Among the skeletal elements of the body, cartilage comprises a group of highly diverse tissues that perform a wide array of functions, including support, growth, and locomotion. Articular cartilage is present at the ends of long bones, where it serves as a load-bearing surface. Although cell distribution within the tissue is sparse, the highly glycosylated proteoglycan molecules of the extracellular matrix bind water, and by rapidly changing the cartilage hydration state, accommodate biomechanical forces. The network of oriented collagen fibers endows the tissue with much of its mechanical strength. During aging and progression of osteoarthritis (OA), there is a net loss of matrix macromolecules (in particular, the proteoglycan aggrecan), disruption of tissue architecture, and a concomitant limitation of joint function (1).
In the axial and appendicular skeleton, much of the bone growth is regulated by the activity of chondrocytes residing in a specialized transient cartilage, the epiphyseal growth plate. The maturing cells in this cartilage undergo a series of phenotype changes that include secretion of a unique set of proteins into the avascular extracellular matrix, up-regulation of alkaline phosphatase, and the release and subsequent mineralization of matrix vesicles (2). We have recently shown that prior to deletion from the growth plate, the mature hypertrophic chondrocyte becomes glycolytic and undergoes functional and immunohistochemical changes that are characteristic of autophagy (3). In addition, we recently demonstrated that hypoxia-inducible factor 1 (HIF-1), a transcription factor that responds to the tissue oxemic state, promotes chondrocyte autophagy (4). We have suggested that autophagy is an intermediate step in the chondrocyte life cycle that permits the cells to assume a mature phenotype prior to elimination by apoptosis (4, 5).
A second HIF isoform, HIF-2, has also been shown to be present in cartilage (6). In contrast to HIF-1, which serves to metabolically adapt chondrocytes to their microenvironment and sensitize them to apoptogens, HIF-2 is cytoprotective. Thus, up-regulation of HIF-2 lowers intracellular levels of reactive oxygen species (ROS) by promoting the activities of the dismutating proteins catalase and superoxide dismutase (SOD) (7). In addition, the small size of HIF-2–knockout animals suggests that there may be an increased rate of chondrocyte apoptosis that serves to impede normal long bone growth.
The overall goal of this study was to test the hypothesis that HIF-2 modulates the induction of autophagy by chondrocytes. We found that HIF-2α was expressed by cells in a number of cartilage types. Furthermore, this expression was decreased with maturation, aging, and onset of disease. Our finding that in cartilage, a decrease in HIF-2 expression was accompanied by an increase in HIF-1 expression, supports the idea that the activities of these 2 isoforms can be viewed as opposing arms of a rheostat that functions to modulate autophagic activity, thereby allowing chondrocytes to complete their life cycle.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
A number of recent studies have shown that local oxygen tension provides a microenvironmental signal that influences skeletal cell function (10, 11). While it is clear that the oxemic response is transduced by HIF-1, there is some evidence to indicate that HIF-2 expression may be a requirement for cartilage function. For example, a recent study of HIF-2α–knockout mice indicates that deletion of this gene (EPAS–/–) resulted in small animals, suggesting an abnormality of chondrocyte function (7). Herein, we showed that the HIF-2 homolog was expressed by chondrocytes of a number of different cartilage types. If HIF-2α has a protective function and serves to promote chondrocyte survival and function, then loss or deletion of this homolog is likely to be associated with development of disease or possibly with aging. Lending support to this notion, we noted that while HIF-2 was expressed abundantly by cells in articular cartilage and in the cartilage of mineralizing vertebrae, expression was reduced in cartilage from older animals and was low in mature end-plate cartilage. Similarly, there was a significant drop in expression in OA chondrocytes compared with healthy cells. Based on these findings, it is plausible to consider that this HIF homolog, together with HIF-1, may play a role in maintaining chondrocyte functional activity and tissue health.
We also examined the expression of HIF-2α in the epiphyseal growth plate and in the N1511 chondrocytic cell line. This HIF homolog was robustly expressed in vivo; in hypoxic culture, there was expression of this transcription factor. When we evaluated ROS generation in HIF-2α–silenced cells, we noted a small, but significant (20–30%), increase in total ROS values as compared with the pSHH controls. Thus, we confirmed earlier findings that indicated that HIF-2 regulates dismutation of ROS. Next, we evaluated the activity of catalase and SOD in HIF-2–silenced cells. These 2 genes encode proteins that serve to decrease levels of intracellular ROS. Predictably, when HIF-2α was suppressed, there was a marked decrease in the activities of both enzymes, and the SOD protein levels were also low.
Since these 2 dismutating enzymes are HIF-2 target genes, it is likely that their expression serves to minimize ROS generation in the chondrocytes. Minimization is of considerable importance, since ROS accumulation can influence structural as well as signaling activities of a number of cell types. The recent observation by Scherz-Shouval et al (12) that H2O2 modifies a conserved cysteine residue in the autophagy protein ATG4 couples HIF-2α activity with the induction of autophagy. We explored this possible relationship by evaluating autophagy and HIF-2 expression in a number of cartilage types. We noted that low levels of HIF-2 or suppression of HIF-2 correlated with a robust autophagic response in OA tissue as well as in aging cartilage; in growth-plate cartilage from HIF-2α–knockout mice, there was elevated autophagy. From this perspective, HIF-1 and HIF-2α have separate and opposing effects on the induction of autophagy.
To further examine the relationship between HIF-2 and autophagy, we probed for LC3 protein expression in the suppressed chondrocytes. There was redistribution of the microtubule-associated protein to form punctate structures that are characteristic of the autophagic state. Since LC3 redistribution was evident under serum-rich conditions, we inferred that autophagy is directly controlled by HIF-2 and is not an artifact induced by the culture system. We also used LysoTracker Red to probe cells for autophagic activity. The increase in LysoTracker fluorescence as a result of an elevation in lysosomal activity confirmed that the silenced cells in serum-replete medium displayed the functional characteristics of an autophagic chondrocyte. In addition, ultramicroscopic analysis indicated the presence of double-membrane vacuoles and isolation membranes, which are morphologic characteristics of an autophagic cell. Based on these observations, we concluded that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Thus, in contrast to HIF-1, which serves to promote autophagy (4, 13), HIF-2 regulates the extent of the autophagic response and can be viewed as acting as a brake for the accelerator function of HIF-1.
Our earlier studies (4), together with those discussed above, suggest that both HIF-1 and HIF-2 regulate chondrocyte maturation and the development of autophagy. Moreover, since the impact of each of these transcription factors is maximal at different stages of chondrocyte maturation and disease (HIF-2α is maximal in prehypertrophic cells, whereas HIF-1α is maximal in cells of the hypertrophic zone of the growth plate), the following question was raised: Does HIF-2 expression regulate HIF-1 activity? While the details of this relationship are fragmentary, one possible indication may come from the contrasting suppressive effects of HIF-1 and HIF-2 on each other (14). Thus, suppression or loss of HIF-2 results in higher expression of HIF-1, a potent inducer of autophagy (4, 13). In young healthy chondrocytes, HIF-2–dependent expression of SOD and catalase would serve to down-regulate ROS generation. Furthermore, HIF-1 is expressed at a minimal level. With aging and the onset of disease, the subsequent lowered expression of HIF-2 would lead to a loss of dismutating activity, an increase in ROS generation, and the promotion of HIF-1 expression. In addition, there was a decrease in mTOR activity and a reduction in the expression of both Akt-1 and the antiapoptotic protein Bcl-xL. These changes would cause an increase in chondrocyte autophagy, as was evident in cells from OA and aging cartilage.
Finally, from the perspective of the oxemic microenvironment, there were no significant differences in total ROS levels between hypoxic control cells and HIF-2–silenced cells. However, under normoxic conditions, there was a significant increase in ROS levels in HIF-2–silenced cells. These observations may explain the refractory nature of hypoxic chondrocytes to apoptogen challenge (8, 15). From a physiologic perspective, the hypoxic environment would allow the cells to complete their developmental and maturation processes in the presence of potential apoptogens generated by matrix degradation (16). We hypothesize that at the chondro-osseous junction, the increase in oxygen tension as a result of vascular invasion would lead to an increase in ROS generation in a manner similar to that reported for ischemia-reperfusion injury. In addition to the elevation in ROS generation, the decrease in HIF-2 levels and the stabilization of HIF-1 would further elevate the autophagic activity of chondrocytes, resulting in sensitization to apoptogen challenges and final removal by apoptosis.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Srinivas had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Bohensky, Terkhorn, Adams, Shapiro, Srinivas.
Acquisition of data. Bohensky, Terkhorn, Freeman, Adams, Garcia, Srinivas.
Analysis and interpretation of data. Bohensky, Terkhorn, Freeman, Adams, Shapiro, Srinivas.
Manuscript preparation. Bohensky, Terkhorn, Freeman, Shapiro, Srinivas.
Statistical analysis. Terkhorn, Srinivas.