Current Pharmaceutical Design - Volume 15, Issue 33, 2009

Within the last decade it became evident that a family of hypoxia-inducible transcription factors (HIFs) are key players in the cellular response to limited O2 supply. From the three HIFs known today, HIF-1 is the best characterized and it appears to be of special interest since it activates transcription of glycolytic enzymes and glucose transporters as well as angiogenic growth factors to improve nutrient and blood supply to meet the energy demands of the cell. Thus, HIFs appear to have a central role in the adaptive cellular program responding to hypoxia in normal tissues. HIFs are heterodimers composed of α- and ß-subunits, the latter also known as the ARNT proteins. While the levels of the β-subunits seem to be quite stable, the α-subunit levels are low under normoxia and upregulated under hypoxia. Thereby transcriptional, posttranscriptional and posttranslational processes are involved. From the latter, the oxygen-dependent hydroxylation of proline 402 and 564 followed by the binding of the von Hippel-Lindau (VHL) protein initiates degradation of HIF-1α by the ubiquitin-proteasome system whereas hydroxylation at asparagine 803 initiates loss of the coactivator p300/CBP. Importantly, hypoxia is also a major feature of a number of diseases and almost all solid tumors contain hypoxic regions in which O2 concentrations are greatly reduced compared to the surrounding normal tissue. Further, a number of studies from various tumor entities showed that increased levels of HIFs, especially HIF-1α are associated with a poor prognosis. Thus, tumor cells survive and proliferate under extreme conditions where nutrients and oxygen are limited, and HIFs initiate the expression of various genes wich increase the risk of patient mortality. In addition, many studies focused on the understanding of cancer progression have shown that HIF α-subunits also respond to growth and coagulation factors, hormones, cytokines or stress factors under non-hypoxic conditions. Altogether this increased the interest in HIF α-subunits, especially HIF-1α, as a drug target and new, specific therapeutic strategies targeting the HIF pathway are intesively investigated.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric protein composed of HIF-1α and HIF-1α subunits, which is activated in response to reduced O2 availability. HIF-1 transactivates genes encoding proteins that are involved in key aspects of the cancer phenotype, including cell immortalization and de-differentiation, stem cell maintenance, genetic instability, glucose uptake and metabolism, pH regulation, autocrine growth/survival, angiogenesis, invasion/metastasis, and resistance to chemotherapy. Increased HIF-1α levels, as determined by immunohistochemical analysis of tumor biopsy specimens, is associated with increased mortality in many human cancers. Drugs that inhibit HIF-1 activity and have anti-cancer effects in vivo have been identified and clinical trials are warranted to establish the contexts in which addition of such agents to therapy protocols will result in increased patient survival.

The hypoxia-inducible factor HIF-1 has been shown to be mandatory for the cellular adaptation to hypoxia. In addition, evidence has been provided that HIF-1 can mediate various stress responses and that it may play an important role under inflammatory conditions even independently of hypoxia. HIF-1 is a heterodimer consisting of an α-subunit which is subject to tight regulation, and a β-subunit, also termed ARNT, which appears to be constitutively expressed. In addition to the complex network controlling the cellular content of HIF-1α at the level of protein stability, recent evidence showed that HIF-1α levels can also be regulated at the mRNA level. In fact, transcriptional regulatory networks seem to be an additional way of controlling HIF-1α levels and may open new therapeutic options to modulate HIF-1α also under non-hypoxic conditions.

The heterodimeric transcription factor HIF-1 (hypoxia-inducible factor-1) induces angiogenesis, a process that is aberrantly elevated in cancer. The HIF-1β subunit is constitutively expressed, but the levels of the HIF-1α subunit are robustly regulated, increasing under hypoxic conditions and decreasing in normoxia. These changes result from rapid alterations in the rates of HIF-1α production and degradation. While the regulation of HIF-1α degradation is understood in significant detail, much less is known about the regulation of HIF-1α biosynthesis. Here, we review recent evidence that HIF-1α production is effectively controlled by post-transcriptional mechanisms. We focus on the RNA-binding proteins (RBPs) and the non-coding RNAs that interact with the HIF-1α mRNA and influence its half-life and translation rate. HIF-1α mRNA-binding factors are emerging as promising pharmacological targets to control HIF-1α production selectively and efficiently.

Recent studies have established that the regulation of microRNAs (miRs) is a feature of the hypoxic response. In this review, we discuss the role of hypoxia-regulated miRs, with an emphasis on miR-210 and miR-373, and anticipate directions for clinical applications. The induction of miR-210 and miR-373 is dependent upon hypoxia inducible factor (HIF), and their up-regulation has been detected in a variety of solid tumors. Both miRs have been associated with adverse prognosis and metastatic potential. The increased expression of miR-210 is linked to an in vivo hypoxic signature. MiR- 210 also participates in endothelial and neuronal cells' response to oxygen deprivation and may possess a role in the regulation of angiogenesis. A variety of miR-210 and miR-373 targets that may be relevant to hypoxia have been validated or proposed. Very recently, targets of these miRs that are implicated in DNA repair have been identified, thus establishing an additional link between the hypoxic tumor microenvironment and DNA damage. Extending beyond cancer biology, some of miR-210 targets are likely involved in the regulation of angiogenesis, and neuronal cell survival. Inactivation of miRs affected by hypoxia presents a promising therapeutic strategy in the case of difficult-to-treat cancers, as well as in other non-cancer-related diseases.

The hypoxia-inducible factor-1 (HIF-1) is a key regulator in the mammalian response to oxygen deficiency under both physiological and pathological conditions such as cancer. A number of studies indicated an association between tumor hypoxia, increased hypoxia-inducible factor (HIF-1α) levels and a poor prognosis. The HIF-1α regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation. However, HIF α-subunits also respond to various growth factors, hormones, or cytokines under non-hypoxic conditions implicating the involvement of different kinase pathways in their regulation thereby increasing the interest in HIF-1α as a new drug target. Herein, we review current knowledge about phosphorylation-dependent HIF-1α regulation, HIF-1α protein-protein interactions and subcellular localization with emphasis on new therapeutic strategies targeting the HIF pathway.

Hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxia-inducible genes involved in the mediation of survival and adaptive responses to insufficient oxygen availability, such as genes involved in hematopoesis, angiogenesis, iron transport, glucose utilization, resistance to oxidative stress, cell proliferation, survival and apoptosis, extracellular matrix homeostasis, and tumor progression. The stability of the HIFα subunit is regulated by oxygendependent prolyl 4-hydroxylation catalyzed by the HIF prolyl 4-hydroxylases (P4Hs). The 4-hydroxyproline residues generated in normoxic conditions facilitate binding of HIFα to the von Hippel-Lindau E3 ubiquitin ligase complex resulting in the attachment of ubiquitin molecules and subsequent rapid proteasomal degradation of HIFα. In hypoxia this oxygen-requiring hydroxylation event is inhibited, HIFα escapes degradation and can translocate to the nucleus and form a functional dimer with HIFβ that triggers the hypoxic response. HIF-P4Hs are considered as promising drug development targets in the treatment of diseases such as myocardial infarction, stroke, peripheral vascular disease, inflammation, diabetes and severe anemias. Studies with HIF-P4H inhibitors in various animal models and ongoing clinical trials support this hypothesis by demonstrating efficacy in many applications.

Protein stability of hypoxia-inducible factor (HIF) α subunits is regulated by the oxygen-sensing prolyl-4- hydroxylase domain (PHD) enzymes. Under oxygen-limited conditions, HIFα subunits are stabilized and form active HIF transcription factors that induce a large number of genes involved in adaptation to hypoxic conditions with physiological implications for erythropoiesis, angiogenesis, cardiovascular function and cellular metabolism. Oxygen-sensing is regulated by the co-substrate-dependent activity and hypoxia-inducible abundance of the PHD enzymes which trigger HIFα stability even under low oxygen conditions. Because HIFα itself is notoriously reluctant to the development of antagonists, an increase in PHD activity would offer an interesting alternative to the development of drugs that interfere specifically with the HIF signalling pathway. Interestingly, among the recently discovered PHD interacting proteins were not only novel downstream targets but also upstream regulators of PHDs. Their PHD isoform-specific interaction offers the possibility to target distinct PHD isoforms and their non-identical downstream signalling pathways. This review summarizes our current knowledge on PHD interacting proteins, including upstream regulators, chaperonins, scaffolding proteins, and novel downstream transcription factors.

