Suppression of REDD1 in osteoarthritis cartilage, a novel mechanism for dysregulated mTOR signaling and defective autophagy O. Alvarez-Garcia, M. Olmer,

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Suppression of REDD1 in osteoarthritis cartilage, a novel mechanism for dysregulated mTOR signaling and defective autophagy O. Alvarez-Garcia, M. Olmer, R. Akagi, Y. Akasaki, K.M. Fisch, T. Shen, A.I. Su, M.K. Lotz Osteoarthritis and Cartilage Volume 24, Issue 9, Pages 1639-1647 (September 2016) DOI: 10.1016/j.joca.2016.04.015 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 REDD1 expression is reduced in human articular cartilage during aging and OA. A) REDD1 RNA expression analyzed by quantitative PCR in articular cartilage from normal (N = 23) and OA donors (N = 14). B) Western blot analysis of REDD1 expression in articular cartilage from normal (N = 6) and OA donors (N = 6). C) REDD1 immunohistochemistry on articular cartilage from normal, aged and OA donors. Six donors were analyzed per group and representative images are shown at two different magnifications. On the right, REDD1 quantitation expressed as percentage of REDD1 immunopositive cells. All values are mean ± 95%CI. Magnification bar = 100 μm. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 REDD1 expression is reduced in mouse articular cartilage during aging and OA. REDD1 immunohistochemistry on articular cartilage of normal (6-month-old), old (27-month-old), and 6-month-old mice subjected to surgical destabilization of medial meniscus (DMM). A total of six mice per group were analyzed and representative images are shown at two different magnifications. On the right, REDD1 quantitation expressed as percentage of REDD1 immunopositive cells. Values are mean ± 95%CI. Magnification bar = 100 μm. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 REDD1 inhibits mTORC1 activity in human and mouse articular chondrocytes. A) Western blot analysis of REDD1, phospho-S6 (p-S6) and phospho-4EBP (p-4EBP) in human primary chondrocytes transfected with siRNA control or specific against REDD1. Values are mean ± 95%CI of eight different human donors. B) Western blot analysis of REDD1, p-S6 and p-4EBP in T-C/28 chondrocytes transfected with siRNA control or specific against REDD1. Values are mean ± 95%CI of four different experiments. C) Western blot analysis of p-S6 and p-4EBP in immature murine articular chondrocytes isolated from Redd1+/+ and Redd1−/− mice. Values are mean ± 95%CI of three independent experiments. D) Western blot of REDD1 and p-S6 levels in human primary chondrocytes transduced with adenovirus expressing GFP (Ad-GFP) or REDD1 (Ad-REDD1) at different multiplicities of infection (MOI). E) Western blot analysis of REDD1 and p-S6 levels in T-C/28 chondrocytes transduced with Ad-GFP or Ad-REDD1 at 10 MOI. Values are mean ± 95%CI of three independent experiments. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 REDD1 regulates autophagy in articular chondrocytes. A) Western blot analysis of REDD1 and LC3 in human primary chondrocytes transfected with siRNA control or specific against REDD1 in the presence of absence of chloroquine (CQ, 25 μM). Values are mean ± 95%CI of five different donors. B) Western blot analysis of REDD1 and LC3 in T-C/28 chondrocytes transfected with siRNA control or specific against REDD1 in the presence of absence of CQ. Values are mean ± 95%CI of three different experiments. C) Western blot analysis of REDD1 and LC3 in immature murine articular chondrocytes isolated from Redd1+/+ and Redd1−/− mice. Values are mean ± 95%CI of three independent experiments. Statistical comparisons are Redd1−/− vs Redd1+/+. D) Western blot analysis of REDD1 and LC3 in human primary chondrocytes transduced with adenovirus expressing GFP (Ad-GFP) or REDD1 (Ad-REDD1) at 10 multiplicity of infection. Values are mean ± 95%CI of four different donors. E) Western blot analysis of REDD1 and p-S6 levels in T-C/28 chondrocytes transduced with Ad-GFP or Ad-REDD1 at 10 MOI. Values are mean ± 95%CI of four independent experiments. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 REDD1 induces autophagy in articular chondrocytes through an mTOR independent mechanism. A) Western blot analysis of REDD1, phospho-S6 (p-S6), and LC3 in human primary chondrocytes transfected with siRNA control or specific against REDD1 in the presence of absence of chloroquine (CQ, 25 μM) and rapamycin (Rapa, 1 μM). Values are mean ± 95%CI of four different donors. B) Western blot analysis of REDD1, p-S6 and LC3 levels in human primary chondrocytes transduced with adenovirus expressing GFP (Ad-GFP) or REDD1 (Ad-REDD1) at 10 multiplicity of infection, in the presence of absence of CQ and Rapa. Values are mean ± 95%CI of four different donors. C) Western blot analysis of REDD1, p-S6 and LC3 in immature murine articular chondrocytes isolated from Redd1+/+ and Redd1−/− mice in the presence of absence of CQ and Rapa. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 REDD1 forms a complex with TXNIP to induce autophagy in articular chondrocytes. A) TXNIP RNA expression analyzed by quantitative PCR in articular cartilage from normal (N = 18) and OA donors (N = 20). Values are mean ± 95%CI. B) TXNIP protein levels in articular cartilage from normal and OA donors assessed by western blot. Eight donors were analyzed per group. C) Co-immunoprecipitation experiment in T-C/28 chondrocytes transduced with adenovirus expressing GFP (Ad-GFP) or FLAG-REDD1 (Ad-REDD1) at 10 multiplicity of infection. Western blot shows REDD1, GFP and TXNIP levels after immunoprecipitation with anti-FLAG antibodies. D) Western blot analysis of REDD1, TXNIP, phospho-S6 (p-S6) and LC3 in human primary chondrocytes transfected with siRNA control or specific against REDD1 or TXNIP as indicated in the presence or absence of chloroquine (CQ, 25 μM). Values are mean ± 95%CI of four different donors. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 1 REDD1 protein expression measured by IHC in articular cartilage from 2-month-old Redd1+/+ and Redd1−/− mice. Magnification bar = 100 μm. Osteoarthritis and Cartilage 2016 24, 1639-1647DOI: (10.1016/j.joca.2016.04.015) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions