Hypoxia and HIF-1 in Chondrogenesis

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1 Hypoxia and HIF-1 in Chondrogenesis ERNESTINA SCHIPANI Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA ABSTRACT: In endochondral bone development chondrocytes undergo proliferation, hypertrophic differentiation, mineralization of the surrounding matrix, death, blood vessel invasion, and finally replacement of cartilage with bone. The chondrocytic growth plate is a unique mesenchymal tissue, as it is avascular but it requires blood vessel invasion in order to be replaced by bone. We have recently provided evidence that the growth plate is hypoxic during fetal development. Adaptation to hypoxia is a critical event in numerous pathological settings, such as tumor progression and survival of tissues in which blood flow has been suddenly interrupted. One of the hallmarks of the response to hypoxia is activation of the transcription factor HIF-1. The von Hippel Lindau (VHL) tumor suppressor protein is a component of a ubiquitin ligase promoting proteolysis of HIF-1. By using a genetic approach, we have demonstrated that VHL and HIF-1 are critical regulators of endochondral bone development. KEYWORDS: hypoxia; HIF-1 ; VHL; VEGF; chondrocytes INTRODUCTION Low oxygen tension has an important role not only in tumor progression or in the biology of tissues in which blood flow has been suddenly interrupted, but also in development and differentiation. 1 2 We have recently provided evidence that the fetal growth plate is hypoxic. In this article, we will briefly summarize our current knowledge about hypoxia, HIF-1, and chondrocytes. Skeletal development depends on two mechanisms, intramembranous and endochondral. 3 5 Intramembranous bone formation, in which mesenchymal cells after condensation differentiate directly into osteoblasts, is involved in the formation of the flat bones of the skull. Endochondral bone formation, which accounts for the development of most of the other bones, is a two-stage mechanism: first chondrocytes shape a template, on which osteoblasts then differentiate to form bone. Address for correspondence: Ernestina Schipani, M.D., Ph.D., Massachusetts General Hospital, Endocrine Unit, 50 Blossom Street, Wellman 501, Boston, MA Voice: ; fax: schipani@helix.mgh.harvard.edu Ann. N.Y. Acad. Sci. 1068: (2006). C 2006 New York Academy of Sciences. doi: /annals

2 SCHIPANI: HYPOXIA AND HIF-1 IN CHONDROGENESIS 67 Endochondral bone development is a fascinating story of proliferation, differentiation, and death. 6 The process recapitulates basic and fundamental mechanisms of cell biology with a highly specific spatial and temporal pattern, and it thus constitutes a useful model for the analysis of such mechanisms. In recent years, the tools provided by modern genetic further strengthened the validity and usefulness of the model. Round proliferative chondrocytes synthesize type II collagen and form a columnar layer; they, then, differentiate into post-mitotic hypertrophic cells, which express predominantly type X collagen. This unique differentiation process is followed by mineralization of the surrounding matrix, death of hypertrophic chondrocytes, blood vessel invasion, and finally replacement of cartilage with primary spongiosa. The fetal growth plate is a unique mesenchymal tissue, since it is virtually avascular, but it requires blood vessel invasion for being substituted by bone. 7 Differently from the cartilage anlage, the perichondrium, a mesenchymal structure that surrounds the growth plate, is vascularized. 8 By using EF-5, which is a marker for bioreductive activity, 9 we have demonstrated that the fetal growth plate has a hypoxic central region, the most highly hypoxic chondrocytes being located in the round proliferative layer near the joint space, in the center of the columnar proliferative layer and in the upper portion of the hypertrophic zone. 10 Notably, the late hypertrophic chondrocytes, which are adjacent to the primary spongiosa, do not appear to be hypoxic; this finding is consistent with the extensive vascularization of the primary spongiosa. The EF5 data prompted us to ask two basic questions: (1) How do chondrocytes survive in a hypoxic milieu? and; (2) Does hypoxia play any role in chondrogenesis per se? The transcription factor HIF-1 plays a critical role in adaptive responses to hypoxia HIF-1 belongs to the PAS (PER- ARNT-SIM) subfamily of basic helix-loop-helix (bhlh) transcription factors. The basic domain is responsible for binding to DNA, while the helixloop-helix and PAS domains are critical for heterodimerization HIF-1 is a heterodimer that consists of the subunit HIF-1, and the subunit HIF- 1. Both HIF-1, and HIF-1 (also known as ARNT, aryl hydrogen receptor nuclear translocator) are constitutively expressed in cells. However, whereas HIF-1 is stable in normoxic conditions, HIF-1 is extremely unstable. Under normoxic conditions, HIF-1 is hydroxylated on specific proline residues by a recently discovered family of prolyl-hydroxylases The von Hippel Landau (VHL) tumor suppressor protein, which is a recently identified novel E3 ubiquitin ligase, 19 recognizes the proline-hydroxylated form of HIF-1 and targets it for polyubiquitination and degradation by the proteasome. Conversely, under hypoxic conditions, oxygen-sensitive prolyl-hydroxylase activity is reduced, and HIF-1 translocates into the nucleus. Within the nucleus, the complex binds to specific hypoxic responsive elements (HRE), thereby initiating the transcription of specific genes. HIF-1 regulates the hypoxic response by controlling genes with metabolic functions, such as glucose

3 68 ANNALS NEW YORK ACADEMY OF SCIENCES transport and metabolism, angiogenic factors like vascular endothelial growth factor (VEGF), proteins of the cell cycle and of the apoptotic machinery. 18,20 22 Mice that lack HIF-1 or VHL as result of homologous recombination die around embryonic days 8.5/ Therefore, in order to study the role of HIF-1 and VHL in endochondral bone development, we performed conditional knockout of HIF-1 and VHL, respectively, in chondrocytes, using a Cre-LoxP system in which Cre recombinase expression is driven by a fragment of the collagen type II promotor. Mice lacking HIF-1 die at birth; the causes of death are cleft palate (E. Zelzer and B. Olsen, unpublished data), and respiratory distress, which is a consequence of partial trachea collapse and malformation of the rib cage. 10 Conversely, absence of VHL in cartilage is not lethal, but leads to severe dwarfism. 27 Interestingly, the conditional deletion of both genes generates a phenotype that is apparently indistinguishable from the phenotype caused by lack of HIF-1 alone, suggesting that HIF-1 is epistatic to VHL in growth plate chondrocytes. 27 HIF-1 IS A SURVIVAL FACTOR FOR HYPOXIC CHONDROCYTES Conditional knockout of HIF-1 has demonstrated that this transcription factor is critical for the survival of hypoxic chondrocytes and negatively regulates chondrocyte proliferation. 10,28 Conditional knockout of VHL in chondrocytes has confirmed an important role of both HIF-1 and VHL in chondrocyte biology. 27 Embryos lacking HIF-1 in chondrocytes have shorter and deformed bones; furthermore, the growth plates are wider and misshapen. In particular, the center of the null growth plate is hypocellular, and occupied by abnormal cellular elements with picnotic nuclei. This spatially localized loss of cell viability, which was confirmed by Tunel and in situ hybridization analysis, was particularly striking in the core of the cartilaginous elements, whereas within few microns from the surface, the specimens appear to be histologically normal. 10 Interestingly, the central cell death phenotype of the HIF-1 null growth plates was not preceded by ectopic or premature hypertrophic differentiation, as shown by the analysis of collagen type X expression, which is a marker of hypertrophic chondrocytes, at different stages of fetal development. 10 This finding indicates that chondrocyte death secondary to lack of HIF-1 does not require hypertrophic differentiation, and, therefore, it is likely to be different at the molecular level from the chondrocyte apoptosis that precedes blood vessel invasion and replacement of cartilage with bone. A potential downstream effector of HIF-1 action as survival factor is VEGF, one of the most potent pro-angiogenic factors characterized by data. 29,30,37 We have clearly demonstrated by in situ hybridization analysis

