CITED2-Mediated Mechanotransduction and Its Use for Chondroprotection

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CITED2-Mediated Mechanotransduction and its use for Chondroprotection

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CITED2-Mediated Mechanotransduction and its use for Chondroprotection

Daniel J. Leong

A dissertation submitted to the Graduate Faculty in Biomedical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2014

Daniel Leong

2014 This manuscript has been read and accepted for the

Graduate Faculty in Biomedical Engineering in satisfaction of the

dissertation requirement for the degree of Doctor of Philosophy.

______Sihong Wang, PhD – Chair of Examining Committee Date Department of Biomedical Engineering, The City University of New York

______Ardie Walser, PhD, Executive Officer Date The City University of New York

Hui (Herb) B. Sun, PhD – Dissertation Advisor Departments of Orthopaedic Surgery and Radiation Oncology Albert Einstein College of Medicine

Luis Cardoso, PhD – Dissertation Advisor Department of Biomedical Engineering, The City University of New York

Mitchell B. Schaffler, PhD – Dissertation Committee Member Department of Biomedical Engineering, The City University of New York

Robert J. Majeska, PhD – Dissertation Committee Member Department of Biomedical Engineering, The City University of New York

Abstract of the Dissertation

CITED2-Mediated Mechanotransduction and its use for Chondroprotection By Daniel J. Leong

Doctor of Philosophy In Biomedical Engineering The City University of New York 2014

Advisors: Hui B. Sun, PhD and Dr. Luis Cardoso, PhD

Novel prevention and therapeutic treatment for cartilage degradation is urgently called

for, as cartilage degradation is a hallmark for arthritic diseases including osteoarthritis (OA) and

rheumatoid arthritis (RA), and there is a high incidence of arthritis-related disability and high

medical costs. While both underuse (e.g. physically inactive lifestyle) and overuse (e.g. high

impact or intense repetitive joint use as seen in certain sports) are risk factors for cartilage

degradation, recent studies highlight that dynamic moderate loading is associated with reduced

incidence of developing OA. Exercise is prescribed in most cases at moderate levels for both OA

and RA patients, and accumulating studies demonstrate appropriate exercise maintains cartilage

homeostasis and may exert a role in chondroprotection, but the mechanisms underlying how

mechanical signals are translated into chondroprotective actions are largely unknown.

Identifying the molecules that mediate mechanotransduction and its mechano-responsive nature

will not only provide a biomechanical basis for developing more effective mechanical loading-

based exercises for chondroprotection, but also can provide novel targets or molecular switches

to develop chemical-based modalities for disease treatment. The overall objectives of this dissertation were to determine the mechanical response of

transcriptional regulator CBP/p300 Interacting Transactivator with ED-rich Tail 2 (CITED2) to various mechanical loading regimes, elucidating CITED2-mediated chondroprotective pathways,

and determining the potential of CITED2 as a target for the prevention and treatment of cartilage

degradation in arthritis. The global hypothesis was that CITED2 mediates a mechanical pathway

of chondroprotection, CITED2 is required for cartilage integrity, and restoration or increasing

levels of CITED2 exerts efficacy in the prevention and treatment of cartilage degradation. This

hypothesis was tested with four specific aims: 1) To determine the gene expression response of

CITED2 to various mechanical loading regimes, and the role of CITED2 in mediating

mechanical regulation of matrix metalloproteinases (MMPs), 2) To determine the CITED2-

mediated loading-induced pathway and test whether it is required for loading-induced

downregulation of MMPs, 3) To determine whether deficiency of CITED2 is a causal factor for

cartilage degradation in arthritic diseases such as OA, and 4) Test the concept of CITED2 as a target for chondroprotection.

The key findings of this dissertation include: 1) CITED2 expression is induced by moderate dynamic loading in chondrocytes in an intensity- and duration-dependent manner, and

the upregulation of CITED2 is sustained for at least 12 hours after loading. The induction of

CITED2 is required for the downregulation of MMPs (i.e. MMP-2, -3, and -13), 2) Dynamic

moderate loading induces CITED2 by activating p38δ, which in turn triggers Sp1 and HIF-1α to

transactivate CITED2. CITED2 competes with MMP transactivator Ets-1 for binding to limiting

amounts of co-factor p300, resulting in repression of MMP expression. 3) Deficiency of

CITED2 is associated with cartilage degradation in human OA and disease progression of post-

traumatic OA in mice subject to destabilization of the medial meniscus (DMM). Experimental knockdown of CITED2 caused cartilage degradation and deletion of CITED2 in adult cartilage not only resulted in an early OA phenotype, but also accelerated the disease progression of cartilage degradation in DMM mice, revealing a chondroprotective role of CITED2, which is required for cartilage integrity maintenance. 4) Restoring levels of CITED2, via gene transfer or small molecule epigallocatechin gallate (EGCG), exerts efficacy in slowing cartilage degradation in RA and OA mouse models. Together, these studies provide feasibility for developing

CITED2-activation-based therapies for the prevention and treatment of cartilage degradation.

Acknowledgements

First and foremost, I would like to thank my mentor Dr. Herb Sun. I am so grateful to

have had the opportunity to learn from him, and appreciate all his contributions of time, ideas,

and funding to make my Ph.D. experience productive and stimulating. Herb’s enthusiasm for

research was contagious and motivational. I am so thankful for the excellent example he

provided to be a successful scientist and leader. I am also very grateful for my co-mentor Dr.

Luis Cardoso for his guidance, support, and generosity. I truly appreciate my dissertation committee Dr. Mitchell Schaffler, Dr. Robert Majeska, and Dr. Sihong Wang who have fostered my scientific growth with their tough questions, insightful comments, and encouragement.

I would like to thank all my colleagues and friends who not only donated their time and expertise to teach me but also provided camaraderie during all those long sessions in the lab, including: Yonghui Li, Ph.D, Zhengzhe Li, PhD (Big Li), Damien Laudier, Phil Nasser, Melissa

Ramcharan, Brad Herman, PhD, David Fung, PhD, Jonathan Lee, MD, Zhiyong He, PhD,

Takuya Ruike, MD, Nelly Andarawis-Puri, PhD, Jedd Seresky, MD, PhD, Stephen Ros MD,

PhD, Marwa Choudhury, Justin Tang, MD, Lin Xu, MD, and Fawzy Saad, PhD.

Last and not least, I would like to thank my family and friends for all their love, encouragement, and unwavering support for all my pursuits.

Table of Contents

List of Figures ……………………………………………………………………………... i List of Abbreviations ……………………………………………………………………… ix Chapter 1. Introduction: Articular joints, cartilage homeostasis, mechanotransduction and CITED2 1.1. Research Significance ………………………………………………………………. 1 1.2. Joint structure and physiology ………………………………………………………. 2 1.3. Cartilage degradation ……………………………………………………………….. 4 1.4. Mechanical loading and joint health ……………………………………………….. 14 1.5. CITED2 …………………………………………………………………………….. 18

Chapter 2. Hypothesis and Specific Aims ……………………………………………….. 24 Chapter 3. Biomechanical response of CITED2 and role of CITED2 in loading- induced downregulation of MMPs Abstract …………………………………………………………………………….. 27 Introduction …………………………………………………………………………. 29 Methods and Materials ……………………………………………………………… 31 Results ……………………………………………………………………………… 33 Discussion …………………………………………………………………………. 35 Figures ……………………………………………………………………………… 38

Chapter 4. Physiologic Loading Of Joints Prevents Cartilage Degradation Through CITED2 Abstract …………………………………………………………………………….. 42 Introduction …………………………………………………………………………. 43 Methods and Materials ……………………………………………………………… 44 Results ……………………………………………………………………………… 51 Discussion …………………………………………………………………………. 59 Figures ……………………………………………………………………………… 63 Chapter 5. CITED2 is required for cartilage homeostasis: deletion of CITED2 in mice leads to osteoarthritis Abstract …………………………………………………………………………….. 71 Introduction …………………………………………………………………………. 73 Methods and Materials ……………………………………………………………… 74 Results ……………………………………………………………………………… 75 Discussion …………………………………………………………………………. 78 Figures ……………………………………………………………………………… 80

Chapter 6. The Use of CITED2 for Chondroprotection Abstract …………………………………………………………………………….. 85 Introduction …………………………………………………………………………. 86 Methods and Materials ……………………………………………………………… 87 Results ……………………………………………………………………………… 89 Discussion …………………………………………………………………………. 92 Figures ……………………………………………………………………………… 96

Chapter 7. Global Discussion …………………………………………………………….. 101 References ………………………………………………………………………………….. 105

List of Figures Page

Figure 3.1. Schematic of intermittent hydrostatic pressure loading device. Cell 38 culture plates sealed in sterile bags were loaded in a pressurized chamber interfaced to an Instron 8511 servo-hydraulic loading frame.

Figure 3.2. Loading intensity-dependent induction of CITED2 and suppression 38 of MMP expression. (A) mRNA and (B) protein expression of CITED2 and

MMP-2, -3, and -13, 1 hour after intermittent hydrostatic pressure (IHP) loading at 1.0, 2.5, 5.0, and 7.5 MPa, at 1Hz for 1 hour, in C28/I2 chondrocytes. n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.3. Time- and magnitude-dependent effects of IHP loading on CITED2 39 and MMP expression. (A) mRNA and (B) protein expression of CITED2 and

MMP-2, -3, and -13 after 1 hour of IHP loading for 0.25, 0.50, 1.0, 3.0, and 6.0 hours of IHP at 2.5MPa and 1Hz, in C28/I2 chondrocytes. n=5/group.

*=p<0.05 compared to unloaded controls.

Figure 3.4. Post-loading effect of IHP on CITED2 and MMP expression. (A) 39 mRNA and (B) protein expression of CITED2 and MMP-2, -3, and -13 at 0.25,

0.5, 1, 3, 6, 12, and 24 hours after IHP for 3 hours at 2.5MPa and 1Hz. n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.5. CITED2 is required for moderate IHP-induced suppression of MMPs 40

(A) mRNA and (B) protein expression of MMP-2, -3, and -13 in chondrocytes treated with CITED2 siRNA with or without IHP (2.5MPa, 1Hz, 1hr).

n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.6. Overexpression of CITED2 mimics the effect of moderate IHP- 40

induced suppression of MMPs. (A) mRNA and (B) protein expression of MMP-

2, -3, and -13 in chondrocytes transfected with plasmids carrying 0.1 or 1.0 μg

of human CITED2 cDNA, or vector controls. n=5/group. *=p<0.05 compared

to untransfected controls.

Figure 3.7. Cited2+/- mice exhibited a reduced level of Cited2 and elevated levels of 41

MMPs compared to age and gender-matched wild-type littermates. The Effect of moderate treadmill running (10m/min, 1hr, 0° incline) on suppressing expression of

MMP-1, -2, -3, and -13 which was observed in wild-type mice, was diminished in

Cited2+/- mice. n=3/group. *=p<0.05.

Figure 4.1. In vivo motion knee joint motion loading device. The schematic diagram 63

of the in vivo device (left). Experimental setup with a rat undergoing the motion and

loading protocol (right).

Figure 4.2. Passive motion loading prevents cartilage degradation. (A) Schematic 64

representation of the Short Protocol, where the rat hind limb is immobilized for six

hours interrupted by 1-hour of passive motion or release from immobilization under

anesthesia, or the (D) Extended Protocol, where the rat limb is immobilized for 7 days

with or without a 1-hour per day passive motion protocol. (B, E) qPCR showing fold- change in mRNA levels of MMP-1 or (C, F) CITED2, and (G) MMP-1 (enzyme)

activity after immobilization (Im), passive motion loading (Pm), sham treatment (Sh),

or no treatment (Con). (H) Safranin O staining and immunohistochemical localization

ii of MMP-1, type II collagen denaturation (Col2-3/4M antibody), and CITED2 in articular cartilage from rats undergoing the Extended Protocol. Statistics: One way

ANOVA and Tukey’s post hoc test, *p < 0.05 versus control, n=5 rats per group. Scale bar = 100 µm.

Figure 4.3. CITED2 is required for load-induced downregulation of MMP-1. (A, B) 65 qPCR showing the effect of intermittent hydrostatic pressure loads (IHP, MPa as shown, 1 Hz, 1 hour) applied to articular cartilage explants (n=5 rats per group) or to

(C, D) chondrocytes on CITED2 and MMP-1 mRNA expression and (E) CITED2 and

MMP-1 protein expression detected by Western blot. (F, H) qPCR and (G) Western blots showing, respectively, the effect of transfecting chondrocytes with pcDNA3.1-

CITED2 or small interfering RNAs for CITED2 (si-CITED2) or scrambled RNA (si-

Scramble) on MMP-1 mRNA and protein expression in response to IHP (2.5 MPa,

1Hz, 1 hour) in the absence or presence of IL-1β. *p < 0.05, versus control, in triplicate.

Figure 4.4. Direct effects of CITED2 and p300 on MMP-1 promoter transactivation. 66

Chondrocytes were co-transfected with CITED2, Ets-1 and p300 in various combinations (shown) with or without siRNA for p300 (si-p300) or IHP (2.5 MPa, 1

Hz, 1 hour). MMP-1 promoter activity was measured by luciferase assay. *p < 0.05, versus vector transfected cells, in triplicate.

Figure 4.5. CITED2 competes with Ets-1 for p300 binding. (A) Immunoprecipitation 67 of protein lysates from chondrocytes subjected to IHP, CITED2 overexpression or

CITED2 knockdown with anti-p300 antibody and Western blot (WB) with either anti-

iii

CITED2 or anti-Ets-1 antibodies to reveal protein-protein interactions between Ets-1,

CITED2 and p300. (B) qPCR showing the effects of overexpressing p300 or

dominant-negative p300 (p300-DN) on the relative expression of MMP-1, CITED2 and

p300 in response to IHP (2.5 MPa, 1Hz, 1 hour) or (C) co-transfection with wild type

CITED2 without IHP. Mean half-maximal inhibitor concentrations (IC50, nM) for (D)

CITED2 on Ets-1-p300 or (E) Ets-1 on CITED2-p300.

Figure 4.6. CITED2 regulates MMP-1 through specific motif. (A) Effect of 68

transfecting chondrocytes with plasmids encoding either wild-type (WT) CITED2 or

CITED2 deleted, truncated, or point mutants on (B) MMP-1 and CITED2 mRNA

(PCR) and/or protein (Western blot) expression in the presence of IL-1β. Abbrev: CR

– conserved region; ∆srj – CITED2 wild-type with deletion of 39-amino acid serine-

glycine rich junction; EPEE – mutated LPEL (aa243-246) motif. (C) Effect of co-

transfecting wild-type Ets-1 or Ets-1 deleted or truncated mutants with CITED2 on (D)

MMP-1 and CITED2 mRNA (PCR) and/or protein (Western blot) expression in the

presence of IL-1β.

Figure 4.7. MAP Kinase p38δ is the upstream mediator of CITED2 in response to 69

moderate loading. qPCR showing the effect of IHP (MPa as shown, 1 Hz, 1 hour) on

the expression of (A) CITED2 and (B) MMP-1. (C) Western blot showing p38δ and

p38α activation, measured as phosphorylation of the p38 substrate, ATF2. (D)

Luciferase activity in chondrocytes transfected with the CITED2 promoter-reporter construct after application of IHP (2.5 MPa, 1 Hz, 1 hour), or co-transfection with wild- type or dominant negative p38δ or p38α, or (E) with sequential deletion constructs of

CITED2 co-transfected with p38δ. *p < 0.05 versus control, wild-type, or previous deletion construct, in triplicate.

Figure 4.8. CITED2 mimics suppression of MMPs 1, 2, 3, and 13 in chondrocytes in 70 the presence of IL-1β by wild-type CITED2 but not functional domain deleted

CITED2. (A) Transfection of wild-type CITED2 but not EPEE mutant suppressed

MMP gene expression and activity in chondrocytes in the presence of IL-1β. Upper panel: Wild-type and CITED2 mutant constructs. EPEE is a mutation of the LPEL motif found within the 224-256 amino acid region. (B) Western blot showed overexpression of wild-type and mutant CITED2. (C) ChIP analysis with Ets-1 binding sites on MMP promoters shows that increased Ets-1 binding after IL-1β treatment was prevented with CITED2 overexpression. n=3/group. *=p<0.05.

Figure 5.1. Conditional knockout of Cited2 gene in the cartilage of adult mice. 80

Intraperitoneal injections of Tamoxifen (1mg/10g/mouse/day for 5 days) into 6- months-old male and female inducible Cited2 conditional KO mice

(Col2a1CreERTxCited2fl/fl)64,65 completely and specifically knocked out the

Cited2 gene in mouse articular cartilage. (A) Mouse crossbreeding strategy to generate Cited2 conditional KO mice. (B) Validation of exclusive Cited2 knockout at the DNA, mRNA and protein levels 12 weeks after Tamoxifen administration. WT: age and gender matched wild type control mice. KO: Col2a1CreERTxCited2fl/fl mice receiving Tamoxifen dissolved in corn oil; Sham: Col2a1CreERTxCited2fl/fl that received corn oil without Tamoxifen. NC: negative control without sample.

Figure 5.2. Reduced levels of CITED2 are associated with increased OA severity, and 81

increased degradation of cartilage matrix components. (A) Safranin O staining, and

immunohistochemistry for CITED2, cleaved aggrecan (NITEGE) and denatured type II

collagen (Col2 3/4M) in human OA (B) and mouse OA (destabilization of the medial

meniscus, DMM) cartilage, compared to age and gender-matched non-OA controls. n=5/group.

Figure 5.3. CITED2 knockdown by intra-articular gene transfer of CITED2 shRNA 82

for 2 weeks in wild-type mice (male, C57Bl/6, 10-12 weeks) (A) or conditional

knockout of Cited2 (Cited2 cKO) in the articular cartilage for 8 weeks (male, Cited2flf

x Col2al-CreERT, 10-12 weeks) (B) led to early OA, and increased the amount of

cleaved aggreacan and denatured type II collagen in the articular cartilage compared to

sham (A) or wild-type littermates (B). DMM in Cited2 cKO mice increased severity of

OA and increased cleaved aggrecan and denatured type II collagen compared to wild-

type littermates (C). n=5/group.

Figure 5.4. (A) Reductions in CITED2 expression were associated with decreases in 83

chondrocytes expressing MMP-13 and ADAMTS-5 in human and mouse OA cartilage, compared to non-OA controls. (B) Experimental knockdown of Cited2 in mouse

articular cartilage by intra-articular gene transfer of CITED2 shRNA or (C) conditional

knockout led to increases in MMP-13 and ADAMTS-5 compared to non-knockdown

controls. n=6/group.

Figure 5.5. CITED2 represses ADAMTS-5 expression by negatively regulating 84

syndecan-4. (A) Level of syndecan-4 was elevated in CITED2 conditional knockout

mice (cKO) which was associated with elevated levels of ADAMTS-5. (B) CITED2

gene transfer reduced the levels of both syndecan-4 and ADAMTS-5 in DMM

cartilage. Cell counts of syndecan-4, ADAMTS-5, and CITED2 in conditional

knockout mice vs wild-type, and in DMM+vehicle vs. DMM+CITED2. n=5/group.

*=p<0.05.

Figure 6.1. Increased expression of CITED2 by gene transfer exerts chondroprotection 96

in CIA rats. Top: Experimental protocol, transfection efficiency of CITED2 gene

transfer, and expression of CITED2 in CIA rats throughout the experiment. Bottom:

CITED2 gene transfer prevented degradative changes within the articular cartilage in

the CIA model. Gene transfer prevented loss of Safranin O staining, and decreased

expression of MMP-13 and ADAMTS-5. n=8/group.

Figure 6.2. Intra-articular CITED2 gene transfer reduced OA severity, increased 97

CITED2 expression, and prevented damage to aggrecan and type II collagen in the

cartilage of DMM mice after 4 weeks, compared to vehicle-treated controls.

n=6/group.

Figure 6.3. Green tea extract, epigallocatechin-3-gallate (EGCG, 100 µM), induces 98

expression of CITED2 in C28/I2 human chondrocytes. *p < 0.05 versus control, n=3.

Figure 6.4. EGCG administration slows progression in early and mid-stage OA in 98

DMM mice. Safranin O staining and OARSI score of sham- or DMM-operated mice treated with vehicle or EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group). Arrows heads indicate the areas of cartilage fibrillation or erosion, and arrows indicate loss of Safranin O staining.

vii

Figure 6.5. EGCG administration reduced the degradation of type II collagen in the 99

articular cartilage matrix. Immunohistochemical staining of type II collagen cleavage

epitope (Col2 3/4M) and relative staining intensity of the articular cartilage matrix of

sham- or DMM-operated mice treated with vehicle or EGCG at 4 (A, B) weeks

following surgery (*p<0.05, ANOVA, n=6/group, Scale bar=100µM).

Figure 6.6. EGCG administration reduced the degradation of aggrecan in the articular 99

cartilage matrix. Immunohistochemical staining of cleaved aggrecan (NITEGE) and

relative staining intensity in the articular cartilage matrix of sham- or DMM-operated mice treated with vehicle or EGCG at 4 (A, B) weeks following surgery (*p<0.05,

ANOVA, n=6/group, Scale bar=100µM).

Figure 6.7. EGCG administration reduced MMP-13 levels in the articular cartilage. 100

Immunohistochemical staining of MMP-13 and percentage of MMP-13 positive cells in the articular cartilage of sham- or DMM-operated mice treated with vehicle or EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group, Scale bar=100µM).

Figure 6.8. EGCG administration reduced ADAMTS5 levels in the articular cartilage. 100

Immunohistochemical staining of ADAMTS5 and percentage of ADAMTS5 positive

cells in the articular cartilage of sham- or DMM-operated mice treated with vehicle or

EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group, Scale

bar=100µM).

viii

Abbreviations ADAMTS A Disintegrin And Metalloproteinase with Thrombospondin Motifs

CITED2 CBP/p300 Interacting Transactivator with ED-rich tail 2

DMM Destabilization of the medial meniscus

ECM Extracellular matrix

IL-1β interleukin-1 beta

IHP Intermittent hydrostatic pressure

MMP Matrix metalloproteinase

OA Osteoarthritis

PG Proteoglycans qPCR Quantitative real-time PCR

RA Rheumatoid arthritis

Chapter 1. Introduction and Background

1.1 Research Significance

Cartilage degradation is a hallmark of joint diseases such as osteoarthritis (OA) and

rheumatoid arthritis (RA).1 These joint diseases result in joint pain accompanied by varying degrees of functional limitation and reduced quality of life. They affect more than 27 million

Americans, and are one of the leading causes of pain and disability worldwide.2,3 In response to

articular cartilage damage as seen in OA or RA, cartilage exhibits increased cell activity and new

tissue production in an attempt to repair the damage. However, the intrinsic repair is limited and

deregulated. Cartilage degradation is usually a continuous process, leading to erosion of the articular surface and eventual joint failure.2,4

There is currently no cure or effective treatment for arthritis. Treatments thus far mainly

focus on the secondary effects of joint diseases such as pain and joint function, but there are no

effective disease-modifying therapies for cartilage degradation.5,6 Potentially useful

pharmacologic therapies (i.e. matrix metalloproteinase inhibitors) have seen limited clinical

benefit, and severe side effects.7 A better understanding of the pathogenesis of cartilage degradation will be required to achieve significant advances in the development of effective strategies for the prevention and treatment of joint diseases such as OA and RA.

Moderate exercise is the most common non-pharmacologic therapy prescribed to patients with arthritis. The Arthritis Foundation promotes an exercise program involving low-impact

physical activity, and participants have reported less pain and fatigue, and increased strength.8

Clinical trials of patients with OA report physical activities including aerobic exercise,

stretching/flexibility, endurance training, aquatic exercise, and muscle strengthening lead to

improvements in pain relief, body weight, and metabolic abnormalities.9 In addition to the symptom-modifying effects of exercise, there is evidence of exercise exerting disease-modifying effects. For example, increased physical activity in the form of aerobic and weight-bearing

exercises results in increased proteoglycan content, one of the major components of the cartilage

extracellular matrix, in the cartilage of OA patients.10 Strength training for 30 months, compared to range of motion exercises alone, leads to a decreased mean rate of joint space narrowing.11

While the clinical benefits of dynamic moderate exercise are significant, mechanisms underlying the effects of physical activity on the joint are not well understood. Experimental studies in animal models of osteoarthritis and human cartilage tissues ex vivo suggest moderate exercise also leads to increased anti-catabolic, anti-inflammatory, and anabolic activity.2

Dynamic stimulation of cartilage explants increases synthesis of cartilage matrix components

and physiologic loading of animals with experimental osteoarthritis suppresses expression of

inflammatory mediators (e.g. interleukin-1β and tumor necrosis factor-α) and enzymes which directly cleave the articular cartilage including matrix metalloproteinases (MMPs) and A

Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS).2 Focusing on elucidating the mechanisms underlying the chondroprotective effects of physiological joint loading may provide novel insights aimed at maintaining cartilage health.

1.2. Joint Physiology in healthy cartilage

Articular joints consist of two bony ends with cartilaginous end plates sheathed by a soft envelope of synovium. The cartilaginous plates, consisting of chondrocytes and at least seven species of collagen, but mostly Type II, cushion the bony ends during repeated compression and

enable them to slide with minimal friction.12 The metabolic needs of the avascular cartilage are

met by nutrients and waste products diffusing through the synovial fluid and into and out of the

synovium and its blood vessels and lymphatics.13 Fat, collagen, and glycosaminoglycans

constitute the deformable synovial sheath. Synovial lining cells synthesize joint lubricants,

matrix molecules, digestive enzymes, and cytokines.12

Cartilage structure and composition

Articular cartilage lines the ends of bones in synovial joints and provides an almost frictionless surface for motion.14 Cartilage is anueral and avascular and consists of one cell

type: the chondrocyte, which is embedded in an extracellular matrix (ECM) of collagen

molecules and proteoglycans (PGs). Chondrocytes account for approximately 5% of the total

volume of the cartilage, while water constitutes 70-80% of the total tissue weight.15 Collagen

molecules, of which type II is the predominant form, constitute about 75% of the dry tissue

weight. They form a matrix with tensile strength, which allows cartilage to resist swelling.15

Aggrecan, which is the most abundant proteoglycan in cartilage, accounts for approximately

25% of the dry tissue weight.16 Aggrecan monomers attach to hyaluronan to form large aggregating complexes within the cartilage fiber meshwork. These monomers consist of a protein core to which numerous negatively charged glycoaminoglycans side chains attach.17 Due

to this negative charge the PGs confer onto the extracellular matrix, the cartilage becomes

hydrophilic, resulting in a swelling of the tissue which aids in load support and tissue recovery

from deformations.18

The structural properties of cartilage vary with its depth and the cartilage matrix can be

categorized into a superficial, middle, and deep zone. In the superficial zone, collagen fibers are

densely packed and aligned parallel to the articular surface, and chondrocytes appear flattened in

shape. Collagen fibers in the middle zone are randomly oriented and chondrocytes appear larger

and more rounded than in the superficial zone. Collagen fibers in the deep zone are oriented

perpendicular to the bone and chondrocytes are aligned in radial columns.19 Aggrecan content is

lowest in the superficial zone, highest in the middle, and decreases in the deep zone.20 The half-

life of aggrecan core protein ranges from 3 to 24 years. The proteoglycans are essential for

protecting the collagen network, which has a half-life of more than 100 years if not subjected to inappropriate degradation.21 Under homeostatic conditions, chondrocytes do not divide, and are responsible for maintaining the cartilage extracellular matrix through the synthesis of matrix components and remodeling of the matrix.22

1.3. Cartilage degradation

Osteoarthritis

Osteoarthritis (OA) is the most common joint disease, affecting the structural and functional integrity of articular cartilage, as well as the adjacent bone and other joint tissues in an estimated 15% of the U.S. population.3 Clinical symptoms associated with OA include joint pain, stiffness, and swelling, which may lead to impaired physical function.23 OA is generally

diagnosed radiographically by bony changes, including joint space narrowing, development of

osteophytes, subchondral sclerosis, and subchondral cyst formation.24 These clinical and radiographic features are most commonly diagnosed in the knee, hip, and hand joints.23

Although the most evident morphological sign of OA is the progressive destruction of articular cartilage, and OA is classified as non-inflammatory arthritis, synovitis may also play a role in the progression of cartilage degradation. Mononuclear cell infiltration and synovial and 4

subchondral angiogenesis are frequently present in OA joints,25-29 resulting in synovial hypertrophy and hyperplasia with an increased number of lining cells.26,30 Increased numbers of immune cells (e.g. activated B cells and T lymphocytes) in synovial tissue are also observed,31

leading to the induction of cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-

α),29 and the production of proteases which target the cartilage extracellular matrix.30 These

cartilage breakdown products can further provoke the release of degradative enzymes from

synovial cells.32 Therefore, in developing therapeutic strategies against cartilage degradation, the

effect of the adjoining joint tissues should be considered.

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease that produces a progressive

degeneration of the musculoskeletal system. RA is characterized by both joint inflammation and

destruction of bone and articular cartilage.33 One of the most prevalent chronic conditions, RA is found in approximately 1% of the adult population in the United States.34 Disabilities associated

with RA can lead to a restriction of regular daily activity and diminished quality of life.35 Even with appropriate drug therapy, up to 7% of patients remain disabled to some extent 5 years after

disease onset and 50% are too disabled to work 10 years after onset.34 Consequently, RA results

in considerable direct and indirect costs.

Etiology and risk factors of OA

The initiating factors of OA are unknown, and the exact temporal sequence of events

involving changes in an early OA articular joint are not well defined. However, risk factors for

developing OA have been defined, and are broadly divisible into 1) biomechanical risk factors including joint injury, occupational/recreational over usage, reduced muscle strength, joint laxity, 5

joint malalignment, 2) constitutional factors such as ageing, gender and obesity, and 3) genetic

factors. Importantly, several risk factors are reversible (i.e. obesity, muscle weakness) or

avoidable (i.e. recreational joint trauma) which has important implications for prevention.36

Injuries of the knee articular cartilage surfaces are frequently observed in athletes and recreational participants of high-impact sports, including football, basketball, and soccer.37-39

These cartilage injuries are frequently associated with other acute injuries, such as ligament or

meniscal injuries, traumatic patellar dislocations, and osteochondral injuries.40-42 For example,

articular cartilage defects are reported in up to 50% of athletes undergoing anterior cruciate

ligament reconstruction.40,43 Besides acute injury, articular cartilage defects can develop in the high-impact athletic population from chronic pathologic joint-loading patterns, such as joint instability or misalignment.40-42 Repetitive intense joint loading is also observed in individuals

with physically demanding occupations, including construction workers, metal workers, farmers,

and pneumatic drill operators. The consequence of either acute or chronic high-intensity loads is

frequently cartilage degeneration, which may eventually lead to osteoarthritis.44-46

With respect to obesity, the prevalent view has been that the mechanical effect of excess body weight is the direct cause of OA. However, recent evidence suggests the systemic effects of metabolic factors associated with obesity are major contributors to OA pathogenesis. Obesity is associated with an increased amount of adipose tissue, which is recognized as having potent endocrine activity and can give rise to chronic or low-grade inflammation.47,48 Leptin, a hormone produced mainly by adipose tissue, has been suggested to be an important factor in the link between OA and obesity.49 Elevated levels of leptin have been found in the synovial fluid in

the knees of patients with OA, and have also been found to be expressed by OA chondrocytes.49

Increased amounts of fat—with elevated levels of arachidonic acid, a precursor to

proinflammatory prostaglandin E2—have been identified in the bone marrow of patients with

endstage OA.50

Aging is the one of the most influential risk factors for developing OA. While 7.6% of

the 18-44 age group, and 29.8% of the 45-64 age group report doctor-diagnosed arthritis, 50% of

persons aged 65 or older are diagnosed with arthritis.51 Although aging is generally not viewed as the cause of OA, aging events within articular chondrocytes may predispose the joint to damage when exposed to mechanical loads. One of the most prominent age-related changes in chondrocytes is the exhibition of a senescent phenotype,52,53 which is the result of several factors

including the accumulation of reactive oxygen species and advanced glycation endproducts.54-58

Compared to a normal chondrocyte, senescent chondrocytes exhibit an impaired ability to

respond to many mechanical and inflammatory insults to the cartilage matrix.59-63 Furthermore,

protein secretion is altered in aging chondrocytes, demonstrated by a decrease in anabolic

activity and increased production of pro-inflammatory cytokines and matrix degrading

enzymes.64,65 Together, these events may make the articular cartilage matrix more susceptible to

damage and lead to the onset of osteoarthritis.

Etiology of RA

The cause of RA is unknown and the pathogenic process may be initiated by several different events. The initial stages of RA involve the initiation and establishment of

autoimmunity, followed by an inflammatory response, angiogenesis to maintain the chronic

inflammatory state, and tissue degradation of the joint.66 During the inflammatory process, the usually thin layer of synovial lining cells becomes greatly enlarged from the influx of monocytes

and lymphocytes from the circulation and from the local proliferation of fibroblasts, giving rise

to a synovial hypertrophy called a pannus.67 This pannus formation contains activated B and T cells, plasma cells, mast cells, and particularly activated macrophages, which are involved in

feeding back to perpetuate inflammation. Central to this inflammatory response in the

rheumatoid joint are high levels of inflammatory mediators, cytokines, and growth factors,68

including TNF-α, IL-1β, IL-17, and transforming growth factor (TGF)-β.69

New blood vessels form following the inflammatory response, in a process known as

angiogenesis, to maintain the inflammatory state by transporting nutrients to the developing

pannus and inflammatory cells to sites of synovitis.70,71 Angiogenesis is regulated by many inducers and inhibitors, including acidic and basic fibroblast growth factors (bFGF), TGF, angiopoietin, placenta growth factor, and vascular endothelial growth factor (VEGF).72 VEGF, a dimeric glycoprotein which induces formation of new blood vessels and increases vascular permeability,72 is highly expressed in the synovial tissues of RA patients, and serum levels of

VEGF correlate with RA disease activity.73 VEGF knockout mice show reduced RA disease activity and synovial angiogenesis in antigen-induced models of arthritis,74 suggesting that

inhibiting VEGF-mediated angiogenesis is likely to suppress rheumatoid inflammation.

Pathology of OA

Articular cartilage, often considered the central focus of OA, plays an essential role in

joint function and has a limited ability for self-repair. The function of articular cartilage is to

distribute mechanical loads across the entire joint surface and provide a nearly frictionless

surface for joint articulation.75 In OA, the loss of articular cartilage leads to joint dysfunction,

resulting in severe disability and pain. Breakdown of the articular cartilage extracellular matrix

(ECM) involves destruction of its two primary components – type II collagen and proteoglycans.

Degradation of the collagen fiber network and the loss of proteoglycans is one of the early events

which precede the loss of cartilage tissue.75 Due to its avascular nature, articular cartilage exhibits a limited ability for repair, and damage to the cartilage matrix may continue to progress into more advanced lesions, resulting in osteoarthritis.

The most common and generally accepted theory of the pathogenic mechanisms of primary OA involves the cumulative effects of continuous mechanical wear and tear on cartilage.

In the model of biochemical cartilage degeneration, the initiation and progression of primary OA is linked to time/age-related modifications of resident cartilage matrix components, as well as age-dependent changes in the properties of newly synthesized and secreted matrix components, which together culminate in a structurally and functionally inferior cartilage matrix.46 It has been hypothesized that the pathophysiological process of OA can be described by several stages.76 At the beginning, the matrix–network degrades on a molecular level. The water content

increases and the size of matrix molecules, namely aggrecan, decreases. The structure of the

collagen network is damaged, which leads to reduced stiffness of the cartilage. In the second

stage, chondrocytes try to compensate the damage by enhanced proliferation and metabolic activity. Cell clusters surrounded by newly synthesized matrix molecules develop, which is referred to as cloning. This condition can remain for several years. In stage three, the

chondrocytes are not able to keep up their repair activity and a complete loss of the cartilage

tissue is the consequence.77

Pathways of cartilage destruction in OA include cell death, cell activation and abnormal

differentiation of remaining chondrocytes, degradation of the extracellular matrix, and

production of inflammatory mediators.21 While the extracellular matrix of articular cartilage is the primary target of osteoarthritic cartilage degradation, chondrocytes have a pivotal role during osteoarthritis, as they are mainly responsible for the anabolic–catabolic balance required for matrix maintenance and tissue function.78 Chondrocytes may exhibit abnormalities during

osteoarthritic cartilage degeneration, such as inappropriate activation of anabolic and catabolic

activities, and alterations in cell number through processes like proliferation and (apoptotic) cell

death.79 As the disease progresses, osteoarthritic chondrocytes can no longer maintain cartilage matrix integrity.

There are many modes of interaction between the cells and the extracellular matrix. The cellular phenotype of chondrocytes is significantly influenced by the derangement or removal of the pericellular matrix or ECM to which these cells are adapted.80 The cells are affected by

biomechanical stimuli that are transferred through the matrix as well as by the direct modulating

function of specific matrix components. For example, fragments of fibronectin, one of the

degraded matrix components typically found in OA joints, have been reported to induce severe

matrix-degrading effects.81 Chondrocytes are also directly influenced by proteolytic enzymes

and their inhibitors, which diffuse into the cartilage matrix from the surrounding synovial space.

The ability of chondrocytes to maintain matrix homeostasis is severely impaired in osteoarthritic

cartilage, in which the chondrocytes not only fail to compensate for matrix damage induced

externally by factors such as mechanical stress or enzymatic degradation through synovial

proteases, but also have a direct role in the degradation process. Osteoarthritic chondrocytes

activate or upregulate the expression of many matrix-degrading proteases, such as the matrix

metalloproteinases (MMPs), which are largely responsible for the breakdown of the collagenous

and noncollagenous cartilage matrix components.78 The major collagen backbone (type II 10

collagen) is cleaved mainly by collagenase 3 (MMP-13) and further degraded by gelatinases

(MMP-2 and MMP-9) as well as by other enzymes.82 As for the primary enzyme that degrades primarily aggrecan, the major proteoglycan present in cartilage tissue, studies using mouse models suggested that ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) might be the most important aggrecanase enzyme involved in cartilage destruction.83,84

In OA, increased proteolytic activity is not sufficiently counterbalanced by an increase in

chondrocyte anabolic activity. Although there is an overall activation of anabolism in

osteoarthritic chondrocytes, this increase in anabolic activity seems to decline and the anabolic

defense reaction eventually fails.85

Suppressing elevated levels of MMPs and ADAMTS in arthritis should be regarded to

have substantial clinical benefit; however, a safe and effective inhibitor has not yet been

developed. MMP inhibitors used in clinical trials have so far failed to show significant efficacy

in human diseases and in some instances resulted in side effects such as joint swelling and

musculoskeletal pain.86 The side effects of MMP inhibitors were attributed largely to their lack

of selectivity because metalloproteinases share structural similarities.87 Unintended MMP

inhibition has been proven to be problematic, because aside from tissue breakdown, MMPs play

critical roles in development, wound healing, and angiogenesis.88,89 For example, MMP-2

knockout mice exhibited more severe clinical and histological arthritis than wild-type mice in an

antigen-induced arthritis model90 and OA development was accelerated in MMP-3 knockout

mice when compared to wild-type mice in a joint destabilization model.91 An alternative non-

invasive approach to suppressing MMP and ADAMTS activity may include moderate

mechanical joint stimulation. Reports have demonstrated the efficacy of physiologic joint

loading such as passive motion therapy in arthritis.92-94 Furthermore, observations that moderate 11

loading can antagonize inflammatory cytokine-induced MMP upregulation indicate that such a

strategy might be therapeutically useful.92,93

Although chondrocytes can occasionally divide, adult articular cartilage is usually

classified as a post-mitotic tissue with very little cellular turnover.95 Adult cartilage has no external cell supply from the vasculature, nor any sort of germinal cell layer to compensate for any form of cell death or damage that occurs in cartilage degeneration. Consequently, any type of cell death results in serious disruption of tissue homeostasis.96 Morphological analysis of

osteoarthritic cartilage has suggested the presence of empty lacunae in the matrix, which has lead

to the long-standing assumption that cell death is a central feature in OA.97,98 However,

apoptosis is presumably a rather rare event in osteoarthritic cartilage, as only a small fraction of

apoptotic cells (<1%) are present in normal-aged or osteoarthritic cartilage at any given time

point.95 By contrast, chondrocyte death in post-traumatic lesions seems to be a prominent

issue.99,100 Although the extent and mechanism of cell death in OA is not fully understood, it is

hypothesized that because the chondrocytes are either dead or have insufficient ability to control

the anabolic–catabolic balance, tissue homeostasis is not well maintained. The majority of

chondrocytes in osteoarthritic cartilage are viable, albeit, to a large extent, severely

degenerated.78

Current treatment strategies for arthritis

There is currently no cure for OA. The most successful intervention for OA continues to

be total joint arthroplasty. More than 300,000 total knee arthroplasties are performed each year

within the United States, and that number is expected to increase significantly in the future.101

While total joint replacements, to a degree restore, joint motion and function, there are a few

drawbacks. First, articular cartilage is replaced with synthetic materials with a finite lifespan,

necessitating a revision surgery if the patient outlives the functional lifespan of the implants.

Secondly, this surgical procedure may have complications such as infection, deep vein

thrombosis, and pulmonary embolus. Finally, arthroplasty will not return patients to a pre-

arthritic level of function because there are no implants that can replicate the biomechanical

characteristics of the native joint.102 For these reasons, it is important to understand the

molecular events that lead to the development of OA, and identify potential targets to slow or

halt disease progression.

Current non-surgical OA treatment strategies mainly focus on mitigating the symptom of

pain.5 Non-steroidal anti-inflammatory drugs (NSAIDs) are effective medications for the

management of mild/moderate OA, due to their analgesic and anti-inflammatory properties, but

have possible gastrointestinal, renal, and cardiovascular side effects.103 Intra-articular injections

are also used in the clinical management of OA, particularly in joints that are easily accessible

such as the knee, shoulder and ankle joints. These injections are typically corticosteroid based or

hyaluronic acid derivatives and may temporarily relieve pain and improve joint lubrication, but have not been clearly demonstrated to exert disease-modifying chondroprotective effects.103

In fact, there are no disease modifying OA drugs (DMOADs) approved by the Food and

Drug Administration or the European Medicines Agency.7 Due to the role upregulated

MMPs/ADAMTS play in mediating the destructive process in arthritis, inhibitors for these

proteolytic enzymes have been explored as therapeutic strategies to treat OA. However, clinical

trials so far have been met with limited success and resulted in side effects including

musculoskeletal pain and inflammation.104-106 These adverse effects have been mainly attributed

to the lack of selectivity of these inhibitors. Matrix metalloproteinases share structural

similarities and are susceptible to regulation by broad-spectrum inhibitors.87 Poor selectivity is

problematic because in addition to matrix remodeling, MMPs/ADAMTS play important roles in

wound healing, angiogenesis, development, morphogenesis, and bone remodeling.88,89 Recent attempts at developing anti-catabolic drugs have focused on compounds with more selective profiles and are in early phase development.7 Clearly, pharmacologic solutions for treating OA

need to define the correct molecular targets and ensure proper modulation of these targets.

1.4. Mechanical loading and joint health

In addition to pharmacologic treatment of OA, non-pharmacologic therapies such as

aerobic exercise and strength training are commonly prescribed, and have been reported to exert

protective effects on the joint. A Cochrane Review of 32 clinical trials comparing land-based

therapeutic exercise (i.e. muscle strengthening, aerobics, manual therapy) to a non-exercise

group found that exercise treatment resulted in moderate improvements in pain and physical

function.107 Although clinical trials evaluating the effect of exercise on joint structure in OA

patients are limited, preliminary results from these studies are promising. A 4 month exercise

program consisting of aerobic and weight-bearing exercises resulted in increased proteoglycan content in the articular cartilage of OA subjects.10 Strength training, compared to range of motion exercises for 30 months decreased the mean rate of joint space narrowing, but the difference was not statistically significant.11 Together, while the evidence report that moderate

levels of physical activity improve symptoms of OA and may also exert chondroprotective

effects, these mechanisms are not well understood.

Non-physiologic joint loading

Although joints maintain homeostasis within a physiological range of mechanical

loading, both reduced loading and overloading have catabolic effects, particularly for the

cartilaginous components. Damage to the knee articular cartilage surfaces are frequently

observed in athletes and recreational participants of high-impact sports, including football,

basketball, and soccer.37-39 Articular cartilage damage can also develop from chronic pathological joint-loading patterns, such as joint instability or misalignment.40-42 These articular cartilage injuries, as a consequence of either acute or chronic high-intensity loads, will frequently result in cartilage degeneration, which may eventually lead to osteoarthritis.44,45

Experimental studies show that mechanical overloading can directly damage the cartilage extracellular matrix and shift the balance in chondrocytes to favor catabolic activity over anabolism. While direct measurements of in vivo cartilage-on-cartilage contact stresses due to overuse in human joints have not been made, experimental evidence suggests a range of non- physiological loading intensities. There appears to be a critical threshold of 15-20 megapascals

(MPa) for cell death and collagen damage due to a single impact load in bovine cartilage explants.108 In another study on bovine cartilage explants, chondrocyte apoptosis occurred at

peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner,

while degradation of the collagen matrix occurred in the 7 to 12 MPa range,109 suggesting

chondrocyte apoptosis may precede cartilage matrix damage. In addition to peak stress, strain

rate appears to be an important parameter implicated in cartilage damage. Bovine cartilage

explants compressed at a strain rate of 0.01 s-1 to a final strain of 50% showed in no measurable effect on biosynthetic activity or mechanical functionality (compressive and shear stiffness) in chondrocytes, although peak stresses reached 12 MPa,110 a stress high enough to cause injurious

effects such as cell death.109 However, compression at higher strain rates (0.1 and 1 s-1), 15

resulting in peak stresses of about 18 and 23 MPa, reduced total protein biosynthesis and

compressive and shear stiffness,110 suggesting that an increase in peak stress and strain rate is

associated with increased injury. Consistent with these results, high strain rates were reported to

result in significant matrix fluid pressurization and impact-like surface cracking with cell death

near the superficial zone in bovine osteochondral explants.111 Studies also found that repetitive

impact loading of 5 MPa at 0.3Hz induces collagen network damage and chondrocyte necrosis

and apoptosis,112,113 suggesting that impact damage is cumulative. Taken together, the data indicate that high levels of peak stress, high strain rates, and long-term injurious mechanical loading are usually correlated with deleterious effects on cartilage. However, caution must be used when extrapolating these in vitro results to the in vivo situation. Indenters used in these experiments are likely to cause large stress amplitudes and gradients at the location of impact, while in normally congruent articular joints, these stresses may be limited due a more even distribution of force.113 Therefore, the range of non-physiological load intensities in vivo should

be greater than those reported in these in vitro studies.

Similarly to overuse, reduced loading, occurring as a result of spinal cord injuries,114

secondary to treatments for acute musculoskeletal injury,115 or from joint diseases such as

116 arthritis, also results in articular cartilage degradation. In vivo studies demonstrate that prolonged joint immobilization causes degradation of the cartilage matrix, characterized by fibrillation, ulceration, and erosion.117,118 Spinal cord injury patients report articular cartilage

atrophy at a rate greater than that in age-associated osteoarthritis.119 Even during short-term

reduced loading conditions (e.g. 7 weeks of partial weight bearing), a significant degree of

cartilage thinning is reported in all compartments of the knee, although no cartilage lesions are

observed.120 16

Cartilage damage from joint overuse and prolonged disuse is often irreversible due to the

avascular nature of articular cartilage, which prevents a physiological inflammatory response.121-

123 Therefore, spontaneous repair of cartilage is limited, often resulting in biochemical and metabolic changes resembling early osteoarthritis, such as an accumulation of degradative enzymes and cytokines, disruption of collagen ultrastructure, increased hydration, and fissuring of the articular surface.124

Moderate loading of healthy joints

While reduced loading and overloading both cause cartilage degradation, moderate levels of activity maintain normal cartilage integrity. Physiologic mechanical stimulation of the articular cartilage generates biochemical signals which increase the anabolic activity of the chondrocytes.125-127 Numerous in vitro studies have found increases in proteoglycan synthesis,

collagen II expression, and cell proliferation after dynamic stimulation of chondrocytes in

cartilage explants, 3D and monolayer cultures.126-129 Cyclic pressure-induced strain increased

expressions of aggrecan and superficial zone protein in monolayer cultures130,131 and hydrostatic

pressure in bovine cartilage explants loading increased both proteoglycan production and

aggrecan mRNA synthesis.132,133

Mechanical loading and arthritis

It has been shown that moderate exercise may protect against cartilage degradation in

animals that spontaneously develop OA. Hamsters which ran 6-11 km/day maintained normal

cartilage integrity while their sedentary counterparts developed fibrillation, pitting, and fissuring

in the articular cartilage during this 3-month study.134 Normal cage activity rats with a

surgically-induced model of OA showed more macroscopic and histologic cartilage degradation

when compared to exercised OA rats which ran 30 cm/s for 30 minutes for 28 days.135

In humans, moderate recreational physical exercise is associated with a decreased risk of severe knee OA, suggesting that exercise has a protective effect against developing cartilage degradation.136 Furthermore, moderate joint loading may improve joint health and function in

OA.10 Accumulating studies have demonstrated the effectiveness of non-drug treatment

modalities, e.g., exercise and physical activity, as an adjunct to drug therapy in patients with OA and RA.137 Consequently, clinical practice guidelines developed to aid health practitioners in

treating osteoarthritis recommend exercise therapy to reduce pain and improve joint function,

based largely on the results of large randomized controlled trials evaluating exercise.138-142

Clinical studies have shown that in RA, moderate exercise has systemic anti-

inflammatory effects by reducing disease activity.143 In vitro studies have provided evidence for a direct mechanosensitive mechanism on joint tissues. Mechanical strain in synoviocytes decreases expression of prostaglandin-E2 (PGE2),144 an inflammatory mediator, and strain and

shear both decreased the expressions of MMP-1 and -13 in RA synoviocytes.145,146 Furthermore,

chondrocytes exposed to moderate levels of intermittent hydrostatic pressure inhibited IL-1β-

induced matrix degradation.147

1.5. CITED2

CBP/p300 Interacting Transactivator with ED-rich tail 2 (CITED2), a transcriptional co- regulator, has been suggested to play a role in cartilage matrix homeostasis by suppressing proteolytic enzyme expression.148 CITED2 was originally cloned and shown to be inducible by

various stimuli including cytokines, serum growth factors, lipopolysaccharide and hypoxia.149,150 18

CITED2 encodes a 28-kDa nuclear protein, and is present in a variety of cell types.150 During

early development, CITED2 is widely expressed in both embryonic and extraembryonic cells,

and is involved in regulation of cell proliferation and embryonic development.151 Loss of

CITED2 was shown to result in senescence of cultured fibroblasts,152 while studies with

CITED2-/- animals revealed roles in many different developmental processes, including the

establishment of left-right body axis, and development of cardiac, neural, liver and lung

tissues.151,153

As a co-transcriptional regulator, CITED2 does not possess intrinsic DNA binding

activity. Instead, it functions as a context dependent transcriptional co-activator or co-repressor

via interactions with other proteins.149,154 It has been shown that CITED2 acts as a co-activator

transcription factors such as Lhx2, TFAP2, PPARα, PPARγ, and Smad 2 through, at least in part,

the recruitment of CBP/p300, to act as a positive regulator of transcription. On the other hand,

CITED2 can also negatively regulate the activation of target genes by competing with

transcription factors for binding to factors such as p300. Such a mechanism was established in

hypoxia response, where CITED2 downregulated HIF-1α-induced transactivation by competing

with HIF-1α for binding to p300 via the CH1 domain.149

That CITED2 is able to exert a wide range of regulatory roles in numerous cellular processes such as cell proliferation and embryonic development is mainly due to the ubiquity of p300 as a transcriptional co-regulator.149 By binding to p300 via the CH1 domain, CITED2 may

interact, and likely compete, with a multitude of transcription factors including HIF-1α, Ets-1,

NFκB, RXRα, STAT2, MDM2, and p53.155 Since both CITED2 and these other CH1 domain binding proteins are differentially expressed in various tissue and cell types, CITED2 can act in a

tissue and stimulus-dependent manner. Therefore, the regulatory roles played by CITED2 should

be understood through its interaction with these function-associated proteins.

Because CITED2 is ubiquitously expressed and regulated by a wide range of stimuli, its

effects vary considerably depending on the nature of the signal and of the cell type. For example,

in chondrocytes, TGF-β induces CITED2 expression, which is consistent with the known role of

TGF-β in downregulating MMP-1 in those cells.148 However, in MDA-MB-231 breast cancer

cells, TGF-β downregulates CITED2 at the post-translational level.156 Similarly, upregulation of

CITED2 under moderate intensities of mechanical loading in chondrocytes leads to downregulation of MMP-1 and MMP-13, but CITED2 knockdown in SW480 colon cancer cells results in a mild downregulation of MMP-1.157,158

While CITED2 interacts with a wide range of transcriptional regulators, its own expression is responsive to several stimuli. The ability of CITED2 to respond to a multitude of stimuli may be explained by the presence of regulatory elements such as Sp1, STAT, and NF-κB binding sites in the CITED2 promoter.159 The Sp1 transcription factor-binding site has previously been identified to be responsive to shear stress and promote gene expression.160 The deletion and site-directed mutations of the Ets-1 and Sp1 site upstream of the start codon are critical for CITED2 expression in fibroblasts. Gel mobility shift and supershift assays performed with Rat1 nuclear extracts identified nucleoprotein complexes binding to the Ets-1 site and the

Sp1 site. In Drosophila SL2 cells, which lack the Sp and Ets family of transcription factors, expression of Sp1, Sp3, and Ets-1 or Elf-1 functionally stimulated CITED2 promoter activity in a synergistic manner.161

Role of CITED2 in Mechanotransduction and MMP Downregulation

CITED2 was considered a likely mediator of mechanically-induced MMP suppression

since it was known to antagonize transcriptional regulators like Ets-1, which have several

binding sites within MMP promoter regions.162 Therefore, Yokota et al. used an immortalized

human chondrocyte cell line C28/I2163 to investigate whether CITED2 was responsive to

mechanical stimuli, and if so, could mediate shear-mediated downregulation of MMP-1 and

MMP-13.148 CITED2 expression at the mRNA and protein levels was found to be inducible by

moderate flow shear. Expression was maximal at 5 dyn/cm2 and basal levels of expression were

found at 0, 10, and 20 dyn/cm2. In contrast, mRNA and protein expression and enzyme activities

of MMP-1 and MMP-13 were upregulated at 0, 10, and 20 dyn/cm2 but were suppressed at 5

dyn/cm2.148 Consistent with these findings, CITED2 expression was recently found to be

inversely related to the expression of MMPs 2, 3, 9 and 13 in fractured bone in a rat mandibular

osteotomy model. Furthermore, overexpression of CITED2 in osteoblasts inhibited the

expression and activity of MMP-2, -3, -9, and -13.164 Taken together, these data provide evidence that CITED2 is a mechanical stimuli responsive gene, and its inverse relationship with

MMPs suggests it may play a regulatory role in load-driven MMP downregulation.

To determine the relationship between load-induced CITED2 expression and MMP downregulation, loss-of-function and gain-of-function approaches were used. Transfecting

antisense CITED2 plasmids into chondrocytes abolished the loading-mediated downregulation of

MMP-1, suggesting that CITED2 is required by load-driven MMP downregulation.

Overexpression of CITED2 in chondrocytes reduced the basal level of MMP-1 expression under

regular culture conditions, and protected cells from IL-1β-induced upregulation of MMP-1 and

MMP-13.148

To elucidate the mechanism of MMP downregulation by CITED2, co-

immunoprecipitation with p300 specific antibody was used to identify potential regulatory

proteins in p300 binding protein complexes. This approach was based on the understanding that

CITED2 is not a DNA binding protein, and its role in MMP regulation has to be based on its

interaction with DNA binding transcription factors of MMPs, either directly or via their co-

regulators such as CBP/p300. Ets-1 was chosen as a primary target since CITED2 and Ets-1 are

known to interact with p300, and Ets-1 is a DNA binding protein which can transactivate MMPs

with p300 as a co-factor.162 C28/I2 chondrocytes were treated with moderate shear at 5 dyn/cm2

or high intensity shear at 20 dyn/cm2. Nuclear extracts were prepared and incubated with

antibody specific for p300 to immunoprecipitate p300 protein complexes. Western blotting

identified equal amounts of p300 in control and treated extracts. In control cells, no p300-Ets-1

or p300-CITED2 complexes were detectable. In contrast, p300-CITED2 complexes were

identified in cells exposed to flow shear at 5 dyn/cm2 and p300-ETS-1 complexes were detected

in cells under 20 dyn/cm2.148 This study supports the hypothesis that CITED2 may mediate the mechanical loading-induced downregulation of MMP by competing with MMP transactivator

Ets-1 for limiting amounts of co-factor p300 protein.

Ets-1 proteins comprise a highly conserved family of transcription factors that share a unique DNA binding domain, the Ets domain.162 The Ets domain specifically recognizes DNA

sequences that contain a GGAA/T core element.165 Ets-1 is a transcription factor that binds to

MMP-1 and other MMP promoter regions.162 The Ets domain also serves as p300 CH1 binding

domain, recruiting p300 as a co-factor in the transactivation of MMPs. The Ets-1 activation

domain or C-domain, is located between amino acids 130–242 and contains a high content of

acidic residues. It is essential for Ets-1 to activate transcription and is also necessary for the interaction of Ets-1 with p300.162

Chapter 2 – Hypothesis and Specific Aims

Mechanical loading has been recognized as the most important factor for cartilage homeostasis. It contributes critically to maintaining joint tissue homeostasis, especially cartilage integrity, and may cause cartilage degradation depending on the intensity, frequency, and duration of loading and form of mechanical loading (e.g. compression, fluid shear, strain, etc.).

Cartilage homeostasis is maintained by balancing anabolic and catabolic activities. Imbalance between anabolic activity and catabolic activity, especially overactivated catabolic activities in cases such as overloading, post-trauma, or aging, induces high levels of pro-inflammatory cytokines and high levels of proteolytic enzymes such as matrix metalloproteinases (MMPs), a predominant enzyme family that degrades components of the cartilage extracellular matrix. In contrast, dynamic moderate loading not only enhances anabolic activities such as production of collagen and aggrecan, the two major matrix components, but also exerts anti-inflammatory activities. It has also been reported that dynamic moderate loading such as intermittent hydrostatic pressure (IHP) suppresses expression and activity of MMP-1, -3, and -13 in chondrocytes, both in vitro and in vivo. However, how dynamic moderate loading suppresses

MMP expression is largely unknown. Yokota et al. demonstrated that moderate intensities of flow shear (5 dyn/cm2) increased CITED2 mRNA and protein levels, and down-regulated MMP-

1 and MMP-13 in chondrocytes. Antisense CITED2 plasmids prevented the shear-induced down-regulation of MMP expression, while overexpression of CITED2 repressed MMP-1 and

MMP-13 mRNA and protein levels.

These findings led to the hypothesis that CITED2 mediates a mechanical pathway in chondroprotection; appropriate levels of CITED2 are critical for cartilage integrity; and

24 restoration, or increasing levels of CITED2 exerts efficacy in prevention and treatment for cartilage degradation in arthritis.

Objectives: The overall objective is to determine and characterize the role of CITED2 as a mechanical inducible transcriptional regulator, identify the novel CITED2-mediated mechanical sensitive pathway in the downregulation of MMPs, and test the potential of CITED2 as a target for the prevention and treatment of cartilage degradation in arthritis. Accordingly, four specific aims are proposed:

Specifically, in Chapter 3 (Specific Aim 1), we will determine whether CITED2 predominately responds to dynamic moderate loading and represses MMPs that are critical for cartilage integrity in chondrocytes. The study will focus on the gene expression response of

CITED2 to intermittent hydrostatic pressure (IHP), the most relevant mechanical loading modality articular cartilage experiences under various loading parameters (e.g. intensity, duration, post-loading time), and determine the role of CITED2 in mediating the mechanical regulation of MMPs.

In Chapter 4 (Specific Aim 2), we will identify the CITED2-mediated mechanical signaling pathway that is required for the moderate loading-induced downregulation of MMPs.

This study will focus on the role of CITED2 with its co-activator p300 and MMP transactivator

Ets-1 in regulating MMP expression, and the pathway / signaling molecules that mediate loading-induced transactivation of CITED2.

In Chapter 5 (Specific Aim 3), we will determine whether CITED2 is required for cartilage integrity maintenance by determining whether CITED2 is a causal factor for cartilage degradation in human specimens and mouse surgical and genetic models. 25

In Chapter 6 (Specific Aim 4), we will conduct a proof-of-concept study that CITED2 is a target for chondroprotection by examining the efficacy of CITED2 in slowing or preventing cartilage degradation via a) intra-articular gene transfer and b) pharmacologically via small molecule epigallocatechin gallate (EGCG), a compound extracted from green tea that activates

CITED2, using two animal models of cartilage degradation: destabilization of the medial meniscus (DMM) and collagen-induced arthritis (CIA).

Chapter 3 – Biomechanical response of CITED2 and role of CITED2 in loading-induced

downregulation of MMPs

Abstract

Mechanical loading is a critical regulator of cartilage homeostasis. While both reduced loading and overloading lead to cartilage degradation, loading at moderate intensities is required for the maintenance of the cartilage matrix. The molecular mechanisms which mediate these loading responses are not well understood. CITED2 has been previously identified as transcriptional regulator which is induced by moderate intensities of flow shear, and mediates the loading-induced downregulation of MMP-1 and MMP-13. In this study, we examined the

mechanobiologic response of CITED2 to mechanical loading in vitro and in vivo. In vitro, we

determined the effects on chondrocytes of intermittent hydrostatic pressure (IHP), which is

considered the predominant mechanical force chondrocytes experience in vivo. CITED2

expression was induced by moderate intensities of IHP, at a range of 1.0 to 5.0 MPa, and peaked

at 2.5MPa, when assessed at the mRNA and protein levels in chondrocytes after 1hr of loading at

1Hz for 1hr. CITED2 expression was induced with 15 minutes of IHP, peaked after 3 hours of

loading, and started declining after 6 hours of loading. The loading-induced response for

CITED2 was transient; CITED2 levels peaked at 3 hours after loading, was elevated until at least

12 hours, and returned to basal levels 24 hours after loading. IHP loading at the range of 1.0 to

7.5 MPa for 1 hr caused the decrease in multiple MMPs, such as MMP-2, -3, and -13, and was

inversely correlated with the CITED2 response at both the mRNA and protein levels under all

the tested loading regimes. The data suggest a role of CITED2 in mediated the loading-induced

downregulation of MMPs, which was further supported by the following evidence: 1)

27 overexpression of CITED2 in chondrocytes mimicked moderate IHP-induced suppression of

MMPs in the presence or absence of IL-1β; 2) Moderate treadmill running (10m/min, 1hr, 0º incline) suppressed MMP-1, -2, -3, and -13 expression in wild-type mice, but this downregulation was not observed in CITED2+/- mice. The response level of CITED2 may vary depending on the type of load, which may be critical in developing a CITED2 activation-based chondroprotection therapies based on mechanical loading.

Introduction

Mechanical loading is considered to be a critical regulator of skeletal homeostasis.2,166,167

Mechanical loading induces the complex interaction of musculoskeletal tissues including bone,

articular cartilage, and ligaments/tendons. These mechanical stimuli can be categorized as

beneficial for tissue development and maintenance, or detrimental, which can result in tissue

breakdown and cause joint diseases such as osteoarthritis (OA).

Articular cartilage functions as a nearly frictionless bearing surface while uniformly transferring loads on underlying bone preventing high stress concentrations. Cartilage consists of one cell type, the chondrocyte, embedded in an extracellular matrix of mainly type II collagen and proteoglycans. Cartilage responds to mechanical loading in an intensity-dependent manner.

Acute or chronic high-intensity loads, as seen in athletes participating in high-impact sports such as soccer, football, and basketball, often predispose the joint to OA.40-42 Joint

injuries, such as anterior cruciate ligament (ACL) tears, similarly increase contact forces in the

knee, and result in degradative changes such as chondral softening and fracture.168,169 Reduced joint loading also creates catabolic responses within articular cartilage.114,115 Animal models of reduced loading report that a decrease in mechanical stimuli leads to atrophy of the tissue,170-173

and ultimately erosion of the articular cartilage.117,118 In humans, reduced loading in the form of partial weight bearing for 7 weeks results in significant cartilage thinning in the knee articular cartilage120 and spinal cord injury patients exhibit a rate of cartilage atrophy greater than that

reported in age-associated osteoarthritis.119

While reduced loading and overloading both cause cartilage degradation, moderate levels of activity maintain cartilage homeostasis. Moderate loading of the articular cartilage generates mechanical signals that increase the synthetic activities of the chondrocyte while suppressing its 29

catabolic actions.125-127 In humans, exercise at moderate intensities is associated with a decreased risk of severe knee OA, suggesting that physical activity has a protective effect against developing cartilage degradation.136 However, the mechanisms underlying the beneficial effects

of mechanical loading on cartilage integrity are not well understood.

Cartilage degradation, or breakdown of the cartilage extracellular matrix (ECM), involves

a variety of degradative enzymes, many of which are matrix metalloproteinases (MMPs).86,174

The human genome has 24 MMP genes, including two duplicated MMP-23 genes. The MMP

family consists of the collagenases, (MMP-1, -8, and -13) which degrade collagens types I, II,

and III, the gelatinases (MMP-2 and -9), which target denatured collagen, the stromelysins

(MMP-3, -7, -10, and -11), which degrade several ECM proteins and are involved in proenzyme

post-translational activation.

Our previous work has implicated CBP/p300-interacting transactivator with ED-rich tail

2 (CITED2), as a mechanosensitive regulator of MMP-1 and -13.148 CITED2, a transcriptional

regulator, is required for the suppression of MMP-1 and MMP-13 in response to moderate shear.

In this study, we hypothesize that CITED2 mediates a chondroprotective signaling pathway in

which moderate mechanical loading downregulates production of several MMPs in response to

intermittent hydrostatic pressure, a loading stimulus used to mimic the chondrocyte mechanical

environment.175 We will determine the loading duration and intensity-dependent nature of

CITED2 and MMPs expression and the role of CITED2 in the mechanoregulation of MMPs

through loss- and gain-of-function strategies

Materials And Methods

Intermittent hydrostatic pressure (IHP) loading

C28/I2 cells, a human chondrocytic cell line,163 were cultured as high-density monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum,

100 μg/ml ascorbic acid, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C.158 Cells were starved in serum-free DMEM 18 hours prior to intermittent hydrostatic pressure (IHP) loading in a custom-made device.158 Culture plates were placed in 37ºC water bath in a stainless steel loading vessel within sterile, heat-sealed bags containing culture medium. The loading vessel was maintained within a temperature controlled chamber set at 37ºC. Pressure was generated within the pressure vessel interfaced with an Instron 8511 servo-hydraulic loading frame (Fig 3.1). Air was evacuated from the system so the application of pressure was purely hydrostatic. Control cultures were maintained under identical conditions in heat-sealed bags in a

37ºC water bath not subject to hydrostatic pressure. After loading, cells were lysed for total

RNA or protein isolation, for quantitative PCR or Western blot, respectively.

Transfection of siRNA and plasmids

To knockdown CITED2, C28/I2 cells were transfected with CITED2 siRNA (Santa Cruz, sc-35959) or a scrambled control (Santa Cruz, sc-37007) 24 hours prior to IHP. To overexpress

CITED2, C28/I2 cells were transfected with pcDNA3.1 plasmids carrying human CITED2 cDNA as described.158

RNA isolation and quantitative PCR

C28/I2 cells were extracted using the RNeasy mini kit (Qiagen, Valencia, CA) with

DNase treatment according to manufacturer’s instructions. RNA was quantified with a

Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), then reverse

transcribed (RT) using oligo(dT) primers. Two nanograms of total RNA were analyzed by real-

time quantitative PCR with SYBR Green (BioRad) to assess relative gene expression using the 2-

ΔΔCt method.176 Target gene expression was quantitated relative to housekeeping gene GAPDH.

Gene (Accession number) Primer Sequence

CITED2 (NM_001168388.2) F: 5’-GCGAAGCTGGGGAATAACAA-3’ R: 5’-ATGGTCTGCCATTTCCAGTCT-3’ MMP-1 (NM_001145938.1) F: 5’-TCTGGAAGGGCAAGGACTCT-3’ R: 5’-TCCGCTTTTCAACTTGCCTTT-3’ MMP-2 (NM_001127891.1) F: 5’-GAGTGCATGAACCAACCAGC-3’ R: 5’-GGGCAGCCATAGAAGGTGTT-3’ MMP-3 (NM_002422.3) F: 5’-TGTTCGTTTTCTCCTGCCTGT-3’ R: 5’-CAGCAGCCCATTTGAATGCC-3’ MMP-13 (NM_002427.3) F: 5’-GCACTTCCCACAGTGCCTAT-3’ R: 5’-AGTTCTTCCCTTGATGGCCG-3’ GAPDH (NM_001256799.2) F: 5’-AATGGGCAGCCGTTAGGAAA-3’ R: 5’-GCGCCCAATACGACCAAATC-3’

Western Blot

Western blot was performed by using antibodies specific for MMP-1 (Abcam, ab52631),

MMP-2 (Abcam, ab86607), MMP-3 (Abcam, ab18898), MMP-13 (Abcam, ab39012), CITED2

(Santa Cruz, sc-21795) and Actin (ab3280). Approximately 10 µg of the extracted proteins were

separated by electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel, and the separated

proteins were transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA).

Membranes were incubated with primary antibodies followed by incubation with secondary

antibodies conjugated to horseradish peroxidase (ECL Western blotting analysis system, GE Life

Sciences, Piscataway, NJ). A mouse antibody specific for β-actin (Sigma-Aldrich, St. Louis,

MO) was used as loading control.

Treadmill running

Cited2+/- carry one allele of Cited2, with the other allele excised and replaced with a neo

cassette.177 These mice were received as a gift from Dr. Yu-Chung Yang. Cited2+/- mice and

wild-type littermates (male, 6 months old) were subjected to 1 hour of treadmill running (1 hour,

10m/min, 0° incline) or regular cage activity. Three hours after this loading regimen, the

articular cartilage from the knee was harvested and flash frozen for RNA isolation. Gene

expression for Cited2, MMPs-1, -2, -3, and -13 were assessed by real-time PCR with GAPDH

used as a housekeeping control.

Statistical Analysis

Results are expressed as mean ± SD. Statistical analysis was carried out using a one-way

ANOVA and Tukey’s test for post hoc analysis with significance set at P < 0.05.

Results

Response of CITED2 and MMPs to IHP loading

IHP induced biphasic changes in the expression of CITED2 and MMP-2, -3 and -13 in

C28/I2 chondrocytes (Fig 3.2A). CITED2 expression by qPCR increased up to 7-fold at 2.5

MPa, returning to basal levels at 7.5 MPa. In contrast, expression of MMP-2, -3, and -13

exhibited biphasic decreases in expression over the same range of IHP. MMP-2 and -13 were

suppressed most strongly at 2.5 MPa IHP and returned to basal levels at 7.5 MPa, while MMP-3

was less sensitive. Maximal inhibition of MMP-3 was seen at 5.0 MPa, and expression remained

significantly below control levels at the highest pressure tested (7.5 MPa). Corresponding

elevations in CITED2 and reductions in MMPs were also seen at the protein level by Western blotting (Fig. 3.2B). Expression of all MMPs and CITED2 were inversely correlated, with correlation coefficients of -0.73, -0.53, and -0.85 for MMP-2, -3 and -13, respectively.

CITED2 mRNA upregulation in response to continued exposure to 2.5 MPa IHP was

rapid; 60% of maximal stimulation was observed after 15 minutes. A plateau of expression

(values not significantly different from the 3 hour maximum value) was achieved by 1 hour and

was sustained through 6 hours, although there appeared to be a trend of declining expression at

the latest time point (Fig 3.3A, 3.3B). Downregulation of MMP-2, -3, and -13 occurred over the

same time interval, although significant reductions were in expression were not seen until 30

minutes of IHP exposure, i.e. following the upregulation of CITED2 (Fig 3.3A, 3.3B). A single

3 hour exposure to IHP induced changes in CITED2 and MMP expression that persisted for up to

12 hr or more, as assessed by real-time PCR and Western blot, respectively (Figs 3.4A and

3.4B).

CITED2 mediated loading-induced suppression of MMPs

The inverse time and dose relationships between CITED2 and MMP expression in

response to IHP suggested that the suppression of MMP expression by moderate loading may

result through the actions of CITED2. To test this possibility, we first suppressed CITED2

expression in C28/I2 chondrocytes with siRNA and found that MMP expression in the absence

of IHP was increased. Moreover, moderate IHP loading (2.5 MPa, 1 hour) failed to suppress 34

MMP expression in chondrocytes treated with si-CITED2 (Fig 3.5A). Suppression of MMPs in

the absence of mechanical load was also dependent on the extent of CITED2 overexpression (Fig

3.6A, B).

Loading-induced MMP suppression reduced in Cited2+/- mice

To determine whether the mechanical effect of moderate loading (treadmill running)

would similarly affect expression of MMPs in vivo, and to test whether CITED2 mediates these

loading-induced effects, wild-type and Cited2+/- mice were subjected to moderate treadmill

running for 1 hour or allowed regular cage activity. Without loading, Cited2+/- mice exhibited

50% less Cited2 expression and elevated MMP expression (Fig 3.7). One hour after this

treadmill running, Cited2 gene expression was elevated and MMP expression was reduced in

wild-type mice, but to a lesser extent in Cited2+/- mice (Fig 3.7).

Discussion

Presently, both resistance and gentle exercises are prescribed for rehabilitation of injured

or arthritic knees. However, the optimal duration and physical loading necessary for achieving

the beneficial effects of exercise are unclear. Defining a physical activity as “moderate or physiological” is a challenge because loading regimes involve many parameters such as intensity, frequency and duration, and a “moderate” physical activity for a particular individual might vary depending on an individual’s age, genetics, gender, and physical activity history.

Therefore, identifying biomarkers associated with moderate loading may provide useful indicators for choosing the right type exercise/physical activity with a precise loading regime and therefore increase efficacy of mechanical-based therapies for cartilage protection and treatment. 35

We examined the response of CITED2, a transcriptional regulator induced by mechanical shear

which exerts an anti-catabolic role by repressing MMP-1 and -13 expression and activity.148 In

this study, we showed CITED2 was upregulated in response to moderate intensities of IHP,

which was directly related to the downregulation of MMP-2, -3, and -13. This suggests there is a

moderate range of loading which is chondroprotective. As CITED2 only responded to moderate

mechanical loading, it is possible CITED2 expression may be utilized as a biomarker to define

which physical activities may be considered “physiologic.” We also examined the effect of

loading duration, and post-loading time in response to moderate IHP. The induction of CITED2

and suppression of MMP expression plateaued after 1 hour of loading. Furthermore, the up-

regulation of CITED2 was sustained until at least 12 hours after loading. Together, this suggests,

with regards to chondroprotection, that 1 hour of moderate physical activity once or twice a day

may be sufficient to maintain an anti-catabolic environment within the articular cartilage.

While chondrocytes in vivo experience many types of mechanical forces, such as shear, strain, and compression, we focused on intermittent hydrostatic pressure loading because it is the predominant force chondrocytes experience in vivo.178 The fact that CITED2 is responsive to

multiple forms of mechanical loading supports the concept that CITED2 is a mechanically

responsive regulator.

In addition to the role CITED2 played in suppressing MMP-1 and -13 expressions, this

study revealed CITED2 is also a transcriptional regulator for MMP-2, and -3. MMP expression

was reduced but not completely suppressed in response to moderate mechanical loading. This

form of MMP modulation may represent a more physiologic method of regulating MMP

36 expression in arthritis, in contrast to complete MMP suppression induced by MMP inhibitors tested in clinical trials.

The overexpression of CITED2 mimicked the anti-catabolic effects of moderate mechanical loading on MMP suppression, suggesting CITED2 may be a molecular target for chondroprotection to suppress excess catabolic activity in arthritis. The activation of CITED2, by mechanical means may be a therapeutic strategy to slow or arrest cartilage degradation.

However, the mechanisms through which mechanical loads transactivate CITED2 are not understood and require further elucidation.

Figures

Fig 3.1. Schematic of intermittent hydrostatic pressure loading device. Cell culture plates sealed in sterile bags were loaded in a pressurized chamber interfaced to an Instron 8511 servo- hydraulic loading frame.

Figure 3.2. Loading intensity-dependent induction of CITED2 and suppression of MMP expression. (A) mRNA and (B) protein expression of CITED2 and MMP-2, -3, and -13, 1 hour

38 after intermittent hydrostatic pressure (IHP) loading at 1.0, 2.5, 5.0, and 7.5 MPa, at 1Hz for 1 hour, in C28/I2 chondrocytes. n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.3. Time-dependent effects of IHP loading on CITED2 and MMP expression. (A) mRNA and (B) protein expression of CITED2 and MMP-2, -3, and -13 after 1 hour of IHP loading for 0.25, 0.50, 1.0, 3.0, and 6.0 hours of IHP at 2.5MPa and 1Hz, in C28/I2 chondrocytes. n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.4. Post-loading effect of IHP on CITED2 and MMP expression. (A) mRNA and (B)

protein expression of CITED2 and MMP-2, -3, and -13 at 0.25, 0.5, 1, 3, 6, 12, and 24 hours

after IHP for 3 hours at 2.5MPa and 1Hz. n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.5. CITED2 is required for moderate IHP-induced suppression of MMPs (A) mRNA

and (B) protein expression of MMP-2, -3, and -13 in chondrocytes treated with CITED2 siRNA

with or without IHP (2.5MPa, 1Hz, 1hr). n=5/group. *=p<0.05 compared to unloaded controls.

Figure 3.6. Overexpression of CITED2 mimics the effect of moderate IHP-induced suppression of MMPs. (A) mRNA and (B) protein expression of MMP-2, -3, and -13 in chondrocytes

transfected with plasmids carrying 0.1 or 1.0 μg of human CITED2 cDNA, or vector controls.

n=5/group. *=p<0.05 compared to untransfected controls.

Figure 3.7. Cited2+/- mice exhibited a reduced level of Cited2 and elevated levels of MMPs compared to age and gender-matched wild-type littermates. Effect of moderate treadmill running

(10m/min, 1hr, 0° incline) on suppressing expression of MMP-1, -2, -3, and -13, which was observed in wild-type mice, was diminished in Cited2+/- mice. n=3/group. *=p<0.05.

Chapter 4 - Physiologic Loading Of Joints Prevents

Cartilage Degradation Through CITED2

Abstract

Both overuse and disuse of joints upregulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiologic) loading maintains cartilage integrity. Here we test whether CITED2, a mechanosensitive transcriptional co-regulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo,

hindlimb immobilization of Sprague-Dawley rats upregulates MMP-1 and causes rapid,

histologically detectable articular cartilage degradation. One hour of daily passive joint motion

prevents these changes and upregulates articular cartilage CITED2. In vitro, moderate (2.5 MPa,

1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and

upregulates CITED2 in human chondrocytes whereas high IHP (10 MPa) downregulates

CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that

CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its co-

activator p300. Furthermore, CITED2 upregulation in vitro requires the p38δ isoform, which is

specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory

pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular

cartilage integrity under normal physical activity levels.

