Erythropoietin, tumours and the von Hippel–Lindau gene: towards identification of mechanisms and dysfunction of oxygen sensing

Michael S. Wiesener and Kai-Uwe Eckardt

Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow Klinikum, Humboldt University, Berlin, Germany

Keywords: erythropoietin; gene expression; hypoxia; hypoxia-inducible transcription factors; renal tumours; von Hippel–Lindau disease

Introduction

One of the central issues of physiology is the adaptation to alterations in supply or need of molecules that are essential for cellular functions. Mammalian and many non-mammalian organisms depend critically on oxygen for generation of energy that is required to maintain cellular structure and function. The adaptation to changes in oxygen supply and consumption includes alterations in oxygen uptake from the environment (respiration), its transport within the body (circulation), and the modulation of alternative pathways for energy production (anaerobic metabolism). Some of the immediate responses to changes in oxygen supply involve alterations in the conductance of ion channels. More sustained adaptation, however, is based on changes in cellular gene expression and protein synthesis. Considerable advances have been made recently in understanding the molecular mechanisms of oxygen dependent gene regulation and have led to exciting insights into their importance in the biology of renal and non-renal cancer.

Hypoxia-inducible transcription factors

The glycoprotein hormone erythropoietin (Epo), which increases red cell production and thus oxygen transport capacity, has long been considered as a ‘prototype’ of an oxygen regulated molecule. Its production rate in liver and kidneys can be upregulated more than hundred-fold within a few hours of hypoxia [1]. The possibility that this induction could be mediated by cellular energy deprivation was considered. However, it was found that molecular oxygen itself is the direct regulator of the transcriptional rate of Epo. A DNA element was identified adjacent to the coding region of the Epo gene, which is essential and sufficient to convey increased gene expression during hypoxia (hypoxia responsive element, HRE). Activation of the HRE requires binding of a specific, hypoxia-inducible transcription factor (HIF) [2]. The role of this transcriptional regulator was soon found not to be limited to the control of Epo production. In vitro, virtually all cells respond to hypoxia with an upregulation of HIF, and besides Epo many other genes have meanwhile been identified as downstream targets of HIF. Amongst others, these include genes involved in the regulation of vascular tone (NO synthase, endothelin-1, adrenomedullin), new vessel formation (vascular endothelial growth factor, VEGF; platelet derived growth factor, PDGF), catecholamine synthesis (tyrosine hydroxylase) and anaerobic glycolysis and cellular glucose uptake [36]. HIF, therefore, appears to be a ubiquitously expressed key regulator of adaptation to reduced oxygen supply.

The HIF molecule is a heterodimer composed of an {alpha}- and a ß-subunit of basic helix-loop-helix PAS proteins, with the {alpha}-subunit representing the oxygen regulated component (Figure 1Go). Interestingly, degradation rather than production of HIF{alpha} is oxygen regulated and determines HIF abundance and function. In the presence of oxygen, newly formed HIF{alpha} molecules are rapidly destroyed by proteasomes, organelles which are responsible for intracellular protein degradation [7,8]. In order to be recognized for proteasomal destruction, proteins have to be ‘labelled’ by the addition of ubiquitin molecules. Not surprisingly, ubiquitination is a tightly controlled process, performed by a cascade of specific ligase complexes. For HIF{alpha}, a previously recognized tumour suppressor gene product is the critical part of an active E3 ubiquitin ligase, conveying target recognition and binding. Loss of function of this protein due to a germline mutation leads to a characteristic cancer syndrome: von Hippel–Lindau (VHL) disease.



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Fig. 1.  Schematic presentation of the O2 dependent regulation of hypoxia-inducible transcription factors. In the presence of oxygen, proline residues of HIF{alpha} are hydroxylated, which allows HIF binding to VHL and subsequent proteasomal degradation (upper part). Lack of oxygen (hypoxia; lower left) prevents HIF proline hydroxylation and, thus, degradation. HIF{alpha} is stabilized, forms the functional heterodimer with HIF ß, and binds and activates hypoxia response elements (HRE). VHL inactivation (lower right) mimics hypoxia, because despite HIF proline hydroxylation, binding to VHL does not occur.

 

The familial VHL cancer syndrome

Von Hippel–Lindau disease is an autosomal dominant genetic disorder named after E. von Hippel and A. Lindau, who described retinal angiomas and their association with central nervous system lesions, respectively. Besides retinal angiomas and cerebellar and spinal haemangioblastomas, affected individuals frequently develop renal cell carcinomas (RCC), which represent their most common cause of death [9]. Pheochromocytomas and pancreatic cysts are further manifestations. The VHL gene, located on chromosome 3, was isolated in 1993 [10] and encodes a 213 amino acid protein (pVHL). Patients with VHL disease carry a germline VHL mutation. They develop tumours when the remaining wild type allele is inactivated in susceptible cells. Although VHL disease is a comparatively rare disorder, the association between pVHL loss of function and tumour development was found to be of much greater relevance since approximately half of the sporadic haemangioblastomas and the majority of sporadic clear cell RCC are also associated with biallelic VHL inactivation [9]. Clear cell or ‘common’ renal carcinoma accounts for 75–80% of RCCs, the tenth most frequent type of cancer [11,12].

