No abstract

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.

Objective. We have previously demonstrated that the transcription factor hypoxia-inducible factor 1 (HIF-1) promotes the onset of autophagy in chondrocytes. The overall goal of this study was to test the hypothesis that another HIF family transcription factor, HIF-2, modulates the induction of autophagy by chondrocytes.Methods. Expression of HIF-1, HIF-2, and light chain 3 (LC3) in human and murine articular cartilage was visualized by immunohistochemistry. Suppression of HIF-2 was achieved using small interfering RNA technology. Assessments of autophagic flux and lysosomal activity, as well as ultrastructural analysis, were performed in chondrocytes in cell culture.Results. HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2␣ was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1 and mammalian target of rapamycin activities, reduced Bcl-x L expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2␣-knockout mouse growth plate showed an elevated autophagic response throughout the plate.Conclusion. Based on these observations, we conclude that HIF-2 is a potent regulator of autophagy in maturing chondrocytes. Our data suggest that this protein acts as a brake on the autophagy-accelerator function of HIF-1.

The overall goal of the investigation was to examine autophagy in the growth plate and to ascertain how this process was regulated. Herein, we show that in the postmitotic maturing zone of the growth plate, chondrocytes express an autophagic phenotype. This robust and particulate immunohistochemical response provides direct evidence that autophagy is a new and transient stage in the chondrocyte maturation pathway. We found that induction of autophagy was regulated by mTOR, a sensor of cellular metabolism. When mTOR was inhibited, changes in LC3 fluorescence indicated that this kinase regulated development of the autophagic state. To determine if AMP kinase was required for chondrocyte autophagy, we suppressed its expression in N1511 cells using siRNA technology. When these cells were serum starved, a condition that triggers autophagy, there was no change in LC3 distribution. This result confirmed that AMP kinase was required for the induction of the autophagic response. Based on the 2 studies described above, and our previous observation that HIF-1 is required for the induction of autophagy, we put forward the hypothesis that autophagy is regulated by the activities of AMP kinase and mTOR in a HIF-1-dependent manner. Once autophagy is activated, the postmitotic chondrocytes would be expected to remain viable in their unique microenvironment and complete their life cycle.

The goal of the study is to examine the relationship between the sensor molecules, Hypoxia Inducible Factor-1 (HIF-1), AMP activated Protein Kinase (AMPK) and mammalian Target of Rapamycin (mTOR) in chondrocyte survival and autophagy. We showed that chondrocytes expressed the energy sensor AMPK-1 and that activation increased with maturation. In addition, we showed that thapsigargin treatment activated AMPK and autophagy in a HIF-1 dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Based on these findings, it is concluded that due to the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1 dependent manner.

The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient deprivation and hypoxic culture, stress conditions that osteocytes encounter in vivo. Furthermore, in response to calcium stress, the transcription factor hypoxia inducible factor 1 regulated MLO-A5 autophagy. Finally, we showed that the more differentiated MLO-Y4 osteocyte-like cells exhibited a significant basal autophagic flux. Based on these findings, we suggest that raising the level of autophagic flux is a mechanism by which differentiated bone cells survive in a stressful environment.

Chondrocytes in the growth plate and articular cartilage and osteocytes subsumed in Haversian bone exist in environmental niches that are characterized by a limited oxygen supply. In these tissues, cells display a hitherto unrecognized state in which there is evidence of autophagy. The autophagic condition serves to promote cell survival. When the response is triggered, the cell cannibalizes itself to generate energy; if extended, then it can activate Type II apoptosis. We opine that survival is dependent on niche conditions and regulated by crosstalk between mTOR, AMPK and HIF-1 and HIF-2. Recent studies suggest that HIF-2 is a potent regulator of chondrocyte autophagy and that this protein acts as a brake to the stimulatory function of HIF-1. Accordingly, the oxemic state of the tissue, its nutrient supply as well as the energetic state of the cells regulates autophagic flux. From a clinical viewpoint, it may be possible to enhance skeletal cell survival through drugs that modulate the autophagic state and prevent the induction of apoptosis.

The overall goal of the investigation was to examine the activity and role of the PIM serine/threonine protein kinases in the growth plate. We showed for the first time that PIM-2 was highly expressed in epiphyseal chondrocytes and that the kinase was required for critical activities linked to cell survival. These activities were independent of those mediated by Akt-1. It was noted that PIM-2 protected chondrocytes from rapamycin sensitized (TOR inhibited) cell death. Since inhibition of mTOR caused autophagy, we examined the autophagic response of PIM-2 silenced cells. We showed that PIM-2 promoted expression and organization of autophagic proteins LC3, and Beclin-1 and enhanced lysosomal acidification. At the same time, PIM-2 modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. We conclude that PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy.