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Biomedical subjects

William G Kaelin

Publications and source records attributed to William G Kaelin.

At least 19 recordsLinked to original sources
PubMed2007

The hypoxia-inducible factor 2alpha N-terminal and C-terminal transactivation domains cooperate to promote renal tumorigenesis in vivo.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor, consisting of an alpha subunit and a beta subunit, that controls cellular responses to hypoxia. HIFalpha contains two transcriptional activation domains called the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD). HIFalpha is destabilized by prolyl hydroxylation catalyzed by EglN family members. In addition, CTAD function is inhibited by asparagine hydroxylation catalyzed by FIH1. Both hydroxylation reactions are linked to oxygen availability. The von Hippel-Lindau tumor suppressor protein (pVHL) is frequently mutated in kidney cancer and is part of the ubiquitin ligase complex that targets prolyl hydroxylated HIFalpha for destruction. Recent studies suggest that HIF2alpha plays an especially important role in promoting tumor formation by pVHL-defective renal carcinoma cells among the three HIFalpha paralogs. Here we dissected the relative contribution of the two HIF2alpha transactivation domains to hypoxic gene activation and renal carcinogenesis and investigated the regulation of the HIF2alpha CTAD by FIH1. We found that the HIF2alpha NTAD is capable of activating both artificial and naturally occurring HIF-responsive promoters in the absence of the CTAD. Moreover, we found that the HIF2alpha CTAD, in contrast to the HIF1alpha CTAD, is relatively resistant to the inhibitory effects of FIH1 under normoxic conditions and that, perhaps as a result, both the NTAD and CTAD cooperate to promote renal carcinogenesis in vivo.

Amino Acid Sequence↗
PubMed2006

Retinoblastoma protein and anaphase-promoting complex physically interact and functionally cooperate during cell-cycle exit.

The retinoblastoma protein (pRB) negatively regulates the progression from G1 to S phase of the cell cycle, in part, by repressing E2F-dependent transcription. pRB also possesses E2F-independent functions that contribute to cell-cycle control--for example, during pRB-mediated cell-cycle arrest pRB associates with Skp2, the F-box protein of the Skp1-Cullin-F-box protein (SCF) E3 ubiquitin ligase complex, and promotes the stability of the cyclin-dependent kinase-inhibitor p27(Kip1) through an unknown mechanism. Degradation of p27(Kip1) is mediated by ubiquitin-dependent targeting of p27(Kip1) by SCF -Skp2 (ref. 4). Here, we report a novel interaction between pRB and the anaphase-promoting complex/cyclosome (APC/C) that controls p27(Kip1) stability by targeting Skp2 for ubiquitin-mediated degradation. Cdh1, an activator of APC/C, not only interacts with pRB but is also required for a pRB-induced cell-cycle arrest. The results reveal an unexpected physical convergence between the pRB tumour-suppressor protein and E3 ligase complexes, and raise the possibility that pRB may direct APC/C to additional targets during pRB-mediated cell-cycle exit.

Anaphase↗
PubMed2006

Failure to prolyl hydroxylate hypoxia-inducible factor alpha phenocopies VHL inactivation in vivo.

Many functions have been assigned to the von Hippel-Lindau tumor suppressor gene product (pVHL), including targeting the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for destruction. The binding of pVHL to HIFalpha requires that HIFalpha be hydroxylated on one of two prolyl residues. We introduced HIF1alpha and HIF2alpha variants that cannot be hydroxylated on these sites into the ubiquitously expressed ROSA26 locus along with a Lox-stop-Lox cassette that renders their expression Cre-dependent. Expression of the HIF2alpha variant in the skin and liver induced changes that were highly similar to those seen when pVHL is lost in these organs. Dual expression of the HIF1alpha and HIF2alpha variants in liver, however, more closely phenocopied the changes seen after pVHL inactivation than did the HIF2alpha variant alone. Moreover, gene expression profiling confirmed that the genes regulated by HIF1alpha and HIF2alpha in the liver are overlapping but non-identical. Therefore, the pathological changes caused by pVHL inactivation in skin and liver are due largely to dysregulation of HIF target genes.

