AUTOPHAGY IN MINERALIZING TISSUES

Abstract

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.

INTRODUCTION

The activities of chondrocytes embedded in a specialized cartilaginous structure called the epiphyseal growth plate are primarily responsible for bone growth. These chondrocytes proliferate and then in the post-mitotic stages of their life cycle undergo a series of rapid temporal and spatial changes that lead to the development of a unique terminally differentiated phenotype. It has also been shown that this differentiation cascade is accompanied by changes in chondrocyte energy generation. Thus, cells in the proliferating zone generate energy by oxidative metabolism, while hypertrophic chondrocytes become anaerobic and utilize glycolysis for ATP synthesis.¹ These metabolic adaptations serve to accommodate cells to the avascular nature of the epiphyseal cartilage. In this unique microenvironment, there is stabilization and activation of the HIF transcription factors and selective modulation of the expression and activity of redox-sensitive genes.^2,3 These adaptations are required to facilitate chondrocyte survival and promote terminal differentiation leading to the synthesis of the calcified extracellular matrix. Prior to osteogenesis, these differentiated hypertrophic cells are deleted from the cartilage through the induction of apoptosis.⁴

THE GROWTH PLATE MICROENVIRONMENT AND CHONDROCYTE AUTOPHAGY

The avascular microenvironment of the growth plate results in complex signaling circuits that are controlled by feed forward and negative feedback loops involving a growing number of regulatory molecules.⁵ Herein, the concept is advanced that the maturing chondrocyte exist in a topographically defined, three-dimensional space, the niche. Within the niche, cells sense the physical environment (nutrient availability, oxygen tension, etc) and generate signals that regulate cell function. Changes in the niche structure or organization has been shown to lead to disregulated function and development of disease.⁶

In earlier studies, we have demonstrated that within this niche, the cellular oxemic state, transduced by HIF-1 enhanced the development of a new and transient maturation stage, autophagy.⁷ It was suggested that HIF-1 mediated regulation of autophagy was a required step for the maturation of chondrocytes prior to their removal from the plate by apoptosis.

The autophagic state differs from conventional apoptosis (Type 1 Programmed Cell Death) in that the cells exhibit a series of morphological feature: of these the most characteristic is the engulfment of cytosolic components and organelles in vacuoles (autophagosomes). In this double membrane vacuole, fused lysosomes degrade the enclosed macromolecules. Studies with yeast indicate that over 20 genes (ATG genes) are involved with autophagolysosome formation.⁸ The autophagic response is seen during stress conditions such as nutrient depletion, hypoxia and aging and may even be implicated in the pathogenesis of diabetes, neurodegenerative diseases, and cancer.^9,10 When the response is triggered, the cell cannibalizes itself, using its own protein and lipid building blocks as nutrients. In autophagosomes, these macromolecules are catabolized to generate energy for the nutritionally-challenged cell. The energy released from these molecules extends the longevity of the cell. Whether autophagy delays cell death is still debated; how autophagy regulates chondrocyte death is a question of considerable importance with respect to normal tissue function and chondrocyte survival.

Since autophagy is closely linked to the induction of the death process, it has been postulated that that the autophagic protein Beclin-1 together with the anti-apoptotic protein Bcl2 act as a rheostat that regulates cell survival.⁹ Based on our studies on the oxemic microenvironment mentioned above, we opine that regulation of the survival-death rheostat is more complex than originally indicated. We put forward the notion that chondrocyte survival is dependent on niche conditions and regulated by crosstalk between HIF-1, HIF-2, mTOR and AMPK.¹¹ If the rheostat is directed towards survival, then the induction of autophagy serves to delay chondrocyte death until completion of the maturation process. Postponement of apoptosis and retention of chondrocytes in the growth plate would prevent their replacement by osteoblasts, and bone growth would enter a stasis mode. Release of autophagy and movement of the rheostat towards apoptosis would be expected to promote chondrocyte death, accelerate cartilage replacement by bone and induce bone growth (saltation). From a clinical viewpoint, suppression of the autophagic response would be expected to lead to disregulated bone growth, slow fracture repair and delayed distraction osteogenesis. Most importantly, since therapeutic agents can influence autophagy and the activities of regulating pathways, there is the possibility that these drugs could be used clinically to enhance mineralized tissue formation and repair.

