXO1 and FOXO3. Numbers of sections correspond to the regions in panel B. Magnification 40×.

Fig. 2

Zonal distribution of total and phospho-FOXO in human articular cartilage. Cartilage sections from maximal and minimal weight bearing regions were stained with antibodies to total FOXO and phospho-FOXO. Representative images for nuclear and cytoplasmic localization of FOXO1 and zonal distribution are shown in panel A. Images for cellular localization are 100×, for zonal distribution is 10× magnification. Results are shown as % positive cells for FOXO1 in panel B and for FOXO3 in panel C. Values are the median and quartile. Data are representative of six normal donors.* = P < 0.05 vs each zone; & = P < 0.05 vs maximal weight bearing.

Fig. 3

Changes in FOXO protein expression in human cartilage from aging and OA-affected joints. A. Cartilage was collected from the femoral condyles of normal (weight bearing regions) and OA knee joints and analyzed by immunohistochemistry with antibodies as indicated. Images are 40×, insets on the right are 100× magnification. B. Quantification of total FOXO, phospho-FOXO in cartilage zones and cytoplasmic vs nuclear localization (shown as % cytoplasmic localization). Data are representative of six young, four aged and four OA donors.

Fig. 4

FoxO protein expression in normal, aging and OA-affected mouse joints. Knee joints were collected from C57/Bl6 mice at the indicated ages or 10 weeks following induction of experimental OA by surgical knee destabilization. A. Representative images normal young, aging and experimental OA cartilage. Images are 10×, insets are 40× magnification. B. Quantification of total FoxO, phospho-FoxO and cytoplasmic localization. Data represent four mice per time point and three for surgical OA.

Fig. 5

Changes in FOXO protein expression in response to stimulation of cultured human chondrocytes. Cells were treated with the indicated stimuli for 2 or 5 days and cell lysates were analyzed by western blotting with the antibodies indicated. Representative western blot images are shown and graphs represent quantification of western blots from three different experiments using cells from different donors, each including all stimuli and time points.

Fig. 6

Changes in FOXO phosphorylation in response to stimulation of cultured human chondrocytes. Cells were treated with the indicated stimuli for 60 min and cell lysates were analyzed by western blotting with the antibodies for phosphorylated FOXOs and Akt as indicated. Representative western blot images are shown and graphs represent quantification of western blots from five different experiments using cells from different donors, each including all stimuli.