Chondrocyte-specific Gene Pten Knockout Mice Leads To Osteoarthritis

Osteoarthritis (OA) is characterized by degeneration of articular cartilage, intra-articular osteophytes formation and periarticular synovitis. Multiple factors are involved in the initiation and progression of OA, including aging, obesity, gender, joint trauma, inflammation, and genetic factors. The molecular mechanisms underlying OA still remain unclear. It has been reported that transforming growth factor-β signaling pathway, Wnt signaling pathway, proteases, microRNAs, transcription factors as well as complements are involved during the initiation and progression of OA.Cell senescence is the major causal factor for many degenerative diseases. Senescent cells are characterized by the change of cell morphology, cell cycle arrest, metabolism recession and increased expression of matrix metalloproteinases. Many extrinsic stresses including oxidative stress could cause cell senescence, which called stress-induced senescence. p53is not only a famous tumor suppresser protein but also involved in the regulation of cell senescence in response to extrinsic stresses. Oxidative stress provokes the activity of p53, which then triggers the signaling cascade of cell senescence via altering the expression of pro-oxidant enzymes and anti-oxidant enzymes to reduce the cellular anti-oxidant capacity, and increasing the expression of cycling-dependent kinase inhibitors p21and p16to inhibit cell cycle progression.The initiation of OA is considered to be closely associated with chondrocytes senescence. Chondrocytes from OA patients exhibit typical cell senescence phenotype. In vitro experiments showed that chondrocytes exposed under abnormal stimuli would generate excessive reactive oxygen species (ROS) resulted from metabolism disorder. When the amount of ROS in chondrocytes exceeds the anti-oxidant capacity of the cell, senescent phenotypes such as shortened telomere and increased expression of MMPs will occur, in consistence with those characteristics observed in OA chondrocytes. Nevertheless, the molecular mechanisms underlying how oxidative stress is triggered and how it aggravate the chondrocyte senescence has not been elucidated in vivo, the causal relationship between chondrocyte senescence induced by oxidative stress and OA initiation and progression also needs further clarification.PTEN (phosphatase and tensin homolog deleted from chromosome10) is known as an important tumor suppressor by negatively controling the PI3K (phosphoinositide3kinase) signaling pathway via dephosphorylation of both serine/threonine residues, therefore participating in the regulation of cell growth, lineage determination, apoptosis and motility. In vitro experiments proved that PI3K/Akt signaling pathway increases the synthesis of extracellular matrix and inhibits cellular apoptosis in cultured chondrocytes. Therefore, the activation of PI3K/Akt signaling pathway is suspected to play a positive role during OA progression. However the most evolutionarily conserved function of PI3K/Akt signaling pathway is the controling of energy metabolism and thus the determination of life span. It has been proved that, in a variety of organisms, inhibition of PI3K/Akt signaling pathway decreases the rate of cell metabolism so as to delay cell senescence. Therefore, one may ask if PI3K/Akt signaling pathway is commonly activated during the initiation and progression of OA and it would be a very intriguing question whether continued activation of PI3K/Akt signaling pathway inhibits the progress of OA by enhancing the capacity of cell repair, anti-apoptosis and extracellular matrix synthesis, or else causes OA by inducing chondrocytes senescence.We found that the expression of phoshpo-Akt and phospho-Pten were generally increased in human OA cartilage samples, indicating that PI3K/Akt signaling pathway was abnormally activated in OA patients. In the rat model of OA induced by destabilization of the medial meniscus (DMM), the expression of phoshpo-Akt and phospho-Pten were also increased rapidly and constantly in articular chondrocytes. These results showed that abnormal activation of PI3K/Akt signaling pathway is common during the initiation and progression steps of OA. In order to make clear whether continuous activation of PI3K/Akt signaling pathway directly initiates OA, we generated chondrocyte-specific Pten knockout mice(Col2a-Cre;Ptefl/fl) by crossing chondrocyte-specific Cre transgenic mice (Col2a-Cre) with conditional Pten alleles mice(Pterfl/fl). PI3K/Akt signaling pathway was activated in Pten-deficient articular chondrocytes. The Pten knockout mice gradually exhibit humpback, faltering steps and anchylosis as they grew up. The human OA-like phenotype of Pten-deficient mice was revealed by histology of Safranin-O staining. The articular cartilage of mutant mice at the age of1month appeared normal, however, at the age of4months, the articular cartilage became thin and rough partial destruction of articular cartilage was evident at the age of8months. At the age of12months, most of the articular cartilage gotlost and thus caused the explosion of subchondral bone. DMM accelerated the OA progression of8-week-old Pten knockout mice. Additionally, we blocked PI3K/Akt signaling pathway by deletion of Aktl in articular chondrocytes. The articular cartilage of chondrocyte-specific Aktl knockout mice (Col2a-Cre;Akt1fl/fl) exhibited no difference with wide type mice. Interestingly, articular cartilage structure of Col2a-Cre;Aktlfl/fl mice kept relatively normal without obvious erosion after the manipulation of DMM, verifying that blocking the activation of PI3K/Akt signaling pathway could effectively inhibit OA. From these compelling results above, we provided the first genetic evidence to address the causal relationship between abnormal PI3K/Akt signaling pathway and OA.We then detected increased protein oxidation in articular chondrocytes of Pten knockout mice as compared with that of wide type mice by OxyIHC and Oxyblot. Obviously increased protein oxidation was also observed in OA mice model induced by DMM. The ubiquitous protein oxidation indicated that oxidative stress occurred in Pten-deficient chondrocytes. Oxidative stress induces cellular senescence. SA-β-gal staining and Western blot detection of the secescence marker phospho-p53disclosed that articular chondrocytesof Pten knockout mice exhibited senescent phenotype as compared with the wide type mice. Chondrocytes senescence were also observed in OA mice model induced by DMM. N-Acetyl-L-cysteine (NAC) is an anti-oxidant that could effectively block the oxidative stress generated in cells. We found that the chondrocytes senescence and the OA phenotype of Pten knockout mice was successfully blocked after continual administration of NAC. These results suggested that oxidative stress induced by the excessive activation of PI3K/Akt signaling pathway is the key causal factor for OA.In this study, we have revealed the causal relationship between abnormal PI3K/Akt signaling pathway and OA under physiological conditions by taking advantage of a series of gene knockout mice. We provided the first genetic evidence to show that excessive activation of PI3K/Akt signaling pathway could cause OA by inducing oxdative stress. Theseresults provided new clues for better understanding the molecular mechnism underlying the initiation of OA.