CHONDROCYTE AUTOPHAGY AND THE HIFs

While the role of HIF-1 in the regulation of chondrocyte function has been extensively investigated, the role of HIF-2 in skeletal tissue biology has received minimal attention. With regard to their ability to transcriptionally regulate specific hypoxia-responsive genes, HIF-1 and HIF-2 have distinct functions. For example, glycolytic genes appear to be predominantly regulated by HIF-1¹² whereas dismutating enzyme expression is regulated by HIF-2.¹³ Target gene selectivity between HIF-1 and HIF-2 may be the result of tissue-specific interactions with other nuclear factors, differential interactions with transcriptional cofactors, or simply reflect tissue- and cell type-dependent differences in the ratios of HIF-α protein levels. Moreover, while HIF-1α is ubiquitously expressed_and serves to metabolically adapt chondrocytes to their microenvironment and sensitizes them to apoptogens¹⁴, the expression and function of HIF-2 needs further rigorous attention.

We and others have previously shown that HIF-2 is present in various types of cartilaginous tissue and can be viewed as serving a cytoprotective function.¹⁵ Thus, upregulation of HIF-2 lowers intracellular ROS levels by promoting the activities of the dismutating proteins, catalase and superoxide dismutase.^16,13 While ROS generation can effect a number of different systems in the cell, new research points to the importance of these species in promoting the development of the autophagic state.¹⁷ Thus, the possibility exists that the autophagic response may be mediated through the regulation of chondrocyte ROS levels by HIF-2. From this perspective, chondrocyte maturation and the vascularity-dependent decrease in oxygen tension might be expected to promote an increase in HIF-2 expression and a concomitant elevation in superoxide dismutase and catalase activities. Together these enzymes would serve to suppress ROS levels in the cartilage. Since REDD-1 is activated by both HIF-1 and ROS to block signals from mTOR, a potent inhibitor of autophagy, the oxygen tension can be viewed as an effective regulator of the autophagic response.

From a broader perspective, it is worth noting that HIF-2 is also expressed in the cells of the end plate cartilage of the intervertebral disc as well as articular chondrocytes (unpublished). Interestingly, the levels of HIF-2 decreases with age and the onset of osteoarthritis. This decrease is associated with a reciprocal increase in the levels of HIF-1 and an increase in the autophagic phenotype in all the cartilaginous tissues examined (unpublished). Based on these observations, we surmise 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.

ROLE OF AMP KINASE AND HIF-1 IN THE REGULATION OF CHONDROCYTE AUTOPHAGY

AMP-activated protein kinase (AMPK) is a key sensor of changes in energy metabolism. The protein is exquisitely sensitive to the intracellular AMP/ATP ratio and when activated, it inhibits enzymes involved in biosynthetic ATP consuming pathways (18). Recent studies suggest that the role of AMPK is not limited to the regulation of metabolic flux; AMPK signaling also modulates biological pathways related to protein synthesis and mitochondrial biogenesis.¹⁹ In addition, the activity of AMPK is modulated by intracellular calcium levels. This is particularly important in the case of mineralizing tissues and is discussed later from the perspective of bone function. Thus, AMPK serves as a checkpoint to sustain energy balance by modulating biological responses to changes in niche conditions.

It has been demonstrated that AMPK is sensitive to the oxemic status of the tissue. This finding suggests that AMPK and HIF are components of a concerted cellular response to maintain energy homeostasis in a low O₂ or ischemic tissue microenvironment. In cartilage, this kinase is activated by a decrease in the intracellular energy charge and results in the inactivation of mTOR (unpublished). Inactivation has been shown to induce autophagy not only in chondrocytes (unpublished), but also in other cells.²⁰ Noteworthy, these studies have demonstrated that the induction of autophagy by active AMPK is dependent on a functional response by HIF-1. Thus, silencing of HIF-1 in chondrocytes, inactivated AMPK, even under nutrient starved conditions, with a suppression of autophagic flux (unpublished). The proposed interplay between the HIFs, AMPK, mTOR and the induction of autophagy in chondrocytes is shown in Fig 1.