Introduction

Mechanical loading is one of the most important environmental factors responsible for

joint homeostasis and there is tremendous concern about how joint motion and load bearing may

impact joint health. Articular cartilage maintains its integrity throughout life under moderate

loading conditions experienced during routine daily activity. In contrast, when cartilage is

subjected either to excessive or insufficient mechanical loading, degeneration occurs. Exposure

of joints to acute and chronic (repetitive) high-intensity loading eventually leads to osteoarthritis

(OA).46,179 On the other hand, the absence of normal mechanical stimulation, as occurs in

paralysis following spinal cord injury, can also cause articular cartilage breakdown through the

induction of a similar catabolic response.171,180 To date, we have begun to define many of the

cellular and molecular processes responsible for these catabolic changes.21 However, the

mechanism through which moderate loads prevent cartilage degradation and maintain functional

capacity remains unknown.

Articular cartilage destruction results from the breakdown of extracellular matrix (ECM)

constituents, primarily type II collagen and aggrecan, whose proteolysis are mediated by matrix

metalloproteinases (MMPs) and a disintegrin-metallo proteinases with thrombo spondin motifs

(ADAMTS), respectively. MMPs comprise a large family of structurally related endopeptidases

and are considered to be the principal mediators of both normal ECM remodeling and pathologic

degradation.181 Of the 26 MMPs identified, MMP 1, 8, and 13 represent the secreted neutral

proteinases capable of cleaving type II collagen.181 MMPs 1 and 13 are highly expressed in the cartilage and synovium of OA and rheumatoid arthritis patients,182-185 though MMP-8 is not

appreciably elevated in OA.186 Other MMPs including MMPs 2, 3, and 9 have also been reported

to play a role in the degradation of cartilage ECM.21,187

MMP expression and function are controlled, both positively and negatively, at multiple

levels including transcription and enzyme activity (e.g. via inactivation by specific inhibitors like

tissue inhibitors of metalloproteinases, or TIMPs).86 Upregulation of MMPs in response to

mechanical stimuli has been documented in a wide range of tissues and cells,188 via multiple

signal transduction pathways often involving members of MAP kinase families, e.g. p38 and

p42/44,189 but much less is known about the downregulation of MMP expression by mechanical loading. In a previous study, we showed that moderate levels of flow shear downregulated expression of MMP-1 and MMP-13 in human C28/I2 chondrocytes, and that this downregulation

coincided with upregulation of the transcriptional regulator CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2),148 suggesting that CITED2 may contribute to MMP regulation

in chondrocytes. In the present study we sought to define the role of CITED2 in the regulation

of MMPs by focusing on MMP-1, a prototypical catabolic enzyme that cleaves type II collagen

and is highly expressed in arthritic articular cartilage.183,190 We then determined whether

CITED2 regulates other MMPs such as MMPs 2, 3, 13, through a similar mechanism as with

MMP-1 using a strategy involving mutagenesis of the CITED2 functional motif and a ChIP

assay.

Materials And Methods

Animal Experiments

Male Sprague-Dawley rats (5-6 months old, 580 ± 35 g) were housed with access to food

and water ad libitum. All procedures were approved by the IACUC of Mount Sinai School of

Medicine. The right hind limbs were immobilized as previously described.191 Briefly, rats were anesthetized with isoflurane and were fitted with a cast made of steel mesh and cotton materials which fixed the knee in full flexion (115°). Animals were either left immobilized for the duration of the experiment, released from casting for 1h under anesthesia (sham) or subjected to motion loading at a frequency of 2 cycles/minute with a range of motion between 65° and 115° using a custom-designed loading device.192 The in vivo loading device consisted of an animal bed and an anesthesia machine integrated into a custom-built knee joint motion/load apparatus, a

linear actuator with micro-stepping driver and digital encoder, a magnetic goniometer for real-

time joint angle readout, a load cell for compressive load monitoring, a USB analog/digital I/O

interface module, and LabView control panel (Fig 4.1). The anesthetized animal was placed

prone over an acrylic bed, and the right hind limb of the animal was placed in the knee joint

apparatus. The knee joint apparatus transformed the linear motion provided by the actuator into

angular displacement at the knee joint. The loading device had adjustable control of motion

frequency and range of angular displacement via a linear actuator with 50 mm travel stroke

(bipolar NEMA 13 Hybrid, Haydon Motion, Waterbury, CT) coupled to an incremental rotary

encoder (E5S, US Digital Corp, Vancouver, WA). After motion loading or release from

immobilization under anesthesia (sham group), the rats were sacrificed or re-cast until the next

motion loading session. The short and extended protocols are described in the ‘Results’ section.

In each protocol, a control group was allowed free cage activity throughout the experiment.

Following sacrifice, cartilage from immobilized knee joints (distal femur, proximal tibia) was

rapidly removed and prepared for MMP activity assays, histology or RNA isolation.

MMP activity assay

Articular cartilage was dissected, flash-frozen in liquid nitrogen and stored at -80°C.

MMP-1 activity was assayed by using the Senosolyte Plus 520 MMP-1 assay kit (Anaspec,

Fremont, CA). Frozen tissue samples were pulverized (Dismembrator, B Braun Biotech,

Germany) in MMP activity assay buffer (Anaspec) containing 0.1% (vol/vol) Triton-X 100, and

then centrifuged for 15 minutes at 10,000 g at 4°C. The supernatant was added to a 96-well plate

containing immobilized MMP-1 antibody in the presence of 4-aminophenylmercuric acetate

(APMA) for 3 hours, and its proteolytic activity was measured by the 5-FAM/QXL 520 FRET

peptide (Anaspec). The fluorescence of 5-FAM (fluorophore) was quenched by QXL 520 in the

intact FRET peptide. Fluorescence signal was monitored at Ex/Em=490 nm/520 nm upon

MMP-1 induced cleavage of the QXL FRET substrate. Fluorescent intensity was measured

using the SpectraMax M5 spectrofluorometer (Molecular Devices, Sunnyvale, CA).

Immunohistochemistry and Safranin O staining

Total knee joints were fixed in formalin, decalcified, paraffin-embedded and sectioned.

For immunostaining, endogenous peroxidase activity was blocked with 3% (vol/vol) H2O2 for 5 minutes. Tissues were incubated overnight at 4°C with either anti-CITED2 (1:300, Santa Cruz,

Santa Cruz, CA), anti-MMP-1 (1:100, Abcam, Cambridge, MA), or Col2-3/4M (1:200, Ibex

Pharmaceuticals, Canada), followed by a 30 minute incubation with anti-mouse or anti-rabbit

secondary antibody (Dako, Carpinteria, CA) and visualization with DAB chromagen for 3

minutes. Negative control sections were prepared using irrelevant isotype-matched antibodies in

place of the primary antibody. Safranin O-fast green staining was carried out to demonstrate

glycosaminoglycans in the articular cartilage.

Cartilage explants, cell culture, and intermittent hydrostatic pressure loading

Articular cartilage from the rat distal femur and proximal tibia were carefully separated from the underlying bone under a dissecting microscope. The cartilage explants and C28/I2 cells

(cultured as high-density monolayers) were each cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/ml ascorbic acid, 100

U/ml penicillin, and 100 μg/ml streptomycin at 37°C.193 Both the rat articular cartilage explants

and C28/I2 cells were starved in serum-free DMEM 18 hours prior to intermittent hydrostatic

pressure (IHP) loading in a custom-made device.194 After loading, cartilage explants pulverized in liquid nitrogen and C28/I2 cells were lysed for total RNA or protein isolation, for quantitative

PCR or Western blot, respectively.

Transfection of siRNA and plasmids

C28/I2 cells were incubated with siRNA for CITED2 (Santa Cruz) or transfected with

pcDNA3.1 plasmids constructed to encode cDNA sequences for wild-type (WT) human

CITED2, Ets-1, or mutant forms of CITED2 and Ets-1 with selected sequences deleted using

restriction enzyme digestion and PCR cloning strategies as described.148 In other studies, WT or

mutated human CITED2 cDNA were co-transfected with either WT p300 or a dominant negative

(DN) p300 form in which the CH1 domain was deleted to prevent CITED2-binding (Upstate

Biotechnology, Lake Placid, NY).

RNA isolation and quantitative PCR

Total RNA from flash frozen articular cartilage or C28/I2 cells was extracted using the

RNeasy mini kit (Qiagen, Valencia, CA) with DNase treatment according to manufacturer’s

instructions. RNA was quantified with a Nanodrop spectrophotometer (Thermo Fisher

Scientific, Wilmington, DE), then reverse transcribed (RT) using oligo(dT) primers. Two

nanograms of total RNA were analyzed by quantitative PCR with SYBR Green to assess relative gene expression, as well as GAPDH and β-actin as housekeepers. PCR primers pairs used were: rat-MMP-1A, forward 5’- ACAGTTTCCCCGTGTTTCAG-3’, reverse 5’-

CCCACACCTAGGTTTCCTCA-3’; human-MMP1, forward 5’-

GGTGACACCAGTGACTGCAC-3’, reverse 5’-TCCACAAATGGTGGGTACAA-3’; rat/human-CITED2, forward 5’- TGCCGCCCAATGTCATAG-3’, reverse 5’-

CTGCTGTTTGCACACGAAG-3’; rat/human-GAPDH, forward 5’-

GAGGACCAGGTTGTCTCCTG-3’, reverse 5’-ATGTAGGCCATGAGGTCCAC-3’; rat/human-β-actin, forward 5’-TTGCTGACAGGATGCAGAAG-3’, reverse 5’-

ACATCTGCTGGAAGGTGGAC-3’. Expression values of GAPDH and ß-actin for each treatment condition were averaged and used as a denominator to determine the relative expression of target genes using the 2-ΔΔCt method.

Western Blot/Immunoprecipitation Assay

Western blot was performed by using antibodies specific for MMP-1 (EMD Chemicals,

Gibbstown, NJ), CITED2 (Santa Cruz). Ets-1 (Santa Cruz), and p300 (Upstate Biotechnology).

Approximately 10 µg of the extracted proteins were separated by electrophoresis (SDS-PAGE)

in a 10% polyacrylamide gel, and the separated proteins were transferred to Immun-Blot PVDF

membranes (Bio-Rad, Hercules, CA). Membranes were incubated with primary antibodies

followed by incubation with secondary antibodies conjugated to horseradish peroxidase (ECL

Western blotting analysis system, GE Life Sciences, Piscataway, NJ). A mouse antibody specific for β-actin (Sigma-Aldrich, St. Louis, MO) was used as loading control.

For immunoprecipitation, 100 µg of nuclear extracts were mixed with 8 µg of p300- specific antibody (Upstate Biotechnology) and incubated with gentle rocking at 4°C overnight.

The immunocomplexes were captured by mixing with protein G-agarose beads for 2 h at 4°C.

The collected agarose beads were washed four times with an ice-cold cell lysis buffer, and the precipitated proteins were eluted by boiling the beads for 5 min in the SDS sample buffer.

Fractions of the supernatant were analyzed by SDS-PAGE and immunoblotting using antibodies against p300, CITED2, and Ets-1.

Promoter Analysis and Luciferase Assay

A 510 bp region from the MMP-1 promoter (-468 to +42 relative to the transcription start site, GenBank M16567) was cloned by amplifying human genomic DNA by PCR and subcloned into the pGL3-Basic Vector (Promega, Madison, WI) immediately upstream of the luciferase reporter gene. Wild-type and serial deletions of the 5′ end of the CITED2 promoter region were generated in separate reactions and subcloned into pGL3-basic vector. For the

MMP-1 promoter assay, chondrocytes were transfected or co-transfected with MMP-1 promoter reporter construct, pcDNACITED2, pCMVp300 (Upstate Biotechnology), pKD-p300-v1 (p300 siRNA), p300 siRNA scramble, and empty vector controls, and/or subjected to IHP loading (2.5

MPa, 1 Hz, 1 hr). For the CITED2 transactivation assay, the cells were transfected or co- transfected with a CITED2 promoter reporter construct, pCMVp38δ WT, pCMVp38δ DN, pCMVp38α WT, pCMVp38α DN (Cell Biolabs Inc., San Diego, CA), p38δsiRNA (Ambion,

Austin, TX), and/or subjected to IHP loading (2.5 MPa, 1 Hz, 1 hr). For the CITED2 promoter

serial deletion analysis, wild type and deleted CITED2 promoter reporters were co-transfected

with pCMVp38δ WT into chondrocytes. DNA constructs were transfected into cells using

Lipofectamine Plus Reagent (Life Technologies, Carlsbad, CA). For luciferase assay, cells were

harvested in Passive Lysis Buffer (Promega). Luciferase activity in the cell lysates was

determined using the Dual-Luciferase Reporter Assay System (Promega). All firefly luciferase

values were normalized for transfection efficiency using the pRL-TK, Renilla-luciferase value.

Peptide Competition Assay

Ets-1 displacement from GST-p300 CH1 (aa302-423) domain by the synthetic CITED2 transactivation domain (TAD) peptide (aa224-255), and CITED2 TAD displacement from the

GST-p300 CH1 domain by Ets-1 TAD peptide (aa153-210) were measured by an ELISA-based peptide competition assay, as previously described.195 Briefly, increasing amounts of CITED2 or

Ets-1 TAD peptide were added to immobilized biotinylated Ets-1 peptide or biotinylated

CITED2 TAD peptide, respectively. A constant amount of GST-p300 CH1 was then added.

Bound GST-p300 CH1 was quantified by time-resolved fluorescence using europium-labeled anti-GST antibody (Perkin Elmer, Waltham, MA). Mean half-maximal inhibitor concentration

(IC50) values were calculated for twenty-five separate experiments after fitting data using a non-

linear regression (GraphPad Prism, La Jolla, CA).

Kinase Assay

The phosphorylation of p38 isoforms were conducted using a nonisotopic p38 MAPK

assay kit (Cell Signaling, Danvers, MA) as previously described.196 Briefly, total cell lysates

were prepared from chondrocytes and equal amounts of total protein (100 μg) were precipitated

using p38α or p38δ MAPK antibody, respectively. The activities of precipitated kinases were 50

then allowed to phosphorylate the p38 substrate, ATF2, in the presence of ATP.

Phosphorylation of ATF2 was analyzed by immunoblotting with an antibody specific for

Phospho-ATF2. The expression of p38α or p38δ was confirmed by immunoblotting with the

isoform-specific antibody.

Chromatin immunoprecipitation (ChIP)

C28/I2 human chondrocytes were treated with IL-1β (10ng/ml) to induce MMP

expression, with or without CITED2 transfection. ChIP was performed using a commercial kit

following manufacturer’s instructions (USB). Cell lystes were immunopreciptated with anti-Ets-

1 (Santa Cruz), and PCR performed using primers specific for Ets-1 binding sites (GGAA/T) in

MMP promoter regions.

Statistical Analysis

Results are expressed as mean ± SD. Statistical analysis was carried out using a one-way

ANOVA and Tukey’s test for post hoc analysis with significance set at P < 0.05.

Results

CITED2 Induction in Articular Cartilage in vivo is Associated With MMP-1 Downregulation

and Anti-catabolic Effects of Joint Motion

To test whether CITED2 mediates the protective effects of physiologic joint motion on cartilage, we first determined the relationship between of CITED2 and MMP-1 expression under different mechanical loading regimens. Two protocols were used. In the first “short” protocol, four groups of rats were treated as follows: (1) no immobilization and no passive motion (Con); 51

(2) continuous immobilization for 6 hours (Im); (3) immobilization for 6 hours interrupted by 1- hour passive motion (Pm); and (4) immobilization for 6 hours interrupted by a 1-hour release from the cast under anesthesia, with no passive motion (Sh) (Fig. 4.2A).

Quantitative PCR (qPCR) analysis of articular cartilage samples dissected immediately after the immobilization period revealed that MMP-1 was upregulated in the immobilization- only group (Fig. 4.2B). The upregulation of MMP-1 was accompanied by a decline in CITED2 mRNA (Fig. 4.2C). In contrast, passive motion loading applied for 1 hour in the midst of the 6- hour immobilization period completely reversed these changes (Figs. 4.2B and 4.2C).

Importantly, simply removing the immobilization stimulus for 1 hour without accompanying passive motion (sham group), did not reverse the immobilization-induced upregulation of MMP-

We next extended the immobilization protocol to 7 days and inserted a 1-hour passive motion loading period each day (“extended” protocol) (Fig. 4.2D). qPCR analysis (Figs 4.2E and 4.2F) revealed that immobilization stimulated MMP-1 while attenuating CITED2 expression, but both of these changes were prevented upon passive motion loading. We also measured the activity of MMP-1 and found that it mirrored MMP-1 mRNA levels under all conditions examined (Fig. 4.2G).

Histological examination of distal femoral articular cartilage revealed evidence of tissue degradation in immobilized rats, and also indicated that these changes were largely prevented by passive motion loading (Fig. 4.2H). In particular, immobilization led to decreased Safranin O staining (indicating reduced proteoglycan content) in the superficial zone of articular cartilage; this decrease was accompanied by increases in the number of MMP-1-positive cells, stronger

staining for denatured type II collagen in the superficial and middle zones of the articular

cartilage, and reduction in the number of cells with positive staining for CITED2. By contrast,

passive motion loading of immobilized limbs prevented loss of proteoglycans, limited the

increases in immunohistochemically detectable MMP-1 and denatured type II collagen, and also

increased the number of cells staining positive for CITED2 (Fig. 4.2H, middle and bottom

panels). Taken together, these results indicate that in vivo moderate loading suppresses the

catabolic response to immobilization by inducing CITED2 and downregulating MMP-1.

CITED2 is Required for Moderate Loading-induced Downregulation of MMP-1

In order to assess the regulatory role of CITED2 in MMP-1 expression in vitro and ex

vivo, intermittent hydrostatic pressure (IHP), which is thought to best mimic the loading environment experienced by articular cartilage in vivo 178, was utilized to mechanically stimulate freshly recovered rat cartilage explants or cultured chondrocytes. As shown in Figures 4.3A and

4.3B, the application of 2.5 and 5.0 MPa, pressures consistent with those experienced by joints during walking,185 caused a profound increase in CITED2 and a decrease in MMP-1 mRNA

expression in rat articular cartilage explants. Parallel in vitro studies with chondrocytes showed

qualitatively similar results to those with cartilage explants, namely elevation in CITED2 mRNA

and reduction in MMP-1 mRNA seen best at 2.5 MPa (Figs. 4.3C and 4.3D), as well as

corresponding changes in protein expression on Western blotting (Fig. 4.3E).

To determine whether CITED2 was indeed required for MMP-1 downregulation in

response to mechanical stimuli, we used CITED2 loss and gain of function strategies. A modest,

but significant, increase in MMP-1 mRNA was noted when CITED2 was suppressed by siRNA

knockdown in the basal state (Fig. 4.3F). As expected, loading lowered MMP-1 mRNA, but

53 importantly, failed to do so in si-CITED2 transfected cells (Fig. 4.3F). Western blotting showed an effective reduction of CITED2 protein in the si-CITED2 transfectants, and also demonstrated their resistance to load-induced MMP-1 downregulation (Fig. 4.3G). Consistently, moderate loading, as well as CITED2 overexpression, also dramatically inhibited the expression of MMP-

1 induced by IL-1β, a key mediator of cartilage degradation in both OA and rheumatoid arthritis

197 (Fig. 4.3H). This load-induced suppression of MMP-1 was again abolished in si-CITED2 transfected cells (Fig. 4.3H). These experiments provide clear evidence that, at least in vitro, the upregulation of CITED2 is required for the load-induced attenuation of MMP-1.

To further demonstrate the critical involvement of CITED2 in the moderate loading- induced downregulation of MMP-1, modulation of MMP-1 at the transcriptional level by

CITED2 was examined by transactivation assays. CITED2 is a transcriptional regulator that does not bind directly to DNA promoter regions, but functions by interacting with other transcription factors or cofactors such as p300.198 For this reason, chondrocytes were co- transfected with a 2.3 kb MMP-1 promoter-luciferase construct and either empty vector or constructs containing CITED2, p300, or si-p300, and the promoter activity was measured using a luciferase assay. In certain conditions, cells were subjected to moderate loading at 2.5 MPa, 1

Hz, for 1 hr. As shown in Figure 4.4, cells transfected with empty vector or si-scramble displayed potent luciferase activity (in agreement with the MMP-1 mRNA results in Fig. 4.3), and the activity level was further stimulated by p300 overexpression. In contrast, the MMP-1 transactivation was substantially reduced in all groups of cells overexpressing CITED2, and the suppressive effect of CITED2 was comparable to those with either loading (2.5 MPa) or p300 knockdown (Fig. 4.4). These findings indicate that MMP-1 transcription is directly mediated by

CITED2 and p300. 54

CITED2 Downregulates MMP-1 by Competing with Ets-1 for p300 Binding

We next took further steps to dissect the detailed molecular events surrounding the

CITED2 regulation of MMP-1 expression. The transcriptional co-regulator p300 is the principal binding partner of both CITED2 and MMP-1 transactivator Ets-1.149,195,199,200 We speculated that

CITED2 may function to downregulate MMP-1 by competing with Ets-1 for p300 binding. To

test this hypothesis, we characterized the p300 complexes in cells with altered expression of

CITED2 by immunoprecipitation assays. As shown in Figure 4.5A, the formation of CITED2-

p300 complexes due to either loading (which upregulated CITED2) or CITED2 overexpression

was accompanied by complete displacement of Ets-1 from its p300 binding site. Conversely,

suppression of CITED2 by transfection with si-CITED2 enhanced Ets-1 binding to p300 (Fig.

4.5A). These results support our premise that the p300-CITED2 complex formed upon loading

is responsible for the effects of CITED2 on MMP-1 expression.

The direct competition between CITED2 and Ets-1 for binding to p300 was supported by

two additional observations. First, an examination of a dominant-negative (DN) p300 lacking

the CH1 domain that binds to CITED2 and Ets-1 indicated that p300 was absolutely required for

the regulation of MMP-1 by CITED2 or Ets-1. As expected, loading at 2.5 MPa reduced MMP-

1 and increased CITED2 expression without affecting p300, both in untransfected and vector-

transfected chondrocytes. In contrast, overexpression of wild-type p300, which led to increased

complex formation between p300 and Ets-1, blocked the load-induced attenuation of MMP-1

without altering CITED2 levels (Fig. 4.5B). However, p300-DN was unable to correct the load-

induced downregulation of MMP-1. Overexpression of CITED2 under these conditions

mimicked the effects of loading (Fig. 4.5C). Second, bound GST-p300 CH1 quantified by time-

resolved fluorescence measurements of a Europium-labeled anti-GST antibody in ELISA-based

competition assays revealed a subtle difference between the half-maximal inhibitory (IC50)

concentrations for reduction of the Ets-1-p300 complex by CITED2 (0.58 nM) and of the

CITED2-p300 complex by Ets-1 (0.90 nM) (Figs. 4.5D and 4.5E). This difference suggests a

potential chondroprotective mechanism that p300, under physiological conditions, may exhibit a

slight preference for binding to its partner, CITED2, over its catabolic co-regulator Ets-1.

To identify specific regions within the binding domains of CITED2 and Ets-1 required

for MMP-1 regulation, we carried out a series of transfection experiments using deletions and

point mutations introduced into each molecule (Fig. 4.6A). In the first set of experiments, we

treated chondrocytes transfected with wild-type or mutant CITED2 with IL-1β and examined

MMP-1 mRNA and protein expression by PCR and Western blotting, respectively. We found

that the CITED2 fragment containing amino acids (aa) 224 to 256 was both necessary and

sufficient for MMP-1 downregulation, whereas a CITED2 fragment lacking this region (i.e.

fragment 1-223) did not (Fig. 4.6B). More specifically, EPEE, a mutation of the LPEL motif

found within the 224-256 amino acid region, completely abolished the downregulation of MMP-

1 expression, suggesting that LPEL was critical for load-induced MMP-1 regulation.

Co-transfection of chondrocytes with wild-type CITED2 and mutated forms of Ets-1

(Fig. 4.6C) showed that overexpression of CITED2 suppressed IL-1β-induced expression of

MMP-1 and that overexpression of wild-type Ets-1 partially reversed the MMP-1 suppression

caused by CITED2. However, this rescue was abolished in the transfectants overexpressing Ets-

1 fragments in which the transactivation domain (TAD, aa 135-243) was removed either by

truncation beyond aa 153 and 165, respectively, or through its deletion, as in the fragments ∆166-

194 and ∆178-210 (Fig. 4.6D). Most importantly, however, the fragment 153-210 fully reversed the effects of CITED2 overexpression, suggesting that the p300-binding domain of Ets-1, which competes with CITED2, is indeed the critical component of the TAD.

p38δ Activation Mediates the Loading-induced Transactivation of CITED2

Finally, we determined the upstream signal(s) necessary for moderate mechanical loads

to induce CITED2 expression. Amongst several known candidates that act as potential

mechanotransducers, the MAP kinases, particularly p38, have received much attention.201,202

However, the roles played by individual p38 isoforms (p38α, p38β, p38γ, p38δ) in cartilage

mechanotransduction have not been investigated. Moderate loads of 2.5 MPa, which upregulate

CITED2 and downregulate MMP-1 (Figs. 4.7A and 4.7B), did not stimulate phosphorylation of

p38α but specifically stimulated the phosphorylation of a p38δ (Fig. 4.7C). In contrast,

chondrocytes loaded at 10 MPa results in the phosphorylation of p38α (Fig. 4.7C), which

coincides with the upregulation of MMP-1 (Fig. 4.7B), a change that typifies a catabolic

response to non-physiological loads. We therefore explored how p38δ regulates CITED2 expression in response to moderate loading by examining the transactivation of the 3.3 kb

CITED2 promoter. Promoter-reporter assays using luciferase constructs transfected into chondrocytes confirmed the stimulation of CITED2 transactivation by both loading and p38δ

overexpression, proving that p38δ is upstream of CITED2 and downstream of loading (Fig.

4.7D). Furthermore, this response was abolished by dominant-negative p38δ and si-p38δ.

Analysis of CITED2 promoter responsiveness to p38δ by means of progressive deletion

constructs showed that removal of the NF-κB-binding site did not appreciably reduce promoter transactivation (Fig. 4.7E). Deletion of the HIF-1α response element in the (-940) CITED2 57 promoter region attenuated transactivation by ~50% (Fig. 4.7E). However, while removal of one

Sp1 site in the -2300-bp CITED2 fragment produced no change in transactivation, the subsequent deletion of three further Sp1 sites almost completely abolished promoter responsiveness to p38δ (Fig. 4.7E). Together, this data suggests that the effect of p38δ on

CITED2 is, at least in part, mediated by HIF-1α and Sp1.

Competing with Ets-1 for p300 is a common mechanism for CITED2 regulation of MMPs 1, 2,

3, and 13

Our results suggest CITED2 may regulate MMP expression by competing with Ets-1 for p300 binding. As MMP-2, -3, and -13 also carry Ets-1 binding sites on their promoter, we tested that CITED2 regulates multiple MMPs through this Ets-1-p300 mechanism. Towards this end, we constructed and transfected the CITED2 motif (LPEL) which binds to p300 and a mutant motif unable to bind to p300 (EPEE), in chondrocytes (Fig 4.8A). Overexpression of the mutated CITED2 motif, in contrast to the wild-type motif, was unable to downregulate MMP expression and activity when overexpressed in C28I2 cells, suggesting that CITED2 suppression of MMP expression is mediated through p300 binding (Fig 4.8B). To provide further evidence and the precise regulatory mechanism that CITED2 binding to p300 prevents Ets-1 mediated transcription of MMPs, ChIP assays were performed using an antibody against Ets-1. IL-1β, which stimulates MMP expression in C28/I2 chondrocytes, increased binding of Ets-1 to at least one putative Ets-1 binding site on the promoter regions of MMPs 1, 2, 3, and 13 (Fig 4.8C).

However, these protein-DNA bindings were significantly decreased when CITED2 was overexpressed in IL-1β treated cells (Fig 4.8C). These data indicate that CITED2 downregulates

the transactivation of MMPs 2, 3, and 13 by preventing Ets-1 from forming productive transcriptional complexes with p300, similar to that observed with MMP-1.

Discussion

The idea that musculoskeletal tissue integrity is best maintained at moderate

(physiologic) levels of mechanical loading has long been appreciated. In muscle and bone, for

example, repair of minor tissue damage from overuse entails breakdown and removal of the

damaged tissue and replacement with newly formed matrix.203,204 On the other hand, tissue breakdown also occurs as a result of loading insufficiency, resulting in muscle atrophy205 and osteopenia or osteoporosis.206 While an understanding of the molecular mechanisms that

transduce mechanical stimuli into increases in bone and muscle mass is emerging, less is known

of how mechanical stimuli generated by joint movement, and the frequency and intensity of such

loads, help to maintain cartilage integrity. Accumulating clinical evidence indicates that, in contrast to excessive or inadequate loads, continuous passive motion along with other forms of physiological joint loading is necessary for proper joint maintenance, and joint motion can protect cartilage from cartilage degradation 92,93,147. In this study, we show for the first time that physiologically relevant passive loading of normal joints prevents cartilage degradation through

the upregulation of CITED2 using the p38δ pathway.

Matrix metalloproteinases (MMPs) are proteolytic enzymes responsible for the destruction of articular cartilage. This study focused on a single MMP (MMP-1) and on its transcriptional control by CITED2. MMP-1 was selected for this study as a prototypical catabolic enzyme because it directly cleaves type II collagen and is highly expressed in arthritic articular 59 cartilage.183,190 Moreover, since MMP-1 has only been identified recently in mice and rats,207,208 the role of MMP-1 is less understood in rodent models. Using a mutagenesis strategy of the

CITED2 p300 binding domain and ChIP assay, we further demonstrated that the CITED2 p300 binding ability is required and sufficient to suppress multiple MMPs including MMPs 1, 2, 3, and 13 by preventing Ets-1 binding to MMP promoter sites to trigger transcription of MMPs, and therefore repressing their expression.

Our study provides several lines of evidence that CITED2 is essential in order to mediate the transcriptional suppression of MMP-1 by moderate hydrostatic pressure loading, and elucidate mechanistic features of this novel pathway. 1) Analysis of CITED2 loss and gain of function revealed that under moderate levels of intermittent hydrostatic pressure, si-CITED2 abolished the reduction in MMP-1 expression and forced overexpression of CITED2 mimicked the effects of moderate loading by downregulating MMP-1. 2) CITED2 competed with a MMP transactivator, Ets-1, and displaced it from the p300-Ets-1 complex to reduce MMP-1 transactivation. 3) The CITED2 nucleotide region encoding the fragment between amino acids

224 to 270 was absolutely required and sufficient to downregulate MMP-1. 4) CITED2 suppressed MMP-1 promoter activation in a process requiring p300. Taken together, these observations strongly support our hypothesis that CITED2 mediates a chondroprotective signaling pathway by competing with Ets-1 for p300 binding to downregulate MMP-1 expression. That our results in vivo and in vitro were in excellent agreement with respect to the inverse relationship between expression of CITED2 and MMP-1 argue strongly that the mechanistic relationships we established in vitro are likely to hold in vivo.

The proposed mechanism of MMP-1 regulation by CITED2, involving competition with

Ets-1 for binding to p300, was consistent with expectations based on published binding

characteristics of CITED2 and HIF-1α for p300.195 Interestingly, however, the competition binding studies indicated a slightly greater affinity of p300 for CITED2 than for Ets-1. To the extent that these relationships translate to in vivo circumstances, the result suggests that this potential regulatory switching mechanism may be slightly biased toward suppression, rather than activation, of MMP transcription. Such a bias might be useful to prevent overaggressive catabolic responses to changes in the mechanical environment.

While our results demonstrated direct transcriptional regulation of MMP-1 by CITED2 in chondrocytes, it is likely that additional regulatory mechanisms may also operate, particularly in vivo. Virtually all cell types present in joint tissues (cartilage, bone, synovium) are mechanoresponsive.148,209-211 Furthermore, mechanical stimulation is known to alter the production of, as well as response to, cytokines known to regulate MMP expression, including

IL-1β and TNF-α 92,93,147.

Another novel finding of this study was that the mechanosensitive upregulation of

CITED2, which occurred in response to moderate levels of mechanical loading in vitro and in

vivo, was associated with the selective activation of a specific isoform of p38, p38δ. Members of

the p38 MAP kinase family are activated in response to mechanical stresses and a wide range of

chemical stimuli,202,212 and have been implicated in the upregulation of MMPs.213 We have

observed that high levels of loading led to neither phosphorylation of p38δ nor CITED2

upregulation. This may partially explain why CITED2 is functional only under moderate loading

with regards to the regulation of MMP expression. Conversely, excessive loading induced

MMP-1 and phosphorylation of p38α, suggesting that the latter is likely involved in the

regulation of MMP-1. Together, these data indicate that distinct members of the p38 family can act within the same cell population to promote and to suppress MMP-based matrix breakdown

depending on the mechanical loading environment.

In summary, we have identified a novel CITED2-mediated pathway in which CITED2,

induced by moderate loads through the activation of p38δ, suppresses MMP-1 transcription by

displacing transactivator Ets-1 from co-activator p300. The identification of this CITED2

pathway as a means to regulate MMP expression in cartilage not only provides a molecular basis

for the prevention of cartilage degradation by physiologic loads, but also offers the possibility

that this pathway could be exploited therapeutically in diseases like arthritis.

Figures

Figure 4.1. In vivo motion knee joint motion loading device. The schematic diagram of the in vivo device (left). Experimental setup with a rat undergoing the motion and loading protocol

(right).

Figure 4.2. Passive motion loading prevents cartilage degradation. (A) Schematic

representation of the Short Protocol, where the rat hind limb is immobilized for six hours

interrupted by 1-hour of passive motion or release from immobilization under anesthesia, or the

(D) Extended Protocol, where the rat limb is immobilized for 7 days with or without a 1-hour per

day passive motion protocol. (B, E) qPCR showing fold-change in mRNA levels of MMP-1 or

(C, F) CITED2, and (G) MMP-1 (enzyme) activity after immobilization (Im), passive motion

loading (Pm), sham treatment (Sh), or no treatment (Con). (H) Safranin O staining and

immunohistochemical localization of MMP-1, type II collagen denaturation (Col2-3/4M

antibody), and CITED2 in articular cartilage from rats undergoing the Extended Protocol. 64

Statistics: One way ANOVA and Tukey’s post hoc test, *p < 0.05 versus control, n=5 rats per

group. Scale bar = 100 µm.

Figure 4.3. CITED2 is required for load-induced downregulation of MMP-1. (A, B) qPCR showing the effect of intermittent hydrostatic pressure loads (IHP, MPa as shown, 1 Hz, 1 hour) applied to articular cartilage explants (n=5 rats per group) or to (C, D) chondrocytes on CITED2 and MMP-1 mRNA expression and (E) CITED2 and MMP-1 protein expression detected by

Western blot. (F, H) qPCR and (G) Western blots showing, respectively, the effect of transfecting chondrocytes with pcDNA3.1-CITED2 or small interfering RNAs for CITED2 (si-

CITED2) or scrambled RNA (si-Scramble) on MMP-1 mRNA and protein expression in response to IHP (2.5 MPa, 1Hz, 1 hour) in the absence or presence of IL-1β. *p < 0.05, versus control, in triplicate.

Figure 4.4. Direct effects of CITED2 and p300 on MMP-1 promoter transactivation. Effect of co-transfecting chondrocytes with CITED2, Ets-1 or p300 in various combinations (shown)

with/without siRNA for p300 (si-p300) or IHP (2.5 MPa, 1 Hz, 1 hour) on MMP-1 promoter

activity in luciferase assays. *p < 0.05, versus vector transfected cells, in triplicate.

Figure 4.5. CITED2 competes with Ets-1 for p300 binding. (A) Immunoprecipitation with anti- p300 antibody and Western blot (WB) with either anti-CITED2 or anti-Ets-1 antibodies to reveal protein-protein interactions between Ets-1, CITED2 and p300. (B) qPCR showing the effects of overexpressing p300 or dominant-negative p300 (p300-DN) on the relative expression of MMP-

1, CITED2 and p300 in response to IHP (2.5 MPa, 1Hz, 1 hour) or (C) co-transfection with wild type CITED2 without IHP. Mean half-maximal inhibitor concentrations (IC50, nM) for (D)

CITED2 on Ets-1-p300 or (E) Ets-1 on CITED2-p300.

Figure 4.6. CITED2 regulates MMP-1 through specific motif. (A) Effect of transfecting chondrocytes with plasmids encoding either wild-type (WT) CITED2 or CITED2 deleted, truncated, or point mutants on (B) MMP-1 and CITED2 mRNA (PCR) and/or protein (Western blot) expression in the presence of IL-1β. Abbrev: CR – conserved region; ∆srj – CITED2 wild- type with deletion of 39-amino acid serine-glycine rich junction; EPEE – mutated LPEL (aa243-

246) motif. (C) Effect of co-transfecting wild-type Ets-1 or Ets-1 deleted or truncated mutants with CITED2 on (D) MMP-1 and CITED2 mRNA (PCR) and/or protein (Western blot) expression in the presence of IL-1β.

Figure 4.7. MAP Kinase p38δ is the upstream mediator of CITED2 in response to moderate loading. (A) qPCR showing the effect of IHP (MPa as shown, 1 Hz, 1 hour) on the expression of

CITED2 and (B) MMP-1. (C) Western blot showing p38δ and p38α activation, measured as phosphorylation of the p38 substrate, ATF2. (D) Luciferase activity in chondrocytes transfected with the CITED2 promoter-reporter construct after application of IHP (2.5 MPa, 1 Hz, 1 hour), or co-transfection with wild-type or dominant negative p38δ or p38α, or (E) with sequential deletion constructs of CITED2 co-transfected with p38δ. *p < 0.05 versus control, wild-type, or previous deletion construct, in triplicate.

Figure 4.8. CITED2 mimics suppression of MMPs 1, 2, 3, and 13 in chondrocytes in the presence of IL-1β by wild-type CITED2 but not functional domain deleted CITED2. (A)

Transfection of wild-type CITED2 but not EPEE mutant suppressed MMP gene expression and

activity in chondrocytes in the presence of IL-1β. Upper panel: Wild-type and CITED2 mutant

constructs. EPEE is a mutation of the LPEL motif found within the 224-256 amino acid region.

(B) Western blot showed overexpression of wild-type and mutant CITED2. (C) ChIP analysis

with Ets-1 binding sites on MMP promoters shows that increased Ets-1 binding after IL-1β

treatment was prevented with CITED2 overexpression. n=3/group. *=p<0.05.

Chapter 5 – CITED2 is required for cartilage homeostasis: deletion of CITED2 in mice

leads to osteoarthritis

Abstract

Joint diseases such as osteoarthritis (OA) are a leading cause of adult disability and characterized by progressive cartilage destruction and damage to the integrity of bone and other joint tissues. The pathogenesis of OA is not well understood, nor is there an effective treatment to cure or even slow its progression. CITED2 (Cbp/p300 Interacting Transactivator with ED rich tail 2), is a transcriptional regulator which plays critical roles in suppressing expression of enzymes which directly cleave the cartilage extracellular matrix, such as matrix metalloproteinases (MMPs-1, -13). In previous chapters, we demonstrated that CITED2 is a mediator required for the loading-induced downregulation of MMPs. CITED2 downregulated

MMP expression by competing with MMP transactivator Ets-1 for binding to co-factor p300.

Reduction of CITED2 expression was associated with immobilization-caused cartilage degradation, suggesting a critical role for CITED2 in maintaining cartilage integrity and homeostasis. In this study, we tested whether deficiency of CITED2 is a casual factor for cartilage degradation and arthritic diseases. We found that reductions of CITED2 expression were associated with cartilage degradation in human and mouse OA cartilage, compared to age- and gender-matched non-OA controls. Experimental knockdown of CITED2 in adult mice, by intra-articular gene transfer of CITED2 shRNA, or by genetic deletion in an inducible, cartilage- specific knockout mouse model, led to early OA-like cartilage pathology. Furthermore, the knockdown of CITED2 accelerated the progression of post-traumatic OA in the destabilization of the medial meniscus (DMM) mouse model, compared to wild-type DMM mice. We find that

CITED2 plays its role in maintaining cartilage integrity, at least in part, by modulating

expression of MMP-13 and ADAMTS5, which are considered the predominant proteolytic enzymes in OA pathogenesis. Restoring CITED2 expression in OA tissues may be a therapeutic strategy for arresting or slowing OA progression.

Introduction

Previous studies have demonstrated that CITED2 is a mechanically responsive transcriptional regulator required to mediate cellular responses to mechanical loading that protect against cartilage degradation.148,158 Of notice, while dynamic moderate loading induced CITED2 expression and suppressed MMPs, immobilization (reduced loading) resulted in cartilage degradation and was associated with reduced levels of CITED2. Furthermore, knockdown of

CITED2 in vitro, abolished the loading-induced MMP downregulation, and caused an elevation of MMP expression above its basal level. These studies allow us to ask the following questions:

Do reduced levels of CITED2 a) correlate with severity of established OA and b) increase susceptibility of developing OA under given circumstances? What are the mechanisms through which CITED2 mediates chondroprotection?

To address these questions, we first investigated whether there was an association between CITED2 expression and the extent of cartilage degradation characterized by OARSI score in human OA cartilage. We then evaluated then expression level of CITED2 during the initiation and progression of OA in post-traumatic OA mice. We further examined whether knockdown of CITED2 in cartilage can cause OA-like pathology. We examined the effect of specifically deleting CITED2 in adult mice, and examined the deficiency of CITED2 involved in

OA development in post-traumatic mice using the DMM model.

Methods

Human OA samples - Human OA specimens were collected from patients undergoing total knee

or hip replacement surgery with written consent in accordance to an IRB-approved protocol

(n=8, avg. age=70±7.3 yrs). Age- and gender-matched non-OA cartilage was obtained from

post-mortem patients without history of joint diseases. Cartilage was dissected, fixed in 10%

formalin, and embedded in paraffin for sectioning.

Induction of osteoarthritis in mice - Destabilization of the medial meniscus (DMM) in 5-6

month-old male C57BL/6 mice was carried out by transecting the medial meniscotibial ligament

(MMTL), (n=6). A 3 mm longitudinal incision over the distal patella to proximal tibial plateau.

The joint capsule immediately medial to the patellar tendon was incised with a # 15 blade and

the joint capsule opened with micro-iris scissors. Blunt dissection of the fat pad over the

intercondylar area was then performed to expose either the intercondylar region, providing

visualization of or the meniscotibial ligament of the medial meniscus. Mild hemorrhage from the

fat pad upon blunt dissection was controlled by pressure from absorption spears. The medial

meniscotibial ligament (MMTL) anchors the medial meniscus (MM) to the tibial plateau. The fat

pad over the cranial horn of the medial meniscus was dissected with Jewelers forceps. The

MMTL was identified running from the cranial horn of the medial meniscus laterally onto the

anterior tibial plateau. Sectioning of medial meniscotibial ligament was performed with a # 11 blade, gave destabilization of the medial meniscus (DMM). Control joints involved a sham surgery in which the ligament was visualized but not transected. The joint capsule was closed

with a continuous 8-0 tapered Vicryl suture and the subcutaneous layer with 7-0 cutting Vicryl.

The skin was closed by the application of tissue adhesive.

CITED2 suppression in vivo - Intra-articular injections of CITED2 shRNA (10 mg/10 uL PBS)

were performed followed by electroporation every 7 days in a separate set of 8- to 10-wks-old male C57BL/6 mice. Contralateral controls received injections of PBS only followed by electroporation (n=3).

Cited2 conditional knockout mice - Col2a1-Cre/ERT x Cited2flf mice were generated by crossing

Cited2-floxed mice with Col2a1-Cre/ERT mice 214,215. Genotyping confirms mice with Cre and

two Cited2-floxed alleles (Figure 1). Daily intraperitoneal injection of tamoxifen

(1mg/10g/mouse/day for 5 days) in skeletally mature, 5-6 month-old male mice resulted in

knockdown of the Cited2 gene in articular chondrocytes when assessed at 8 weeks.

Immunohistochemistry and Safranin O staining - Sections were incubated overnight at 4°C with

anti-NITEGE (cleaved aggrecan), anti-Col2 3/4M (denatured type II collagen), anti-CITED2,

anti-MMP-13 anti-ADAMTS5, or irrelevant isotype-matched antibodies as negative controls,

followed by a secondary antibody and visualization with DAB chromagen. Histological scoring

was performed by Safranin O/Fast green.

Results

Loss of CITED2 correlates with cartilage degradation in human OA and DMM mice.

In human OA cartilage samples, “healthy,” regions with strong Safranin O staining were

correlated with high levels of CITED2 expression, and low levels of aggrecan cleavage and type

II collagen denaturation. On the other hand, areas of cartilage destruction, indicated by local loss

of Safranin O staining, were correlated with low levels of CITED2 expression, and strong

staining for cleaved aggrecan and type II collagen denaturation (Fig 5.2A). Similarly, CITED2

was detected in healthy mouse cartilage, but was expressed at low levels in degenerated cartilage

4 weeks after OA induction by DMM (Fig 5.2B).

CITED2 suppression produces OA-like changes in normal mouse cartilage.

We next determined the direct effect of CITED2 suppression on cartilage homeostasis

after intra-articular injection of CITED2 shRNA in normal mouse knees followed by electroporation. Two weeks following CITED2 knockdown, a loss of Safranin O staining was detected in the articular cartilage along with a loss of CITED2 expression, an increase in the

amounts of cleaved aggrecan and denatured type II collagen, compared to the PBS-injected

contralateral controls (Fig 5.3A).

Deletion of CITED2 in adult mice results in early OA

Deletion of Cited2 in the mouse articular cartilage of Cited2 conditional KO (cKO) mice

was validated at the DNA, mRNA and protein levels 8 weeks after tamoxifen administration

(data not shown). Cited2 cKO mice developed early OA as evidenced by histologic analysis

(OARSI score 1 in cKO vs 0 in wild-type) and Safranin O staining (Fig 5.3B) while the cartilage

in wild-type controls were healthy. Cited2 cKO exhibited higher levels of denatured type II

collagen, aggrecan cleavage as revealed by Col2 ¾M and NITEGE, respectively (Fig 5.3B).

CITED2 deletion accelerates post-traumatic OA disease progression

As reduced levels of CITED2 may be associated with the pathogenesis of OA, we

compared OA disease progression in wild-type and Cited2 KO mice subject to DMM. Cited2

cKO exhibited more severe cartilage degradation and further elevated levels of type II collagen

denaturation (Col2 3/4M) and aggrecan cleavage (NITEGE) (Fig 5.3C).

CITED2 suppression upregulates MMP-13 and ADAMTS-5 expression.

Since CITED2 suppression led to increased damage to both type II collagen and aggrecan

in the cartilage extracellular matrix, we examined the role of CITED2 in regulating the

expression of both MMP-13 and ADAMTS-5, which respectively are the most efficient

proteolytic enzymes which cleave the above mentioned matrix components. In human and

mouse OA, reduced levels of CITED2 were associated with increased levels of both MMP-13

and ADAMTS-5 (Fig 5.4A). Knockdown of CITED2, by intra-articular gene transfer of

CITED2 shRNA (Fig 5.4B) or conditional deletion of CITED2 (Fig 5.4C), led to an increase in

the number of chondrocytes expressing MMP-13 and ADAMTS-5.

CITED2 represses ADAMTS-5 expression by negatively regulating syndecan-4.

As CITED2 deletion significantly elevated ADAMTS-5 expression, we explored the potential mechanism that CITED2 represses ADAMTS-5 expression, focusing on syndecan-4,

which is critical for the regulation of ADAMTS-5.187 Immunohistochemical analysis showed

syndecan-4 is elevated in the cartilage of Cited2 cKO mice and was associated with the increase

of ADAMTS-5 (Fig 5.5A). Importantly, gene transfer of Cited2 reduced both syndecan-4 and

ADAMTS-5 in the articular cartilage, suggesting Cited2 represses ADAMTS-5 by, at least in

part, negatively regulating syndecan-4 (Fig 5.5B).

Discussion

In this chapter, we showed that levels of CITED2 expression is lower in the cartilage of

OA patients compared to age- and gender-matched controls. We provide the first evidence that

reduced CITED2 is associated with cartilage degradation in human OA. By examining the level

of CITED2 expression in post-traumatic OA, we found that reduction of CITED2 expression is

associated with the progression of cartilage degradation. The consistent results of reduced

CITED2 expression associated with cartilage degradation in both human and mouse OA suggests

deficiency of CITED2 may be an important common factor in OA initiation and disease

progression. The loss-of-function studies by knocking down CITED2 by intra-articular gene

transfer in mice provide preliminary, experimental, and direct evidence that deficiency of

CITED2 is a casual factor of OA. Furthermore, using genetic deletion of CITED2 in tamoxifen

inducible, cartilage-specific CITED2 adult knockout mice, we found that deletion of CITED2

resulted in early OA development after 8 weeks. Of notice, deletion of a gene specifically from

the adult cartilage alone seldom generates phenotypic changes.216 This study provides clear and strong evidence which indicates that CITED2 is required for cartilage integrity maintenance.

Cartilage degradation such as in OA is a complex pathologic process. The initiation and progression of which can be, and usually results from more than “one hit” from disease risk factors such as aging, overuse, genetics and joint injuries. Studies have shown that reduced

CITED2 expression is a function of aging in tendon stem cells, and preliminary studies also

indicate that levels of CITED2 expression decline with aging in chondrocytes, both in human

and in mice, suggesting that CITED2 may be an underlying mechanism of aging as a risk factor

for cartilage degradation in OA.

Post-traumatic injuries are a critical risk factor for OA. Studies show that up to 50% of individuals develop OA 10-12 years after injuries to the meniscus or ACL. In a well established post-traumatic OA model based on a meniscus injury, we found that disease progression is associated with reduced levels of CITED2 expression, suggesting that deficiency of CITED2 plays a role in post-traumatic injury-induced OA. Our data show that deletion of CITED2 in adult cartilage combined with a meniscus injury accelerates OA initiation and progression and results in a more severe cartilage degradation and OA pathology than CITED2 deletion or DMM injury alone. Taken together, these data suggest that deficiency of CITED2, which may be a factor underlying aging and injuries for its role as a cartilage degradation susceptibility factor, or may be also a genetic factor which accelerates cartilage degradation when combined with other environmental, intrinsic, or extrinsic factors.

Cartilage degradation in joint diseases such as OA is characterized by an imbalance between the anabolic and catabolic activities of chondrocytes, yet the set of regulatory mechanisms governing that balance remain incompletely understood. The finding that deficiency of CITED2 is associated with or casually related to upregulation of MMPs, such as

MMP-13 and ADAMTS such as ADAMTS5 in human and mouse OA suggest CITED2 plays an anti-catabolic role via, at least in part, downregulation of these proteolytic enzymes.

In previous studies, we have identified CITED2 as a transcriptional repressor of MMPs.

The finding that the upregulation of ADAMTS5 in the CITED2 knockout mice suggest aggrecanases such as ADAMTS5 may be another target of CITED2 in its chondroprotective pathway. To this end, our preliminary data indicate CITED2 may downregulate ADAMTS5 in chondrocytes by repressing ADAMTS5 activation via syndecan-4. In addition to its role in

79 suppressing expression of MMPs/ADAMTS reported in this study, CITED2 plays a critical role in cell fate determination. As age is a major risk factor for developing OA, and chondrocyte senescence has been suggested to lead to cartilage degradation, further elucidating the role of

CITED2 in this chondroprotective pathway may not only provide a new avenue for the insight of cartilage degradation in arthritic disease, but potential targets for disease prevention and treatment.

Figures

Figure 5.1. Conditional knockout of Cited2 gene in the cartilage of adult mice.

Intraperitoneal injections of Tamoxifen (1mg/10g/mouse/day for 5 days) into 6-months-old male and female inducible Cited2 conditional KO mice (Col2a1CreERTxCited2fl/fl)64,65 completely and specifically knocked out the Cited2 gene in mouse articular cartilage. A.

Mouse crossbreeding strategy. B. Validation of exclusive Cited2 knockout at the DNA, mRNA and protein levels 12 weeks after Tamoxifen administration. WT: age and gender matched wild type control mice. KO: Col2a1CreERTxCited2fl/fl mice receiving Tamoxifen

80 dissolved in corn oil; Sham: Col2a1CreERTxCited2fl/fl that received corn oil without

Tamoxifen. NC: negative control without sample.

Figure 5.2. Reduced levels of CITED2, detected by immunohistochemistry and real-time qPCR are associated with increased OA severity, and increased degradation of cartilage matrix components aggrecan (NITEGE) and type II collagen (Col2 3/4M) in human OA (A) and mouse

OA (destabilization of the medial meniscus, DMM) cartilage (B), compared to age and gender- matched non-OA controls.

Figure 5.3. CITED2 knockdown by intra-articular gene transfer of CITED2 shRNA for 2 weeks in wild-type mice (male, C57Bl/6, 10-12 weeks) (A) or conditional knockout of Cited2 (Cited2

cKO) in the articular cartilage for 8 weeks (male, Cited2flf x Col2al-CreERT, 10-12 weeks) (B)

led to early OA, and increased the amount of cleaved aggreacan and denatured type II collagen

in the articular cartilage compared to sham (A) or wild-type littermates (B). DMM in Cited2 cKO mice increased severity of OA and increased cleaved aggrecan and denatured type II collagen compared to wild-type littermates (C).

Figure 5.4. (A) Reductions in CITED2 expression were associated with decreases in

chondrocytes expressing MMP-13 and ADAMTS5 in human and mouse OA cartilage, compared 83

to non-OA controls. (B) Experimental knockdown of Cited2 in mouse articular cartilage by

intra-articular gene transfer of CITED2 shRNA or (C) conditional knockout led to increases in

MMP-13 and ADAMTS5 compared to non-knockdown controls.

Figure 5.5. CITED2 represses ADAMTS-5 expression by negatively regulating syndecan-4.

(A) Level of syndecan-4 was elevated in CITED2 conditional knockout mice (cKO) which was

associated with elevated levels of ADAMTS5. (B) CITED2 gene transfer reduced the levels of

both syndecan-4 and ADAMTS-5 in DMM cartilage. Cell counts of syndecan-4, ADAMTS-5,

and CITED2 in conditional knockout mice vs wild-type, and in DMM+vehicle vs.

DMM+CITED2. *=p<0.05.

Chapter 6 – The Use of CITED2 for Chondroprotection

Abstract

There is currently no cure for degenerative joint diseases which feature the progressive degradation of articular cartilage. So far, most pharmacologic treatments mainly concentrate on

secondary effects of such diseases, such as relieving pain and improving joint function, but fail

to address the underlying disease progress. CITED2, induced by moderate mechanical loading,

is a transcriptional regulator which suppresses the expression of multiple MMPs and ADAMTS,

enzymes which directly cleave the cartilage extracellular matrix. Cartilage degradation, as seen

in OA, is associated with elevated levels of MMPs and ADAMTS, and reductions in CITED2

expression. Experimental knockdown of CITED2 by intra-articular gene transfer of CITED2

shRNA or genetic deletion of CITED2 in the articular cartilage, led to early cartilage degradation

and damage to the cartilage extracellular matrix components aggrecan and type II collagen. In

this study we tested the hypothesis that restoration of CITED2 would slow progression of

cartilage degradation. Gene transfer of CITED2 in two models of cartilage degradation,

collagen-induced arthritis, and destabilization of the medial meniscus, slowed progression of

cartilage breakdown. Immunohistochemistry revealed CITED2 gene transfer also prevented

degradation to the type II collagen and aggrecan components of the cartilage matrix.

Furthermore, EGCG, a green tea extract, was a small molecule inducer of CITED2, and daily

administration of EGCG slowed progression of post-traumatic OA. Taken together, our studies

suggest CITED2-based therapies have the potential to arrest or slow cartilage degradation in

diseases such as osteoarthritis and rheumatoid arthritis.

Introduction

There is currently no cure for degenerative joint diseases which feature the progressive

degradation of articular cartilage.77 So far, most pharmacologic treatments mainly concentrate

on secondary effects of such diseases, such as relieving pain and improving joint function, but fail to address the underlying disease progress.217

Based on the studies in the previous chapters, we have established that: 1) with a unique

mechanical loading response characteristic and via a newly identified pathway, CITED2 plays a

novel role in mediating loading-induced MMP downregulation (Chapters 3&4), 2) CITED2 is

required for cartilage integrity maintenance and CITED2 deficiency was associated with

cartilage degradation in human OA and post-traumatic OA in mice, knockdown of CITED2

through intra-articular gene transfer of shCITED2 RNA caused OA like pathologic changes, and

most importantly, deletion of CITED2 specifically from adult mouse cartilage resulted in not

only in OA-like phenotypic changes, but also accelerated the progression of post-traumatic OA

(Chapter 5). 3) Furthermore, over-expression of CITED2 represses MMP expression in cultured

chondrocytes in vitro, suggesting CITED2 is a transcriptional repressor of MMP expression and

its anti-catabolic action can be triggered by not only mechanical loading, but also by other

physical or chemical means to protect cartilage. Together, this suggests that CITED2-based

therapies may have efficacy in preventing and/or treating cartilage degradation in arthritic

diseases.

In this study, we tested the hypothesis that restoration of CITED2 would slow progression of

cartilage degradation. To test this, we used intra-articular CITED2 gene transfer in two cartilage

degradation models: destabilization of the medial meniscus (DMM), a model of post-traumatic

OA, and collagen-induced arthritis (CIA), a model of rheumatoid arthritis. Furthermore, we

examined the efficacy of CITED2 in chondroprotection, by administrating epigallocatechin 3-

gallate (EGCG),,218 a hypothesized small molecule activator of CITED2, to mice in the post- traumatic OA model. EGCG increases phosphorylation of p38δ,219 a transactivator of CITED2.

EGCG has also been reported to reduce levels of pro-inflammatory cytokines,220 inhibit production of inflammatory mediators,221,222 and suppress expression of several MMPs223 and

ADAMTS. 224 However, whether EGCG has the capacity to alter OA progression is unknown.

Methods and Materials

CITED2 gene transfer in mice - CITED2 gene transfer was carried out by weekly intra-articular

injections of a plasmid encoding CITED2 cDNA (25µg) followed by electroporation (200V, 0.10

ms pulse length, 4 pulses each polarity) into the DMM knee while the vehicle controls received injections of an empty vector followed by electroporation.

Destabilization of the medial meniscus - One day after the first gene transfer, surgical induction

of OA (destabilization of the medial meniscus) was performed in the right hind limbs of wild-

type mice (male, 20-24 weeks) as described in Chapter 5. Immediately after surgery, mice were allowed normal cage activity.

Collagen-induced arthritis (CIA) - Bovine type II collagen solution (Chondrex) was emulsified

in Freud’s complete adjuvant at 4°C. Under an IACUC-approved protocol, male Sprague-

Dawley rats (6-8 wk) were immunized by two intradermal injections of 100µl emulsion (1µg

collagen/µl) at the base of the tail given one week apart.

In vivo gene transfer efficiency - In order to determine transfection effiency of in vivo gene

transfer, 25µg of pcDNA3.1-GFP was injected into the intra-articular cavity of the right knees of

rats (n=2), followed by electroporation (250V, 0.10 ms pulse length, 4 pulses each polarity).

Contralateral limbs were injected with an empty vector followed by electroporation. Forty-eight

hours after transfection, rats were sacrificed and their hind limbs were fixed in 10% formalin,

decalcified, and embedded in paraffin. Sections were cut (5-7μM), mounted on glass slides, coverslipped, and visualized under a microscope with a GFP filter.