Hypoxia-inducible factors (HIFs) are heterodimeric oxygen-sensitive basic helix-loop-helix transcription factors that play central roles in cellular adaptation to low oxygen environments. The von Hippel-Lindau tumor suppressor (pVHL) is the substrate recognition component of an E3 ubiquitin ligase and functions as a master regulator of HIF activity by targeting the hydroxylated HIF-alpha subunit for ubiquitylation and rapid proteasomal degradation under normoxic conditions. Mutations in pVHL can be found in familial and sporadic hemangioblastomas, clear cell carcinomas of the kidney, pheochromocytomas and inherited forms of erythrocytosis, illustrating the importance of disrupted molecular oxygen sensing in the pathogenesis of these diseases. Tissue-specific gene targeting of pVHL in mice has demonstrated that efficient execution of HIF proteolysis is critically important for normal tissue physiology, and has provided novel insights into the functional consequences of HIF activation on the cellular and tissue level. Here we focus on the contribution of individual HIF transcription factors to the development of VHL phenotypes and discuss how the pVHL/HIF axis could be exploited pharmacologically.

Cellular and systemic oxygen homeostasis is regulated by an oxygen-sensitive signalling pathway centred on a transcription factor known as Hypoxia Inducible Factor (HIF). Regulation of HIF activity and protein stability is mediated by a family of hydroxylases that act as oxygen sensors due to the dependence of the hydroxylation reaction on oxygen. The transcriptional activity of HIF is at least in part determined by asparaginyl hydroxylation by Factor Inhibiting HIF (FIH) of a C-terminal residue that regulates co-activator recruitment. The activity of FIH on HIF is limiting; emerging data suggest this may be due to competition from a large family of alternative FIH substrates that act as a ‘sink’ for FIH activity. These alternative substrates are targeted for hydroxylation at conserved Asn residues within a protein interaction domain known as the Ankyrin Repeat Domain (ARD). Many ARD-containing proteins bind to FIH more tightly than does HIF. Furthermore, ARD proteins are common within the proteome and in some cases are highly abundant. Since ARD substrates bind to FIH in a similar manner to HIF it is thought that these properties of the ARD family lead to competitive inhibition of FIH-dependent HIF hydroxylation. We summarise the current literature here and discuss the possible role of cross-talk between the FIH, HIF and ARD systems in fine tuning hypoxia responses.

Stem cell based therapies for cerebral ischemia (CI) utilize different cell sources including embryonic stem cells (ESCs), neural stem cells (NSCs), umbilical cord blood cells (UCBCs), mesenchymal stem cells (MSCs), and some immortalized cell lines. To date, experimental studies showed that all of these cell sources have been successful to some extent in attenuating the ischemic damage and improving functional recovery after brain injury. Bone marrow derived MSCs seem to be the most widely used and well characterized cell source, which can be also employed for autologous transplantation. Currently, there are two main theories behind the therapeutic effect of stem cell transplantation for treating CIs. The first concept is cell replacement theory in which transplanted stem cells differentiate into progenitor and specialized somatic cells to supersede dying cells. The other hypothesis is based on immuno-modulatory, neuro-protective and neuro-trophic abilities of stem cells which help reducing stroke size and increasing the recovery of behavioral functions. Recent studies focusing on alternative stem cell sources have revealed that dental stem cells (DSCs), including dental pulp stem cells (DPSCs) and dental follicle cells (DFCs) possess properties of MSCs and NSCs. They differentiate into neural linage cells and some other cell types such as osteocytes, adipocytes, chondrocytes, muscle cells and hepatocytes. This review is intended to examine stem cell therapy approaches for CI and emphasize potential use of DSCs as an alternative cell source for the treatment of brain ischemia.