Figure 1. Microenvironmental factors regulating autophagy on mineralizing cells.

Under conditions of hypoxic stress, HIF-1 is stabilized leading to an energy charge dependent activation of AMPK. In addition, there is an increase in HIF-1 mediated REDD-1 expression. Thus, mTOR is inactivated resulting in a stimulation of autophagic flux. In parallel, an increase HIF-2 expression results in the dismutation of ROS, potent activators of autophagy. The reciprocal activities of HIF-1 and HIF-2 influence cell survival-death decisions.

AUTOPHAGY IN BONE CELLS

Almost 95% of the resident cells of bone are terminally differentiated osteocytes. During terminal differentiation, there is a functional shift from synthesis and mineralization of the extracellular matrix by osteoblasts, to a maintenance state characterized by minimal osteocytic secretory and mineralization activities.²¹ The fully differentiated osteocyte is contained within the mineralized matrix of bone and out of direct contact with the vascular supply. Earlier studies suggest that oxygen tension plays a key role in regulating the bone cell phenotype.^22,23,24 Furthermore, the low metabolic activity of osteocytes could result in a slowed rate of delivery of gases and nutritional factors.

We had previously shown that HIF-1 was required for the ordered developmental progression of a preosteocyte to the terminally differentiated non-mineralizing osteocyte.²⁴ Since, HIF-1 regulates autophagic flux.^7,25 it is not unreasonable to suggest that from the microenvironmental perspective, osteocytes, generate at least a portion of their metabolic needs through the induction of autophagy. Indeed, we can now demonstrate that osteocytes undergo autophagy in vivo (unpublished). In addition, we have shown that HIF-1 is required for this process to occur. While autophagy is active in many osteocytes and may well serve to generate metabolic energy, the relationship of this process to biomechanical and molecular stress has not been elucidated. Directly relevant to this point, we have recently demonstrated that ER stress induces osteoblast/osteocyte autophagy. Furthermore, our studies show that ER stress also induces HIF-1, and that HIF-1 is required for the induction of autophagic activity resulting from ER stress (unpublished). In chondrocytes, this latter activity involves HIF-1 mediated activation of AMPK; whether a similar mechanism is operative in osteoblasts and osteocytes is not known at this time.

CONCLUSIONS AND FUTURE DIRECTIONS

Paradoxically, as mentioned above, autophagy is closely linked to both survival as well as the induction of the death process. To explain how the cell can distinguish between these two activities, studies have been performed evaluating the role of two proteins: Bcl2 a pro-survival factor and Beclin-1 a critical autophagic molecule. These two proteins form the molecular rheostat that was mentioned earlier. If the rheostat is directed towards survival, then the induction of autophagy serves to delay chondrocyte or osteoblast/osteocyte death and would sustain matrix deposition. Further, promotion of autophagy and movement of the rheostat towards apoptosis would be expected to promote cell death. From a clinical viewpoint, it may be possible to utilize the rheostat concept and modulate survival-death activities. For example in osteoarthritis, if the rheostat can be directed towards survival then the condition would be ameliorated or even prevented. Most importantly, since a number of therapeutic agents are known to influence the autophagic response and the activities of autophagy regulating pathways, there is the real possibility that these drugs could be used clinically to treat not just osteoarthritis but other conditions where cells enter an autophagic state. One area of importance is the control of post menopausal osteoporosis; in this condition, there is a major loss of cell function that involves both osteoblasts as well as osteocytes. Experiments are in progress to monitor this condition and to assess whether the rheostat movement towards apoptosis can be retarded through the use of drugs that promote development of the basal autophagic state.