Regulation of intercellular adhesion molecule-1 gene expression involves multiple mRNA stabilization mechanisms: effects of interferon- gamma and phorbol myristate acetate

Although the intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at a low level on a subpopulation of hematopoietic cells, on vascular endothelium, on fibroblasts, and on certain epithelial cells, it is dramatically increased at sites of inflammation. Interferon-gamma (IFN-gamma) and phorbol myristate acetate (PMA) are known to increase the expression of ICAM-1 on many cell types. Because both human and murine ICAM-1 mRNAs contain putative destabilizing AUUUA sequences in their 3' untranslated regions (UTRs), we examined the role of mRNA stability in the regulation of ICAM-1 gene expression. The treatment of the murine monocytic cell line P388D1, which constitutively expresses ICAM-1 mRNA at a low level, with IFN- gamma or PMA rapidly enhanced the level of ICAM-1 mRNA and dramatically prolonged its half-life. To determine whether the putative destabilizing sequences are responsible for this effect of IFN-gamma and PMA, fibroblast L cells were transfected with either the full- length ICAM-1 cDNA or a truncated form (ICAM-1 delta 3) lacking the putative destabilizing AUUUA sequences. Although ICAM-1 delta 3 mRNA was more stable than the full-length ICAM-1 mRNA, IFN-gamma treatment induced the accumulation of both mRNA species and prolongation of their half-lives. The transplantation of the ICAM-1 delta 3' UTR into a stable ICAM-2 mRNA rendered it unstable, and it was unresponsive to IFN- gamma. Therefore, the treatment with IFN-gamma stabilizes the otherwise labile ICAM-1 mRNA, but the IFN-gamma-responsive sequence may at least in part reside within the protein coding region. PMA also upregulated ICAM-1 gene expression by mRNA stabilization. However, unlike IFN- gamma, PMA treatment only increased the level of the full-length, but not of the truncated, ICAM-1 mRNA. This shows that the PMA-responsive element is located within the 3'UTR. Furthermore, the effect of PMA on ICAM-1 delta 3 mRNA was recovered by ligating multiple AUUUA sequences derived from a heterologous gene fragment. The stability of this chimeric mRNA and the full-length ICAM-1 mRNA was markedly increased by PMA treatment, indicating that the AUUUA multimers in the 3'UTR are important in the PMA-induced upregulation of ICAM-1 mRNA.     

pVHL19 is a biologically active product of the von Hippel–Lindau gene arising from internal translation initiation

The von Hippel–Lindau (VHL) gene encodes a protein consisting of 213 amino acid residues with an apparent molecular mass of 30 kDa (pVHL₃₀). Here we show that cells also produce a VHL protein (pVHL₁₉) that appears to arise as a result of internal translation from the second methionine within the VHL ORF. pVHL₃₀ resides primarily in the cytosol, with less amounts found in the nucleus or associated with cell membranes. In contrast pVHL₁₉, in biochemical fractionation experiments, is equally distributed between the nucleus and cytosol and is not found in association with membranes. pVHL₁₉, like pVHL₃₀, can bind to elongin B, elongin C, and Hs-Cul2 in coimmunoprecipitation assays and can inhibit the production of hypoxia-inducible proteins such as vascular endothelial growth factor (VEGF) and GLUT1 when reintroduced into renal carcinoma cells that lack a wild-type VHL allele. Thus, cells contain two biologically active VHL gene products.     