4 SCHIPANI: HYPOXIA AND HIF-1 IN CHONDROGENESIS 69 that the fetal growth plate has two distinct domains of VEGF expression. 27,31 The domain with low level of VEGF expression comprises the center of the round, columnar and upper hypertrophic layers, respectively, that is the hypoxic zones of the growth plate. Conversely, the domain with high level of VEGF expression overlaps with the nonhypoxic layer of late hypertrophic chondrocytes that is adjacent to bone. Numerous studies have elegantly shown that expression of VEGF by late hypertrophic chondrocytes is critical for blood vessel invasion and replacement of cartilage by bone. 7,8,32 34 Notably, conditional knockout of HIF-1 in chondrocytes still allows detectable expression of VEGF in this domain. 10,31 This finding indicates that VEGF expression by late hypertrophic chondrocytes is largely HIF-1 independent. The transcription factor Runx2 has been reported to be an important regulator of VEGF in this zone. 35 Conversely, VEGF expression in the hypoxic areas of the fetal growth plate is controlled by HIF-1. 27,31 Numerous lines of evidence support this conclusion. In particular, in fetal growth plates in which HIF-1 transcriptional activity is upregulated as a result of VHL conditional knockout, the outside-inside pattern of expression of VEGF mrna is perturbed, VEGF mrna is now detectable throughout the whole growth plate, and is upregulated in round proliferative chondrocytes located in close proximity to the articular surface. 27 Furthermore, consistent with these data, hypoxia strikingly increases VEGF accumulation in chondrocytes in vitro, and this increase is HIF-1 dependent. 28 Lastly, conditional knockout of VEGF in chondrocytes, using the same Cre-LoxP strategy adopted for both HIF-1 and VHL conditional knockouts, leads to a central cell death phenotype in the center of the proliferative and upper hypertrophic zones that very closely mimics the one observed in HIF- 1 conditional knockout. 31 However, this phenotype is definitively milder than that observed in HIF-1 null growth plates. 10,31 Similar findings were also reported in mice that lack the soluble forms of VEGF, i.e., VEGF120 and VEGF164, as result of a universal knockout. 36 Taken together, these data show that VEGF is important, even if very likely not the only mediator of the central cell death observed in HIF-1 -deficient growth plates. Consistent with the hypoxic status of the growth plate, mrna encoding phosphoglycerokinase (PGK), a key enzyme of the anaerobic glycolysis pathway, is strikingly upregulated in the fetal chondrocytic growth plate in comparison with the surrounding tissues, and, like VEGF mrna, it displays and outside-inside pattern of expression that resembles the pattern of oxygenation of the growth plate. 10 Notably, lack of HIF-1 decreases PGK mrna expression to virtually background levels; 10 conversely, increased HIF-1 transcriptional activity in VHL null growth plates leads to a generalized expression of PGK mrna throughout the whole growth plate, with loss of the outside inside pattern and upregulation in the round proliferative chondrocytes adjacent to the articular surface. 27 Impaired regulation of metabolic pathways is likely thus to be an additional pathogenetic event causing the central cell death phenotype observed in HIF-1 -deficient growth plates. Glycolysis and apoptosis have