Animals↗
PubMed2006

Molecular pathways in renal cell carcinoma--rationale for targeted treatment.

The VHL tumor-suppressor gene is mutated or silenced in most clear cell renal carcinomas (RCCs). pVHL loss results in the stabilization of the heterodimeric transcription factor hypoxia-inducible factor (HIF) and enhanced transactivation of HIF target genes. Downregulation of HIF is both necessary and sufficient for pVHL to suppress the growth of human renal carcinoma cells in preclinical models. HIF itself has been difficult to inhibit with drug-like molecules although a number of agents that indirectly inhibit HIF, including mammalian target of rapamycin (mTOR) inhibitors, have been identified. Moreover, a number of drugs have been developed that target HIF-responsive gene products, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), implicated in tumorigenesis. Many of these targeted therapies have demonstrated significant activity in kidney cancer clinical trials and represent substantive advances in the treatment of this disease. How these agents should be combined with one another and with conventional agents is the subject of current trials.

Antineoplastic Agents↗
PubMed2006

Recent insights into the molecular pathogenesis of pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas are rare tumors derived from chromaffin cells. These tumors can arise in the context of hereditary cancer syndromes such as von Hippel- Lindau disease, multiple endocrine neoplasia type 2, and neurofibromatosis 1. Recent studies indicate that germ line mutations of genes encoding specific succinate dehydrogenase (SDH) subunits also predispose individuals to pheochromocytomas and paragangliomas. This review focuses on the genetics of these tumors and suggests a possible link between familial pheochromocytomas/paraganglioma genes and control of neuronal apoptosis during embryological development.

Adrenal Gland Neoplasms↗
PubMed2006

Clusterin is a secreted marker for a hypoxia-inducible factor-independent function of the von Hippel-Lindau tumor suppressor protein.

Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose people to renal cancer, hemangioblastomas, and pheochromocytomas in an allele-specific manner. The best documented function of the VHL gene product (pVHL) relates to its ability to polyubiquitinate, and hence target for destruction, the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF). pVHL mutants linked to familial pheochromocyctoma (type 2C VHL disease), in contrast to classical VHL disease, appear to be normal with respect to HIF regulation. Using a simple method for identifying proteins that are differentially secreted by isogenic cell line pairs, we confirmed that the HIF targets IGBP3 and PAI-1 are overproduced by pVHL-defective renal carcinoma cells. In addition, cells lacking wild-type pVHL, including cells producing type 2C pVHL mutants, were defective with respect to expression and secretion of clusterin, which does not behave like a HIF target. Decreased clusterin secretion by pVHL-defective tumors was confirmed in vivo by immunohistochemistry. Therefore, clusterin is a secreted marker for a HIF-independent pVHL function that might be especially important in pheochromocytoma development.

Adrenal Gland Neoplasms↗
PubMed2005

Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production.

Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIFalpha subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules.

Animals↗
PubMed2005

The von Hippel-Lindau protein, HIF hydroxylation, and oxygen sensing.

The heterodimeric transcription factor HIF (hypoxia-inducible factor), consisting of a labile alpha-subunit and a stable beta-subunit, is a master regulator of genes involved in acute or chronic adaptation to low oxygen. Studies performed over the past 5 years revealed that HIFalpha-subunits are enzymatically hydroxylated in an oxygen-dependent manner. Hydroxylation of either of two conserved prolyl residues targets HIFalpha for destruction by a ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein whereas hydroxylation on a C-terminal asparagine affects HIF transactivation function. Pharmacological manipulation of HIF activity might be beneficial in diseases characterized by abnormal tissue oxygenation including myocardial infarction, cerebrovascular disease, and cancer.