Functions of the von Hippel–Lindau tumour suppressor protein

Kaelin WG, Iliopoulos O, Lonergan KM, Ohh M (Dana-Farber Cancer Institute, Brigham and Women's Hospital, Boston, MA, USA). Functions of the von Hippel–Lindau tumour suppressor protein (Minisymposium: MEN & VHL). J Intern Med 1998; 243 : 535–9.
Von Hippel–Lindau disease (VHL) is caused by germline mutations in the VHL tumour suppressor gene. Tumour development in this setting is due to loss or inactivation of the remaining wild-type VHL allele. The VHL gene product (pVHL) resides primarily in the cytoplasm. A frequently mutated region of pVHL can bind to complexes containing elongin B, elongin C and Cul2. Loss of pVHL leads to an inappropriate accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This finding is most likely to account for the hypervascular nature of VHL-associated neoplasms. Current studies are focussed on understanding if and how binding to elongins and Cul2 is linked to the ability of pVHL to regulate hypoxia-inducible mRNAs. In this regard, it is perhaps noteworthy that elongin C and Cul2 are homologous to yeast proteins Skp1 and Cdc53. These latter proteins participate in the formation of complexes that target certain proteins for ubiquitination.     

Homotypic association between tumour-associated VHL proteins leads to the restoration of HIF pathway

The von Hippel-Lindau (VHL) tumour suppressor gene encodes a substrate-specifying component of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) alpha subunits for degradation under normoxia. The VHL protein is composed of an N-terminal HIFalpha-binding beta domain and a C-terminal alpha domain, which is necessary and sufficient for the formation of the E3 multiprotein enzyme. A large number of disease-causing mutations in either the alpha or beta domain renders HIFalpha stable irrespective of oxygen tension, leading to the upregulation of numerous HIF-target genes, such as GLUT1 and VEGF. Here, we show that VHL forms a self-associated complex in vivo, but not in vitro, and demonstrate that coexpression of two different VHL missense mutants -- one in the alpha domain and the other in the beta domain -- restores HIF-mediated gene expression profile. These findings indicate that VHL homotypic complexes can function in vivo in a complementary fashion to target HIFalpha for ubiquitin-mediated proteolysis, and potentially explain why VHL-associated tumours with a missense mutation-carrying VHL allele is almost invariably accompanied by a second VHL allele harbouring a gross truncation or deletion.     

pVHL's kryptonite: E2-EPF UCP

E2-EPF ubiquitin carrier protein (UCP) is a member of an E2 family of enzymes that catalyzes the ligation of ubiquitin to proteins targeted for destruction by the proteasome. UCP is overexpressed in common human cancers, suggesting its involvement in oncogenesis, but a physiologic target of UCP has not been identified. In a recent report published in Nature Medicine, Jung et al. identified von Hippel-Lindau (VHL) tumor suppressor protein, which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction, as a bona fide substrate of UCP and demonstrated a potential pVHL-HIF pathway-dependent role for UCP in cancer development.     

Beyond the hypoxia-inducible factor-centric tumour suppressor model of von Hippel-Lindau

PURPOSE OF REVIEW: To provide an overview of the recent advances in the understanding of the molecular mechanisms governing the tumour suppressor functions of the von Hippel-Lindau protein. RECENT FINDINGS: von Hippel-Lindau is a vital component of an E3 ubiquitin ligase complex involved in the oxygen-dependent targeting of hypoxia-inducible factor for ubiquitin-mediated destruction. Recent reports have linked von Hippel-Lindau to the regulation of diverse biological processes including cell adhesion, extracellular matrix assembly and ciliogenesis in a manner dependent and/or independent of hypoxia-inducible factor. SUMMARY: The tumour suppressor function of von Hippel-Lindau has remained hypoxia-inducible factor-centric since the discovery of von Hippel-Lindau as a bona fide negative regulator of the ubiquitous oxygen-sensing pathway. Emerging evidence supports this hypothesis with the elucidation of fundamental cellular processes deregulated upon the inactivation of the von Hippel-Lindau-hypoxia-inducible factor pathway, but has also proved compelling on the hypoxia-inducible factor-independent tumour suppressor role of von Hippel-Lindau. These and continuing studies into the molecular pathways and mechanisms governing the tumour suppressor functions of von Hippel-Lindau will ultimately afford new avenues for anticancer strategies for the improved treatment of a diverse array of cancers.