No abstract

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 ␣ subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the ␣ subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3␣ locus as follows: hHIF-3␣1, hHIF-3␣2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3␣3, hHIF-3␣4, hHIF-3␣5, and hHIF-3␣6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3␣1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.

Embryonic cells from transgenic mice with targeted disruption of metallothionein I and II genes expressed no detectable metallothionein either constitutively or after treatment with cadmium, in contrast to cultured cells that were wild type or heterozygous for the loss of the metallothionein genes. Metallothionein null cells were most sensitive to the cytotoxic effects of cadmium, the membrane permeant oxidant tert-butylhydroperoxide, and the redox cycling toxin paraquat. No marked differences were seen among the wild type, heterozygous, or metallothionein null cells in glutathione levels or in the activity of CuZn-superoxide dismutase, glutathione peroxidase, or catalase. Nevertheless, metallothionein null cells were more sensitive to tert-butylhydroperoxide-induced oxidation as ascertained by confocal microscopic imaging of dichlorofluoroscein fluorescence. These results indicate basal metallothionein levels can function to regulate intracellular redox status in mammalian cells.

Metallothioneins (MTs) are major intracellular, zinc-binding proteins with antioxidant properties. Mouse embryonic cells null for MT due to loss of functional MT I and II genes (MT-/-) were more susceptible to apoptotic death after exposure to tert-butyl hydroperoxide or the anti-cancer agents cytosine arabinoside, bleomycin, melphalan, and cis-dichlorodiammineplatinum(II) compared with wild-type mouse embryonic cells (MT+/+). We measured basal levels of the tumor suppressor protein p53 and the death effector protein Bax and found the basal levels of both proteins were higher in MT null cells compared with MT+/+ cells. After treatment with the DNA-damaging agent cis-dichlorodiammineplatinum(II), p53 protein levels were induced in both MT+/+ and MT-/- cells with MT null cells always maintaining the highest p53 levels. The elevated sensitivity to apoptosis was not restricted to embryonic cells. Primary pulmonary fibroblasts were isolated from distinct litters of MT null, heterozygous, and wild-type mice, and all had undetectable basal MT levels. Zinc exposure increased MT levels in the wild-type and heterozygous fibroblasts but not in the MT null fibroblasts. Consistent with the induced MT levels, we found MT+/+ and MT+/- embryonic cells were less sensitive to cis-dichlorodiammineplatinum(II)-induced apoptosis compared with MT-/- cells. Our results implicate MT as a stress-responsive factor that can regulate apoptotic engagement.

Metallothionein (MT), a low molecular weight, cysteine-rich metal binding protein, has been associated with cytoprotection from heavy metals and cellular oxidants. As MT has the ability to scavenge hydroxyl radicals, MT may control intracellular redox status. In the present study, we examined whether MT regulates the activity of nuclear factor-U UB (NF-U UB), which is one of the redox-regulated transcription factors, using the MT null embryonic cell lines established from MT null mice. We first found that tumor necrosis factor (TNF)-induced activation of the binding of NF-U UB protein to DNA in wild type MT+/+ cells was lower than that in MT3 3/3 3 cells. The NF-U UB activation in MT-expressing cells established from MT3 3/3 3 cells by the transfection of mouse MT-I gene was also significantly lower than that in MT3 3/3 3 cells. In addition, transfection of the MT gene inhibited TNF-induced IU UB degradation and suppressed NF-U UB-dependent gene expression induced by TNF. These results demonstrate that MT may function as a negative regulator of NF-U UB activity.z 1999 Federation of European Biochemical Societies.