Animals↗
PubMed2005

Binding of pRB to the PHD protein RBP2 promotes cellular differentiation.

pRB can enforce a G1 block by repressing E2F-responsive promoters. It also coactivates certain non-E2F transcription factors and promotes differentiation. Some pRB variants activate transcription and promote differentiation despite impaired E2F binding and transcriptional repression capabilities. We identified RBP2 in a screen for proteins that bind to such pRB variants. RBP2 resembles other chromatin-associated transcriptional regulators and RBP2 binding tracked with pRB's ability to activate transcription and promote differentiation. RBP2 and pRB colocalize and pRB/RBP2 complexes were detected in chromatin isolated from differentiating cells. RBP2 siRNA phenocopied restoration of pRB function in coactivation and differentiation assays, suggesting that pRB prevents RBP2 from repressing genes required for differentiation. In addition, two bromodomain-containing proteins were identified as RBP2 targets that are transcriptionally activated by pRB in an RBP2-dependent manner. Our results suggest that promotion of differentiation by pRB involves neutralization of free RBP2 and transcriptional activation of RBP2 targets linked to euchromatin maintenance.

Animals↗
PubMed2005

The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase.

The c-Jun and c-Myc oncogenic transcription factors are highly unstable proteins due to polyubiquitination. Similar to c-Myc, we report here that phosphorylation of c-Jun by GSK3 creates a high-affinity binding site for the E3 ligase Fbw7, which targets c-Jun for polyubiquitination and proteasomal degradation. In keeping with this, we found that c-Jun levels were inversely related to GSK3 activity in mammalian cells that had entered the cell cycle. Importantly, phosphorylation of c-Jun by GSK3 requires a priming phosphorylation event at Ser-243. Ser-243 is mutated to phenylalanine in v-Jun and allows it to escape recognition by Fbw7. These findings explain the enhanced stability and oncogenicity of v-Jun relative to its cellular counterpart and reveal that GSK3 and Fbw7 coordinately regulate c-Jun and c-Myc.

Binding Sites↗
PubMed2005

Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer.

Germline NF1, c-RET, SDH, and VHL mutations cause familial pheochromocytoma. Pheochromocytomas derive from sympathetic neuronal precursor cells. Many of these cells undergo c-Jun-dependent apoptosis during normal development as NGF becomes limiting. NF1 encodes a GAP for the NGF receptor TrkA, and NF1 mutations promote survival after NGF withdrawal. We found that pheochromocytoma-associated c-RET and VHL mutations lead to increased JunB, which blunts neuronal apoptosis after NGF withdrawal. We also found that the prolyl hydroxylase EglN3 acts downstream of c-Jun and is specifically required among the three EglN family members for apoptosis in this setting. Moreover, EglN3 proapoptotic activity requires SDH activity because EglN3 is feedback inhibited by succinate. These studies suggest that failure of developmental apoptosis plays a role in pheochromocytoma pathogenesis.

Adrenal Gland Neoplasms↗
PubMed2005

ROS: really involved in oxygen sensing.

The role of reactive oxygen species (ROS) in the cellular response to cellular oxygen sensing has been controversial. Three papers in this issue of Cell Metabolism (Brunelle et al., Guzy et al., 2005; Mansfield et al., 2005) used genetic tools to establish that ROS produced by mitochondria are required for the normal induction of HIF (hypoxia-inducible factor), which is a master regulator of oxygen-sensitive gene expression, by low oxygen.

Animals↗
PubMed2005

Bioluminescent imaging of ubiquitin ligase activity: measuring Cdk2 activity in vivo through changes in p27 turnover.

Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by different ubiquitin ligases are often modular and can be used to target foreign proteins for destruction in cis. We recently fused the Cdk inhibitor p27, which is polyubiquitylated by a Skp2-containing ubiquitin ligase if phosphorylated by cdk2 to firefly luciferase. The resulting fusion protein, p27-Luc, was induced by cdk2 inhibitors in living cells grown in culture or in nude mice. This article describes protocols for validation of p27-Luc in cell culture using siRNA against cdk2 (or its partner cyclin A) and for imaging cells producing p27-Luc grown in transparent hollow fibers after treatment with cdk2 inhibitory drugs in vivo. These approaches should be generalizable to other ubiquitin-ligase substrate pairs.

Animals↗
PubMed2005

The concept of synthetic lethality in the context of anticancer therapy.

Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells. Synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents. This paradigm has not been exploited in the past because there were no robust methods for systematically identifying synthetic lethal genes. This is changing as a result of the increased availability of chemical and genetic tools for perturbing gene function in somatic cells.

Animals↗
PubMed2005

Proline hydroxylation and gene expression.

Hypoxia-inducible factor (HIF) is a master transcriptional regulator of hypoxia-inducible genes and consists of a labile alpha subunit (such as HIF1alpha) and a stable beta subunit (such as HIF1beta or ARNT). In the presence of oxygen, HIFalpha family members are hydroxylated on one of two conserved prolyl residues by members of the egg-laying-defective nine (EGLN) family. Prolyl hydroxylation generates a binding site for a ubiquitin ligase complex containing the von Hippel-Lindau (VHL) tumor suppressor protein, which results in HIFalpha destruction. In addition, the HIFalpha transcriptional activation function is modulated further by asparagine hydroxylation by FIH (factor-inhibiting HIF), which affects recruitment of the coactivators p300 and CBP. These findings provide new mechanistic insights into oxygen sensing by metazoans and are the first examples of protein hydroxylation being used in intracellular signaling. The existence of three human EGLN family members, as well as other putative hydroxylases, raises the possibility that this signal is used in other contexts by other proteins.

Animals↗
PubMed2004

Role of VHL gene mutation in human cancer.

Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the von Hippel-Lindau hereditary cancer syndrome, and somatic mutations of this gene have been linked to the development of sporadic hemangioblastomas and clear-cell renal carcinomas. The VHL tumor suppressor protein (pVHL), through its oxygen-dependent polyubiquitylation of hypoxia-inducible factor (HIF), plays a central role in the mammalian oxygen-sensing pathway. This interaction between pVHL and HIF is governed by post-translational prolyl hydroxylation of HIF in the presence of oxygen by a conserved family of Egl-nine (EGLN) enzymes. In the absence of pVHL, HIF becomes stabilized and is free to induce the expression of its target genes, many of which are important in regulating angiogenesis, cell growth, or cell survival. Moreover, preliminary data indicate that HIF plays a critical role in pVHL-defective tumor formation, raising the possibility that drugs directed against HIF or its downstream targets (such as vascular endothelial growth factor) might one day play a role in the treatment of hemangioblastoma and renal cell carcinoma. On the other hand, clear genotype-phenotype correlations are emerging in VHL disease and can be rationalized if pVHL has functions separate from its control of HIF.

Adrenal Gland Neoplasms↗
PubMed2004

Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist.

Vascular endothelial growth factor (VEGF) and its receptors have been implicated as key factors in tumor angiogenesis that are up-regulated by hypoxia. We evaluated the effects of DNA-binding small molecules on hypoxia-inducible transcription of VEGF. A synthetic pyrrole-imidazole polyamide designed to bind the hypoxia response element (HRE) was found to disrupt hypoxia-inducible factor (HIF) binding to HRE. In cultured HeLa cells, this resulted in a reduction of VEGF mRNA and secreted protein levels. The observed effects were polyamide-specific and dose-dependent. Analysis of genome-wide effects of the HRE-specific polyamide revealed that a number of hypoxia-inducible genes were down-regulated. Pathway-based regulation of hypoxia-inducible gene expression with DNA-binding small molecules may represent a new approach for targeting angiogenesis.

Base Sequence↗