CITED2 gene transfer in CIA rats - After the collagen booster injection, and at weekly intervals

thereafter, rats received 25µg of pcDNA3.1 encoding human wild-type CITED2 cDNA by intra-

articular injection followed by electroporation. Intra-articular injections of an empty vector were

administered to the contralateral limbs followed by electroporation. Animals were sacrificed 0,

8, 11, 14, 17, 21, and 28 days after the first injection (n=2/timepoint). Articular cartilage from

distal femur and tibial plateau were harvested, flash frozen, and lysed for Western blots. At Day

28, parallel articular cartilage samples were fixed in formalin, decalcified and processed for

histology (n=3/group).

Induction of osteoarthritis in mice and EGCG treatment - Immediately after the DMM surgery in

male, 5-6 months mice, 100 μl of EGCG (25mg/kg, Sigma, dissolved in PBS) or vehicle was administered via intraperitoneal injection 5 days/week for 4 weeks. At the end of the experiment, hind limbs were fixed in formalin, decalcified and embedded in paraffin

(n=6/group).

Immunohistochemistry, Safranin O staining, and OARSI score evaluation - Paraffin sections (5-

7μM) were incubated overnight at 4°C with antibodies against cleaved aggrecan (NITEGE,

Ibex), denatured type II collagen (Col2-3/4M, Ibex), MMP-13 (Abcam), and ADAMTS-5

(Abcam) followed by incubation with anti-mouse or anti-rabbit secondary antibody and

visualization with DAB chromagen (Vector Laboraties). Negative controls were stained with

irrelevant isotype-matched antibodies. Safranin O-fast green staining was used to visualize

proteoglycans in the articular cartilage. OA was severity evaluated by OARSI scoring system225.

Results

CITED2 gene transfer prevents cartilage degradation in CIA model

To determine whether electroporation of CITED2 would prevent cartilage degradation,

we first established the effectiveness of gene transfer into rat articular cartilage. Forty-eight hours after intra-articular gene transfer of a GFP plasmid, we found GFP-producing chondrocytes in all three zones of the articular cartilage (Fig 6.1). Following electroporation,

CITED2 levels assessed by Western blot increased until Day 14, then remained high throughout the experimental period (Fig 6.1). Collagen-induced arthritis rats treated with CITED2 gene transfer, compared to vehicle control, exhibited increased expression of CITED2 in articular chondrocytes (81±6% vs. 22±3%, p<0.05), diminished loss of Safranin O staining (OARSI score

0.18±0.20 vs. 1.12±0.33, p<0.05), lower levels of denatured type II collagen and a reduced number of chondrocytes positive for MMP-13 (79±5% vs 40±3%) and ADAMTS5 (89±6% vs.

51±4%, p<0.05) (Fig 6.1).

CITED2 gene transfer slows cartilage degradation in DMM model

Four weeks after DMM, the articular cartilage in the DMM limb in the vehicle-treated mice

exhibited Safranin O loss, cartilage fibrillation, and an average OARSI score of 2.5±0.5 (Fig

6.2). In contrast, the cartilage in the DMM limb in the CITED2 gene transfer mice exhibited less

Safranin O loss and cartilage fibrillation, and a lower OARSI score (1.1±0.4, p<0.05) compared

to vehicle-treated controls (Fig 6.2).

Immunohistochemical staining showed that CITED2 gene transfer strongly reduced levels of

cleaved aggrecan (NITEGE) and fragmented/denatured type II collagen (Col2 3/4M), in

comparison to vehicle-treated controls (Fig 6.2). These data further confirms that CITED2 gene transfer improves the integrity of the articular cartilage by preserving both collagen and aggrecan components in post-traumatic OA mice.

In vivo administration of EGCG slows progression in DMM OA mice

To confirm EGCG induces expression of CITED2, we treated human C28/I2 chondrocytes

with 100µM EGCG. Three hours after treatment, we found expression of CITED2 at the mRNA

and protein levels was increased (Fig 6.3).

Four weeks after DMM, the articular cartilage in the DMM limb in the vehicle-treated mice

exhibited a mild-OA pathological change characterized by Safranin O loss, cartilage fibrillation,

and an average OARSI score of 2.0±0.5 (Fig 6.4A, B). In contrast, the cartilage in the DMM

limb of EGCG-treated mice exhibited less Safranin O loss and cartilage fibrillation, and the

mean OARSI score (1.2±0.4) was significantly lower compared to vehicle-treated controls

(p<0.05, Fig 6.4A, B). Sham-operated mice receiving either vehicle or EGCG treatment did not

exhibit pathologic changes in the articular cartilage and had OARSI scores of 0.15±0.25 and

0.12±0.31, respectively (Fig 6.4A, B).

Immunohistochemical staining showed that EGCG treatment strongly reduced the levels of

the type II collagen cleavage epitope (Col2 3/4M) in DMM mice compared to vehicle-treated

DMM mice (Fig 6.5). Based on the immunostaining intensities of 6 randomly selected areas of the articular cartilage at 4 weeks following DMM, type II collagen cleavage in vehicle-treated

controls increased to 1.18-fold above sham-treated vehicle mice, and was reduced to 0.97-fold in

EGCG-treated animals (p<0.05, Fig 6.5A, B). Sham animals treated with vehicle or EGCG had

no significant immunostaining for type II collagen degradation at 4 or 8 weeks (Fig 6.5A,B).

Immunohistochemical staining similarly showed that EGCG treatment reduced the levels of

cleaved aggrecan (NITEGE) in DMM mice compared to vehicle-treated DMM mice (Fig. 6.6).

At 4 weeks after DMM, the intensity of aggrecan cleavage was reduced to 1.07-fold in the

EGCG-treated compared to 1.29-fold in the vehicle control (p=0.08, Fig 6.6A, B). Sham animals

treated with vehicle or EGCG did not exhibit significant immunostaining for cleaved aggrecan at

4 weeks (Fig 6.6A,B).

Cartilage matrix degradation is mainly mediated by two major families of proteolytic

enzymes, namely MMPs and ADAMTS 21. In particular, MMP-13 is the most potent enzyme in

cleaving type II collagen, the major form in articular cartilage, while ADAMTS-5 is the

primarily proteolytic enzyme that cleaves aggrecan, the major proteoglycan in cartilage 77. We

therefore examined whether reduction of MMP-13 and ADAMTS5 could underlie the

chondroprotective effect of EGCG using immunohistochemistry.

At 4 weeks following DMM, the percentage of MMP-13 positive cells was reduced from

60% in vehicle-treated mice to 22% in EGCG-treated mice (p<0.05). MMP-13 positive

chondrocytes in the vehicle-treated DMM mice were distributed in all three zones of the articular

cartilage of the femoral and tibial condyles. In contrast, the MMP-13 positive chondrocytes in

EGCG-treated mice were localized mainly in the middle and deep zones (Fig 6.7A, B). Levels of MMP-13 positive cells in the articular cartilage of EGCG-treated sham mice were at a level

(4%) similar to those in sham vehicle mice (5%) (Fig 6.7A, B).

Similarly, at 4 weeks following DMM surgery, EGCG reduced the percentage of

ADAMTS5 positive cells from 61% in vehicle-treated mice to 22% in the articular cartilage

(p<0.05, Fig 6.8A, B). EGCG treatment appeared to exert no measurable effect on the levels of

ADAMTS5 in chondrocytes in the articular cartilage in the sham animals treated either with vehicle (5%) or EGCG (4%) (Fig 6.9A, B). EGCG did not significantly alter the percentage of

ADAMTS5 positive cells in sham-operated mice (Fig. 6.8A,B).

These data further suggest that EGCG treatment improves the integrity of the articular cartilage by preserving both collagen and aggrecan components in post-traumatic OA mice, and that EGCG exerts chondroprotection, at least in part, by suppressing MMP-13 and ADAMTS5 expression.

Discussion

Cartilage degradation is mediated directly by enzymes which cleave the cartilage extracellular matrix, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). Inhibitors for these proteolytic enzymes have been explored as therapeutic strategies to treat OA. However, clinical trials with

MMP inhibitors so far have been met with limited success and resulted in side effects including musculoskeletal pain and inflammation.104-106 These adverse effects have been mainly attributed to the lack of selectivity of these inhibitors, and over suppression of their targets. Matrix

metalloproteinases share structural similarities and are susceptible to regulation by broad-

spectrum inhibitors.87 Poor selectivity is problematic because in addition to matrix remodeling,

MMPs/ADAMTS play important roles in wound healing, angiogenesis, development, morphogenesis, and bone remodeling.88,89 Clearly, pharmacologic solutions for treating OA need to define the correct molecular targets and ensure proper modulation of these targets.

The first aims of the studies in this chapter were to test the concept that gain-of-function of

CITED2 is able to prevent or slow the progression of RA and OA. It has been established that

the etiology of OA and RA are different.21 Cartilage degradation in RA is due to high levels of

inflammatory cytokines. Different from RA, cartilage degradation in OA can be caused by

complex risk factors, including genetic, aging, and overuse/trauma.77 While the initiating

mechanisms for cartilage degradation differ in RA and OA, cartilage destruction is the hallmark

of both diseases. In both, activation of MMPs and ADAMTS, the predominant enzyme families

in directly cleaving the cartilage extracellular matrix, is shared in the molecular pathology.2 Our

previous studies established a CITED2-mediated pathway in downregulating MMP expression.

In this study, overexpression of CITED2 not only significantly reduced expression of MMP-13,

but also ADAMTS-5, a proteolytic enzyme that degrades aggrecans. Together, the findings in

this study indicate restoring or elevating CITED2 in inflammatory arthritic joints or in post-

traumatic OA joints can significantly slow cartilage degradation in early phases of the disease,

but the efficacy of CITED2 in treating cartilage degradation after disease onset remains to be

tested.

Our findings suggest that CITED2 exerts great potential for chondroprotection in both RA

and OA cases; 2) CITED2 plays its role, at least by targeting two major proteolytic enzymes

MMP-13 and ADAMTS-5, and thus preserves cartilage matrix components type II collagen and 93

aggrecans. The action of targeting multiple catabolic enzymes may be an underlying mechanism

for CITED2 as an effective treatment in chondroprotection. Furthermore, CITED2 selectively

repressed, but did not completely suppress expression of MMP-13 and ADAMTS-5. Therefore,

CITED2-based therapeutic strategies may be a promising treatment that can avoid the adverse

effects of MMP inhibitors while exerting efficacy in preventing or slowing cartilage degradation.

Overexpression of CITED2 in articular cartilage proved the concept that CITED2 can be

used as a molecular target to turn on anti-catabolic action by suppressing not only MMPs but

also ADAMTS. However, gene transfer-based therapies are neither patient friendly, or risk-free.

Furthermore, as inhibiting cartilage degradation in OA and RA may require decades-long

treatment, long-term use, safety, and convenience are critical criteria for any treatment strategy.

Based on the multiple-stimuli responsive nature of CITED2, we thought to develop a

nutraceutical treatment based on the activation of CITED2 and examined efficacy of

nutraceutical activation in a post-traumatic model of OA. Through a targeted screening

approach, we found EGCG, a green tea extract, is an activator of CITED2, and slowed

progression of post-traumatic OA. It is unclear whether chondrocytes may be densensitized to

daily stimulation of EGCG, but this possibility can be tested by monitoring CITED2 levels with

treatment time.

EGCG has been used as a general nutritional supplement for anti-cancer, and anti-aging, and

the dosage of EGCG for such purposes has been proven to be safe and non-toxic.218 In human

subjects, daily doses of 800mg EGCG for 4 weeks have been reported to be safe and well

tolerated.226 EGCG is mostly absorbed by the small intestine, and may undergo gastrointestinal inactivation.226 Therefore, oral administration of EGCG may reduce its bioavailability.

Accordingly, in our study, we chose to administer EGCG via intraperitoneal injection, which has 94 been shown to lead to a higher bioavailability compared to oral consumption.226 Future trials should consider optimizing EGCG bioavailability if given orally. Taking together, this study provides direct evidence that CITED2 activation reached by nutraceutical compounds may be a valid approach for the prevention and treatment of cartilage degradation.

Figures

Figure 6.1. Increased expression of CITED2 by gene transfer exerts chondroprotection in CIA rats. Top: Experimental protocol, transfection efficiency of CITED2 gene transfer, and expression of CITED2 in CIA rats throughout the experiment. Bottom: CITED2 gene transfer prevented degradative changes within the articular cartilage in the CIA model. Gene transfer prevented loss of Safranin O staining, and decreased expression of MMP-13 and ADAMTS-5.

Figure 6.2. Intra-articular CITED2 gene transfer reduced OA severity, increased CITED2 expression, and prevented damage to aggrecan and type II collagen in the cartilage of DMM mice after 4 weeks, compared to vehicle-treated controls.

Figure 6.3. Green tea extract, epigallocatechin-3-gallate (EGCG, 100 µM), upregulates expression of CITED2 in C28/I2 human chondrocytes. *p < 0.05 versus control, n=3.

Figure 6.4. EGCG administration slows progression in early and mid-stage OA in DMM mice.

Safranin O staining and OARSI score of sham- or DMM-operated mice treated with vehicle or

EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group). Arrows heads

indicate the areas of cartilage fibrillation or erosion, and arrows indicate loss of Safranin O

staining.

Figure 6.5. EGCG administration reduced the degradation of type II collagen in the articular cartilage matrix. Immunohistochemical staining of type II collagen cleavage epitope (Col2

3/4M) and relative staining intensity of the articular cartilage matrix of sham- or DMM-operated

mice treated with vehicle or EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA,

n=6/group, Scale bar=100µM).

Figure 6.6. EGCG administration reduced the degradation of aggrecan in the articular cartilage matrix. Immunohistochemical staining of cleaved aggrecan (NITEGE) and relative staining intensity in the articular cartilage matrix of sham- or DMM-operated mice treated with vehicle or

EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group, Scale bar=100µM).

Figure 6.7. EGCG administration reduced MMP-13 levels in the articular cartilage.

Figure 6.8. EGCG administration reduced ADAMTS5 levels in the articular cartilage.

Immunohistochemical staining of ADAMTS5 and percentage of ADAMTS5 positive cells in the articular cartilage of sham- or DMM-operated mice treated with vehicle or EGCG at 4 (A, B) weeks following surgery (*p<0.05, ANOVA, n=6/group, Scale bar=100µM).

100

Chapter 7 – Global Discussion

Mechanical loading is the most important factor for cartilage homeostasis while overloading and underloading are considered to be non-physiologic and lead to cartilage

degradation. Moderate loading is required for the maintenance of cartilage integrity, but

mechanisms mediating these effects are largely unknown. Based on the literature review and our previous studies, we hypothesized that CITED2 mediates a mechanical pathway in

chondroprotection; appropriate levels of CITED2 are critical for cartilage integrity; and

restoration, or increasing levels of CITED2 exerts efficacy in prevention and treatment for

cartilage degradation in arthritis. Accordingly, the gene expression response of CITED2 to

mechanical loading, the CITED2-mediated mechanically sensitive anti-catabolic pathway, and

the potential use of CITED2 for chondroprotection were investigated at the molecular, cellular,

and tissue level in vitro and in vivo in this dissertation.

To understand the role of CITED2 in mediating loading-induced MMP downregulation,

we analyzed CITED2 and MMP expression in immobilized joints with daily, 1hr, remobilization.

The results that CITED2 downregulation in immobilized limbs was associated with MMP-1

upregulation and cartilage degradation, and the upregulation of CITED2 associated with the

downregulation of MMP-1 and chondroprotection was the first hint that CITED2 may play a

critical role in maintaining cartilage integrity. We further determined the machinery of this

mechanical regulation and found that CITED2 regulates MMP expression by competing with

MMP transactivator Ets-1 for binding to limited amounts of co-activator p300. The

mechanotransduction pathway involved the phosphorylation of p38δ, which transactivated

CITED2 through the HIF-1 and Sp1 binding sites on its promoter region.

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Upon the establishment and characterization of the biomechanical response of CITED2 and its critical role in mediating loading-induced downregulation of CITED2 in chondrocytes both in vitro and in vivo, we thought that CITED2 deficiency may be a casual factor for cartilage degradation, especially considering the case that immobilization caused cartilage degradation and was associated with reduced CITED2 expression levels. This notion was further supported by the association of reduced levels of CITED2 and increased levels of MMP-13 and ADAMTS-

5 in human and mouse OA and knockdown of CITED2 via intra-articular injection of CITED2 shRNA caused OA-like changes in mice (Chapter 5). We therefore established an inducible and cartilage-specific CITED2 knockout mouse model and examined the cartilage to investigate this possibility. The finding that deletion of CITED2 in adult mice results in early OA and further, that CITED2 deletion accelerates post-traumatic OA disease progression, provides direct evidence that CITED2 is essential for cartilage integrity maintenance and deficiency of CITED2 is a casual factor of cartilage degradation. The results also suggest that deficiency of CITED2 leads to cartilage degradation in OA, at least in part, by reducing the chondrocyte stress-response capacity.

Based on our findings, CITED2 is required for cartilage integrity maintenance, and several gain-of-function studies such as overexpressing CITED2 can mimic the effects of moderate loading by downregulating expression of MMPs. We therefore hypothesized that

CITED2 is a molecular switch / target and activation of CITED2 may prove to be a novel strategy for chondroprotection which can be used for preventing and therapeutic treatment of cartilage degradation in arthritic disease. To this end, in Chapter 6, we first conducted experiments to prove the concept in inflammatory arthritis by using the collagen-induced arthritis model, and we found that weekly intra-articular gene transfer of CITED2 significantly reduced 102

cartilage degradation. Next, we examined the CITED2 chondroprotection effect in post-

traumatic OA and found a similar efficacy. Interestingly, gene transfer of CITED2 reduced

expression of MMP-13 and ADAMTS-5 levels and immunohistochemistry showed such treatment reduced aggrecan and type II collagen degradation. Further, we examined the efficacy of a CITED2 activator, EGCG, an extract derived from green tea in OA prevention. We found

EGCG treatment slowed progression of OA, and prevented degradation to the cartilage matrix components aggrecan and type II collagen, at least in part, by suppressing expression of MMP-

13 and ADAMTS. This suggests CITED2 can be used as a molecular target via mechanical or other means of activation for chondroprotection.

The unique biomechanical response of CITED2 and the novel pathway that CITED2 mediates provides novel insight in understanding the underlying mechanisms of cartilage homeostasis maintenance, pathogenesis of cartilage degradation, and may be more importantly, a potential strategy and molecular target for prevention and treatment of cartilage degradation in

OA, RA, and other arthritic diseases.

Based on the studies in this thesis, four major conclusions can be drawn.

1. CITED2 was induced in an intensity-dependent manner in response to hydrostatic pressure loading, and was required to mediate the loading-induced down-regulation of MMP-1, -2, -3, and

13. The overexpression of CITED2 mimicked these anti-catabolic actions of moderate

mechanical loading.

2. Physiological joint loading prevented disuse-caused cartilage degradation associated with

induced expression of CITED2. CITED2 exerted its chondroprotective actions by competing

103 with MMP transactivator Ets-1 for binding to limited amounts of co-activator p300, and is transactivated by p38δ, through HIF-1α and Sp1 binding sites on its promoter region.

3. Deficiency of CITED2 is associated with loss of cartilage integrity in human and mouse OA, and experimental knockout of CITED2 led to up-regulation of not only MMP-13 and but

ADAMTS-5 and resulted in cartilage degradation and OA.

4. Restoration of CITED2 slowed progression of cartilage breakdown in post-traumatic OA model in mice, and collagen-induced arthritis model in rats, two models of cartilage degradation.

Activating CITED2, with small molecule EGCG, exerted significant efficacy in chondroprotection in also showed slow down the disase progression in post-traumatic OA mice model.

This thesis study focused on the anti-catabolic role of CITED2. Of notice, as a co-factor,

CITED2 may also play a role in cartilage homeostasis by regulating chondrocyte fate such as chondrocyte hypertrophy senescence, and apoptosis. Future studies will focus on elucidating these cell fate-related mechanisms and determining their role in chondroprotection, which may reveal novel insight for understanding the regulatory mechanisms of cartilage homeostasis and strategies / targets for the prevention and treatment of cartilage degradation.

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