Forkhead box O transcription factors control postnatal articular cartilage growth, maturation and homeostasis and expression
of proteoglycan 4
Tokio Matsuzaki1, Oscar Alvarez-Garcia1, Sho Mokuda1, Kohei Miyata1, Merissa Olmer1, Hiroshi Asahara1,2, Martin K. Lotz1
1
The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA, USA
2
Department of Systems Biomedicine, Tokyo Medical and Dental University, Tokyo, Japan
Disclosures: Authors declare no conflict of interest.
INTRODUCTION: Forkhead box O (FoxO) transcription factors regulate expression of diverse genes and protein functions, related with cellular processes,
including cell cycle, oxidative stress response, and metabolism [1-4]. Previous studies showed that FoxOs promote antioxidant gene expression and
autophagy in chondrocytes in vitro and that FoxO expression in cartilage is reduced with aging and OA [5, 6]. However, the role of FoxOs in cartilage in
vivo has not yet been examined. To investigate the role of FoxOs in joint development and homeostasis, cartilage-specific FoxO1/3/4 knockout (KO) mice
were analyzed.
METHODS: We generated mice with cartilage-specific deletion of individual deletion of FoxO1, 3 or 4 and mice with deletion of these three FoxOs (triple
KO) in cartilage by intercrossing with Col2a1-Cre transgenic mice with FoxO1flox/flox, FoxO3aflox/flox, and FoxO4flox/flox mice. These mice were analyzed from
birth up to 6 months of age. The growth plate and articular cartilage in knee joints were examined histologically. Mechanistic studies were performed with
immature articular chondrocytes (IMACs), adipose tissue-derived mesenchymal stem cells, and RNA and protein of cartilage from FoxO KO mice. To
examine the relationship between FoxO and proteoglycan 4 (Prg4), we overexpressed FoxO and analyzed Prg4 mRNA induction after TGF stimulation and
during chondrocyte differentiation from mesenchymal stem cells (MSCs).
RESULTS: FoxO1,3,4 triple KO and FoxO1 single KO mice showed a significantly increased total body length and tail length at 4 weeks. In addition, triple
KO and FoxO1 single KO mice at postnatal day 7 and 1 month showed an increase in the length of the proliferative zone of the growth plate, and FoxO1 KO
mice showed a wider hypertrophic zone. Articular cartilage in triple KO and FoxO1 KO mice was significantly thicker than in control mice at 1 and 2
months. Articular cartilage cell density in triple KO and FoxO1 KO mice was significantly reduced. OA-like changes developed between 4-6 months in
triple KO and in FoxO1 KO mice with increased cartilage, synovial, meniscus and bone scores. IMACs from triple KO showed increased cell death under
hydrogen peroxide challenge and increased cell metabolic activity, increased BrdU incorporation and increased numbers of S phase cell. Cell numbers in the
superficial zone in FoxO triple KO mice were significantly reduced and Prg4 expression was downregulated in cartilage. FoxO1 overexpression increased
Prg4 mRNA levels and Prg4 expression was decreased during the differentiation using FoxO deficient MSCs. The TGFβ-mediated increase in Prg4
expression was blunted in FoxO deficient cells and co-immunoprecipitation studies showed that FoxO1 interacts with Smad2/3 in a TGFβ-dependent manner.
DISCUSSION: Our finding show that the loss of FoxOs, in particular, FoxO1 in cartilage leads to elongation of the growth plate, abnormal cartilage
formation, and spontaneous OA development.
SIGNIFICANCE: FoxOs are essential to control cartilage growth. The direct transcriptional control of Prg4 suggests a novel mechanism by which agingrelated reduction of FoxO expression in cartilage can initiate OA.
REFERENCES:
[1] Cantley LC. Science. 2002;296(5573):1655-7. [2] Stahl M, Dijkers PF, et al. J Immunol. 2002;168(10):5024-31. [3] Klotz LO, Sanchez-Ramos C, et al.
Redox Biol. 2015;6:51-72. [4]Paik JH, Ding Z, et al. Cell Stem Cell. 2009;5(5):540-53. [5] Akasaki Y, Alvarez-Garcia O, et al. Arthritis Rheumatol.
2014;66(12):3349-58. [6] Akasaki Y, Hasegawa A, et al. Osteoarthritis Cartilage. 2014;22(1):162-70.
FIGURES:
Fig. 1 (A) Histological sections in 4- and 6-month-old mice. (B) Summed OARSI
scores for the medial femoral condyle and tibial plateau. (C) Synovial and
meniscus scores in 6-month-old mice. Numbers of mice were as follows: 2month-old, Control (n=11), Triple KO (n=17), FoxO1 KO (n=15), FoxO3 KO
(n=8), FoxO4 KO (n=8). 4-month-old, Control (n=10), Triple KO (n=10), FoxO1
KO (n=13), FoxO3 KO (n=7), FoxO4 KO (n=11). 6-month-old, Control (n=14),
Triple KO (n=20), FoxO1 KO (n=15), FoxO3 KO (n=22), FoxO4 KO (n=11).
*p<0.05, **p<0.01.
Fig. 2 (A) Real-time PCR analysis for Prg4 in adenoviral FoxO1-AAA or
Ad-GFP transfected human chondrocytes, SW1353 chondrosarcoma cells
and mouse IMACs. (B) Adipose tissue derived MSCs from FoxO triple
floxed mice with or without adenovirus-Cre were cultured as pellets in
chondrogenic medium. Real time PCR for Prg4 was performed at day 0, 1
and 2 weeks. (C), (D), (E) Real-time PCR analysis of Prg4 expression, (C)
in mouse chondrocytes from WT and FoxO triple KO mice treated with
10ng/ml TGFß1 or vehicle for 6 hours, (D) in ATDC5 cells transfected with
GFP or FoxO1AAA and treated with 10ng/ml TGFß1 or vehicle for 24
hours, and (E) in ATDC5 cells transfected with GFP, FoxO1H2015RAAA
and treated with 10ng/ml TGFß1 or vehicle for 24 hours. (F) Representative
images of western blot analysis of SMAD2/3 in ATDC5 cells transfected
with GFP or FoxO1AAA and treated with 10ng/ml TGFß1 or vehicle for 24
hours after immunoprecipitation using FLAG antibody.*p<0.05, **p<0.01.
ORS 2017 Annual Meeting Paper No.0073