Osteoarthritis is one of the most common problems associated with aging, and although there are therapies to treat the pain that results from the breakdown of the cartilage that cushions joints, there are no available therapies to modify the course of the disease.
However, working in a mouse model of the disorder, researchers at Washington University School of Medicine in St. Louis have found that a molecule previously linked to diabetes, cancer and muscle atrophy also seems to be involved in the development of osteoarthritis and may be a useful treatment target.
When the gene involved, FoxO1, is knocked out in mice, the animals develop osteoarthritis. But when the researchers increase the levels of the FoxO1 molecule in mice that are developing osteoarthritis, the animals exhibit less cartilage damage.
The study is available online in Proceedings of the National Academy of Sciences.
“Osteoarthritis, or joint degeneration, is a disease that affects more than 32 million people in the U.S. alone but that does not have a medical therapy to alter its progression,” said senior investigator Regis J. O’Keefe, MD, Ph.D., the Fred C. Reynolds Professor of Orthopedic Surgery and head of the Department of Orthopedic Surgery.
“A better understanding of the fundamental processes involved in osteoarthritis and the degeneration of cartilage is required if we’re going to be more successful in treating this very common and very expensive disorder.”
O’Keefe said that commonly, people with osteoarthritis have suffered knee injuries that damaged the knee’s meniscus. Over time, arthritis then can develop in the joint.
“Unlike skin or bone or other organs that can regenerate in response to injury, cartilage has very little regenerative potential,” he said.
However, when the mice in these experiments had elevated levels of the FoxO1 molecule, osteoarthritis’s progress was slowed or even reversed.
The researchers believe the molecule interferes with cartilage damage and the development of arthritis by enhancing a process called autophagy in the arthritic joint.
Autophagy is the body’s way of clearing out damaged tissue.
In these experiments, the researchers found that autophagy was disrupted in the mice with reduced levels of FoxO1 and that the process was enhanced in animals with higher levels of the molecule.
“In other words, maintaining a higher level of autophagy seemed to be beneficial to maintaining these cartilage cells and, thus, maintaining a healthy knee joint,” said co-corresponding author Jie Shen, assistant professor of orthopedic surgery.
O’Keefe said that raises the possibility of delivering FoxO1 to arthritic joints through nanotechnology as a way to regulate autophagy and keep joints healthier.
“In mice with injuries that typically progress to become osteoarthritis, the knee joints still appear normal about a week after injury,” O’Keefe explained.
“But when we measure autophagy in the cartilage after injury to those same knee joints, although the joints themselves look fine, the autophagy process already is shut off. The injury completely turns it off, and once autophagy is off, the cartilage begins to degenerate.”
He said if FoxO1 can alter that process in people, protecting cartilage from damage as it does in mice, it eventually may be possible to prevent or delay millions of future knee and hip replacement surgeries.
Articular cartilage is an integral component of the musculoskeletal system and its main function is to absorb compressive and shear forces during joint movement (1). Aging or trauma related damage to cartilage is a principal event in the pathogenesis of osteoarthritis (OA), the most prevalent joint disease (2).
While tissue level and cellular changes in cartilage aging have been characterized (3), mechanisms that are responsible for cellular homeostasis and reasons for their failure in aging remain to be discovered.
Recent findings support the concept that cartilage aging and the development of structural changes are related to the failure of cellular homeostasis mechanisms, such as autophagy and oxidative stress responses (4).
The FoxO proteins are an evolutionarily conserved family of transcription factors with important functions in development, aging and longevity (5).
In mammals, the FoxO family is comprised of four members (FoxO1, FoxO3, FoxO4, and FoxO6) with distinct and overlapping functions (6). FoxO1, FoxO3, and FoxO4 are ubiquitously expressed whereas FoxO6 expression is largely restricted to the brain (7). The triple deletion of FoxO1, 3, and 4 leads to more severe phenotypes than deletion of individual FoxO (8, 9).
However, each FoxO regulates gene expression in a tissue specific pattern (10). In bone, FoxO1 is more abundant than FoxO3 and FoxO4 (11) and modulates osteoblast differentiation by interacting with the Runx2 promoter (12).
In humans, FoxO3 single nucleotide polymorphisms are associated with exceptional longevity (13). The role of FoxO in regulating lifespan is thought to be through control of cellular homeostasis and maintenance of stem/progenitor cells populations during aging (7, 14).
FoxO expression and activity are induced under oxidative stress conditions (14) and FoxO transcriptionally induce expression of several antioxidant enzymes such as catalase and manganese superoxide dismutase (15). FoxO proteins also regulate two major intracellular clearance mechanisms, autophagy and the ubiquitin-proteasome system, to eliminate damaged and aggregated proteins (14).
Dysregulation of FoxO expression or activity contributes to the pathogenesis of age-related diseases in several different tissues, including bone (11) and muscle (16).
We reported earlier that the expression of autophagy genes which are regulated by FoxO were reduced in OA (17) and that autophagy activation by rapamycin reduced the severity of OA (18). Subsequently, we found that the expression of FoxO is reduced in aging and OA-affected human and mouse cartilage (19).
Our previous findings that FoxO1, FoxO3 and FoxO4 mRNA and protein expression are reduced in aging and OA-affected cartilage in humans and mice (19) motivated the present study to analyze consequences of chondrocyte specific FoxO deletion. First, we used the Col2a1-Cre mouse strain with Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes (26).
We analyzed mice during postnatal growth and maturation from P1 until 18 months of age. The most profound phenotypic changes in the epiphyseal growth plate, articular cartilage and other joint tissues were observed in Col2Cre-TKO mice and to a very similar extent in Col2Cre-FoxO1 KO mice. In addition, deletion of FoxO3 led to more severe and earlier onset of age-related OA-like changes at 18 months.
The role of FoxO in endochondral ossification was recently studied using the same gene targeting strategy to create Col2Cre-TKO mice (27). Neonatal mice showed elongation of the hypertrophic zone of the growth plate and had increased overall body and tail length at eight weeks of age (27). The present study revealed that FoxO1 appears to be the FoxO isoform that is largely responsible for the growth plate abnormalities.
The epiphyseal cartilage in the KO mice appeared normal at birth, and there were no apparent changes in tissue volume, cell density, size or organization. However, the articular cartilage was significantly thicker in Col2Cre-TKO mice at 1 month and Col2Cre-FoxO1 KO mice at 2 months.
The cartilage of these mutant mice also exhibited increased cell proliferation and abnormal expression of chondrocyte differentiation markers. These findings indicate that FoxO deletion leads to increased cartilage thickness by regulating chondrocyte proliferation and differentiation.
Articular cartilage lesions developed spontaneously in Col2Cre-TKO and Col2Cre-FoxO1 KO mice between 2 and 6 months of age. As these degradative changes could be at least in part due to abnormal cartilage growth and maturation during the postnatal period, we deleted FoxO1/3/4 in skeletally mature mice using the Aggrecan-CreERT2 knockin mice (22).
These mice started to show OA-like changes within 2 months after FoxO deletion and full thickness cartilage defects after 5 months. Surgical destabilization of the knee joint in mice (28) is associated with mechanisms and features similar to posttraumatic OA in humans (29).
We also used a treadmill running model with a protocol, which leads to mild cartilage damage in wild-type mice (30). In both models, the mice with postnatal FoxO deletion showed more severe cartilage damage, suggesting that FoxOs have protective functions in the response of cartilage to joint trauma and mechanical overload.
The earliest morphological changes in the FoxO KO mice were irregularities in the superficial zone and a depletion of the elongated superficial zone cells (31). We observed reduced expression of Prg4 in FoxO KO mice, which is consistent with a previous study in T-lymphocytes (32).
The Prg4 gene (33) encodes a mucin-like, O-linked glycosylated protein, termed lubricin (34) or SZP (35). It is produced by cells in the articular cartilage surface (35), meniscus (36), and synovium (37) and present in the extracellular matrix of the superficial zone (38) and in synovial fluid (39).
Lubricin functions as a boundary lubricant in articular cartilage to decrease friction and wear and the accumulation of lubricin at the surface of cartilage are thought to be important for joint homeostasis (31). Mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading (40).
Prg4 deficient mice also develop superficial and upper mid zone chondrocyte apoptosis and cell loss (41, 42). Human cartilage aging is characterized by decreased mechanical function in the superficial zone, with reduced tensile integrity and surface wear, reduced cellularity and a decrease in matrix glycosaminoglycan content (43). The findings observed in FoxO KO mice are thus consistent with changes that were seen in lubricin deficient mice and during early stages of human joint aging.
The present findings support a direct role of FoxO1 in upregulating the expression of Prg4. Transfection of a constitutively active form but not of a DNA binding deficient form of FoxO1 increased Prg4 expression. TGFβ is a main stimulus of Prg4 (23, 24) and the chondrocyte response to TGFβ decreases with aging (44).
As the present results show that FoxO1 synergizes with TGFβ1 to increase Prg4 gene expression, the aging-related loss FoxO is a likely factor in the reduced Prg4 expression and potentially in other compromised TGFβ induced cellular responses.
While the FoxO deficiency-related reduction in Prg4 expression appears to be an early and important event in initiating structural changes in the superficial zone, other chondrocyte functions are abnormal in FoxO deficient mice and are likely to contribute to rapid and severe cartilage destruction.
In addition to reduced Prg4 expression, we observed lower levels of important cellular homeostasis genes in FoxO deficient mice. Genes involved in antioxidant defenses (Sesn3 and Gpx3), autophagy (Map1lc3b, Atg4b, Becn1, Gabarapl1, Bnip3), redox regulation (Txnip) and adaptation to energy stress (Prkaa2) were reduced in cartilage of FoxO KO mice.
Notably, expression of several of these genes including autophagy proteins (17), Txnip (45), sestrins (46), Prkaa2 (47) are reduced in aging and OA cartilage, raising the possibility that this is due to the reduced expression of FoxO.
To test this and examine the potential therapeutic benefit of targeting FoxO in OA, we cultured OA chondrocytes, which show abnormal gene expression patterns. Overexpression of FoxO1 in OA chondrocytes increased autophagy genes, reduced inflammatory mediators and cartilage-degrading enzymes and antagonized the effect of IL-1β stimulation. These results indicate that reduced FoxO expression in OA chondrocytes is at least in part responsible for abnormal expression of homeostasis genes and mediators of OA pathogenesis.
OA is a disease that affects all joint tissues and FoxO KO mice showed changes not only in cartilage but also in synovium and subchondral bone. It should be noted that the Col2a1-Cre driver used in the present study targets not only articular and growth plate chondrocytes but cells in other joint tissues, including cells in synovium (48).
We observed OA-like changes in synovium and subchondral bone in 6-month-old Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Downregulation of FoxO1 is involved in synoviocyte survival and synovial hyperplasia in rheumatoid arthritis (49). Consistent with this FoxO function, synovial hyperplasia was the main manifestation of FoxO deficiency observed in the present study.
FoxOs are also key regulators of bone formation and remodeling (50). However, the subchondral bone changes observed in the mice with chondrocyte specific deletion of FoxO are probably secondary to initial changes in the articular cartilage. Mouse models of OA are characterized by a close association of changes within the osteochondral unit (51).
The results from the present study revealed overlapping and distinct functions of FoxO isoforms. A likely explanation for the mild changes observed in FoxO4 deficient mice is that this isoform is expressed in chondrocytes at much lower levels than FoxO1 or FoxO3 (19). The most severe phenotype during postnatal articular cartilage development was seen in the FoxO TKO mice.
FoxO1 KO mice had similar changes as the TKO mice but all changes in the TKO mice were consistently more severe than in the FoxO1 KO mice, suggesting that FoxO3 also has some, although lesser effects on these processes. In this regard, FoxO3 KO mice also had changes in cell cycle regulators.
The main difference between FoxO1 and FoxO3 KO mice was that only FoxO1 KO mice deficient Prg4 expression and this is a likely explanation why cartilage in these mice rapidly degenerated. The main reason for the aging-related OA development in the FoxO3 KO mice appears to be the reduction in protective genes (autophagy, antioxidants). These differences in FoxO function are in part related to different sets of interacting proteins (52).
In summary, these studies identify FoxOs as essential transcription factors regulating postnatal articular cartilage growth and homeostasis. The role of FoxO in cartilage growth is mainly mediated by FoxO1 and is related to effects on chondrocyte proliferation, survival and regulation of chondrocyte differentiation.
The role of FoxOs in maintaining postnatal articular cartilage integrity is mediated by their role in activating cellular defense mechanisms and in regulating the expression of PRG4, an essential protein in cartilage lubrication and superficial zone protection. These findings support the pathogenic significance of the reduction of FoxO in aging and OA-affected cartilage and suggest that maintaining or restoring FoxO expression can prevent OA onset and delay disease progression.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204214/
More information: Cuicui Wang et al. FoxO1 is a crucial mediator of TGF-β/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2017056117