5 70 ANNALS NEW YORK ACADEMY OF SCIENCES been considered for long time to be major but independent pathways that are critical for cell survival. A recent report provides evidence that the proapoptotic protein BAD and the glycolytic enzyme glucokinase integrate both pathways as part of a common network in mitochondria. 38 HIF-1 may contribute to integrating glucose metabolism and apoptosis by regulating expression of enzymes of the glycolytic pathway. HIF-1 directly regulates both pro- and anti-apoptotic genes. 21,29 42 It will thus be interesting to now investigate which other molecular mechanisms mediate the role of HIF-1 as a survival factor in chondrocytes, in addition to its regulation of anaerobic glycolysis and VEGF. HIF-1 AND VHL SHAPE UP THE GROWTH PLATE BY MODULATING CHONDROCYTE SIZE AND PROLIFERATION It has been reported that hypoxia leads to the arrest of cell proliferation, at least in part through upregulation of HIF-1 transcriptional activity. 43,44 VHLdeficient growth plates display both hypocellularity and a striking decrease in chondrocyte proliferation rate, which are concomitant to an increase of HIF-1 accumulation, and are likely to be the cause of the dwarfism of mice lacking VHL in chondrocytes. 27 It is paradoxical that in fetal growth plates the lack of this tumor-suppressor gene inhibits chondrocyte proliferation. 27,45 However, the finding is consistent with the inhibition of cell proliferation by the upregulation of HIF-1 transcriptional activity. 44 Interestingly, to further support the model that lack of VHL inhibits cell proliferation through upregulation of HIF-1 transcriptional activity, viable chondrocytes at the periphery of HIF- 1 -deficient fetal growth plates show a striking increase in the proliferation rate, i.e., chondrocytes lacking HIF-1 die if located in the center of the fetal growth plate, whereas they proliferate at a higher rate at its periphery. 10 Proliferating chondrocytes of VHL-deficient growth plates are enlarged with a high cytoplasm to nucleus ratio. 27 Our current working hypothesis is that decreased proliferation is the pathogenetic event causing both the hypocellularity and the appearance of these somehow atypical cells in the VHL null growth plates. 27 Interestingly, this unique cartilage phenotype of VHL null growth plates, which is characterized by reduced proliferation rate and increased cell size, has not been described for other knockout models in which chondrocyte proliferation has been reported to be severely impaired. 46 Therefore, it is possible that the increased cell size observed in the VHL-deficient growth plates is not the necessary or direct consequence of the decreased cell proliferation per se. Lack of VHL in chondrocytes may uncouple cell size from cell proliferation by regulating a yet unknown molecular mechanism, which may or may not involve HIF-1.

6 SCHIPANI: HYPOXIA AND HIF-1 IN CHONDROGENESIS 71 CONCLUSIONS Taken together, our findings lead to a working model in which hypoxiadependent upregulation of HIF-1 transcriptional activity is critical for the survival of hypoxic chondrocyte, and also significantly contributes to shaping up the growth plate by inhibiting chondrocyte proliferation and probably regulating cell size. It will be now interesting to investigate the potential role of hypoxia and HIF-1 in early chondrogenesis. In any event, HIF-1 may not be the only downstream effector of hypoxia in cartilage, and vice versa hypoxia may not be the only regulator of HIF- 1 transcriptional activity in chondrocytes, as also shown in other cellular systems. 31 In addition, it is still an open and important question whether indeed VHL actions in cartilage are exclusively mediated by HIF-1. One of the best-documented consequences of hypoxia in vivo is increased angiogenesis. It is thus quite intriguing and almost paradoxical that hypoxia and HIF-1 are of critical importance in the biology of cartilage, which is a classical avascular tissue. The apparent paradox suggests that the hypoxia/vhl/hif pathway may turn on a genetic program that could be specific for chondrocytes, as also shown for other cell types. 43 Unveiling this program could expand our understanding of the molecular mechanisms underlying the hypoxic response in development. ACKNOWLEDGMENT This work was supported by NIH grant AR REFERENCES 1. GIACCIA, A., M. SIMON & R. JOHNSON The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease. Genes Dev. 18: CHEN, E., M. FUJINAGA & A. GIACCIA Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development. Teratology 60: KARSENTY, G The complexities of skeletal biology. Nature 423: ERLEBACHER,A.et al Toward a molecular understanding of skeletal development. Cell 80: KRONENBERG, H Developmental regulation of the growth plate. Nature 423: SCHIPANI, E. & S. PROVOT PTHrP, PTH, and the PTH/PTHrP receptor in endochondral bone development. Birth Defects Res. Part C Embryo Today 69: VU, T. et al MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 93:

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