No abstract

The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1␣ (HIF-1␣) that binds von Hippel-Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-␣ and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylationdependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygensensitive cells of the carotid body and pheochromocytomaderived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFa ubiquitination, increased accumulation of HIFa proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoterreporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

A multi-modality treatment programme, where stereotactic methods were used preferentially, gave results in a consecutive series of craniopharyngiomas, not inferior to those reported after microsurgical removal. Fourty-two patients with a follow-up range of 10-23 years are reported.