VHL targets HIF for O2-dependent degradation

The VHL gene behaves both genetically and functionally as a classical tumour suppressor gene. Conforming to Knudson's two hit hypothesis [13], a double hit of both alleles is necessary for tumorigenesis. Restoration of VHL function in tumour cells that lack a wild type VHL allele suppresses tumour formation in nude mice [1416]. In addition, certain features of the tumours associated with VHL disease have long suggested a role of pVHL in oxygen regulated gene expression. Tumours lacking VHL function are abundantly vascularized and further analysis indicated that this is driven at least in part by overexpression of VEGF [9]. In addition, both brain and kidney tumours occasionally overproduce Epo and can lead to erythrocytosis [17]. In fact, isolated cells lacking pVHL exhibit stable high level expression of several hypoxia-inducible genes independent of ambient oxygen tensions [15,18]. Following these observations it was shown that pVHL forms a complex with HIF{alpha}. Cells lacking intact pVHL are unable to degrade the transcription factor in the presence of oxygen, i.e. lack of function of pVHL mimics hypoxia [19] (Figure 1Go). Direct binding occurs between the ß domain of pVHL and a subdomain of HIF{alpha} which has been shown to be both necessary and sufficient for its oxygen-dependent degradation [8]. Very recently, investigators in Oxford and Boston have shown independently that enzymatic hydroxylation of a proline residue within this domain is a critical step in the oxygen sensing mechanism [20,21]. Hydroxylation of proline 564 is essential for binding to pVHL, and this protein modification depends on the presence of molecular oxygen. The two known functional subdomains of pVHL, termed {alpha} and ß, also represent the hot-spot regions of VHL mutations in tumours [22]. The ß domain is responsible for target recognition, and the {alpha} domain binds the multiprotein complex necessary for ubiquitination (at least elongins B and C, Cul2 and Rbx1). Interestingly, not only mutations in the ß-domain, but also in the {alpha}-domain, can lead to impaired HIF{alpha} degradation [23]. It is possible that the latter occurs either because it affects pVHL conformation at a distance or because it impairs binding to the ubiquitination protein complex, which is essential for HIF degradation.

VHL loss of function exemplifies the importance of HIF

When tumour cell clusters grow beyond a size of a few millimetres, their supply of oxygen and nutrients becomes growth limiting. Only invasion with blood vessels and enhanced energy metabolism allow for further proliferation. Several lines of evidence indicate that microenvironmental hypoxia within growing tumours is the key signal that mediates these adaptations and that upregulation of HIF plays a critical role in this process [24,25]. In support of this concept, focal expression of HIF has been demonstrated in several types of cancer [26,27]. Preferential expression of HIF at the margin between viable and necrotic tumour tissue and at the invading edges of tumours of different origin is compatible with regional stimulation through hypoxia. In contrast, in tumours associated with VHL loss of function (i.e. haemangioblastomas and clear cell RCCs), due to a presumed genetic rather than microenvironmental stabilization, virtually every tumour cell accumulates HIF [26,28,29]. As a consequence, VHL negative tumours express HIF more frequently and at higher levels [29]. In addition, we were able to demonstrate that HIF-1{alpha} protein levels in renal tumours correlate closely with the expression of its target genes, suggesting that upregulation of the transcription factor is of dominant functional significance [29]. Tumours associated with VHL loss of function thus exemplify the role of HIF for tumour biology. This is more difficult to demonstrate in other tumours, where HIF-activation is only regional and in which HIF is rapidly degradable. Moreover, overexpression of HIF offers a unifying mechanistic hypothesis for apparently unrelated characteristics of renal tumours, such as hypervascularity, high metastatic potential and occasional erythrocytosis.

HIF as molecular target for intervention

The mechanisms by which loss of pVHL induces tumour development are still unclear. Whether they are related to stabilization of HIF or other potentially independent effects remains to be investigated. It is very likely, however, that upregulation of HIF and increased transcription of its target genes promotes tumour growth. HIF{alpha}, therefore, appears as a very attractive candidate for therapeutic intervention. In patients with tumours, blocking of HIF activity would be the goal, and such attempts may be particularly worthwhile in metastatic renal cancer of clear cell histology. On the other hand, the association between HIF activity and vascularization, as is evident in VHL negative tumours, suggests that temporary overexpression of HIF could be a great advantage in several types of ischaemic diseases. Further unravelling of critical steps in HIF regulation will undoubtedly help to tailor and pursue strategies in each direction.

Note added in proof

Proline hydroxylases responsible for oxygen dependent hydroxylation of HIF have recently been identified [30,31].

Notes

Correspondence and offprint requests to: Prof. K.-U. Eckardt, Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. Email: kai\|[hyphen]\|uwe.eckardt{at}charite.de Back

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