Hypoxia-inducible factor (HIF)-3alpha is subject to extensive alternative splicing in human tissues and cancer cells and is regulated by HIF-1 but not HIF-2.

The hypoxia-inducible transcription factors (HIFs) play a central role in the response of cells to hypoxia. HIFs are alphabeta dimers, the human alpha subunit having three isoforms. HIF-3alpha is unique among the HIF-alpha isoforms in that its gene is subject to extensive alternative splicing. Database analyses have predicted the generation of six HIF-3alpha splice variants that utilize three alternative transcription initiation sites. None of these variants is likely to act as an efficient transcription factor, but some of them have been reported to inhibit HIF-1 and HIF-2 functions. We analyzed here for the first time in detail whether these six variants are indeed generated in various human tissues and cell lines. We identified four novel variants, named here HIF-3alpha7 to HIF-3alpha10, whereas we obtained no evidence for the predicted HIF-3alpha3 and HIF-3alpha5. Distinct differences in the expression patterns of the variants were found between human tissues, the levels being particularly low in many cancer cell lines. Hypoxia upregulated transcription from all three alternative HIF-3alpha promoters. siRNA experiments showed that this induction is mediated specifically by HIF-1 and not by HIF-2. The tissue-specific differences in the expression patterns and levels of the HIF-3alpha variants can be expected to modulate the hypoxia response of various tissues and cell types to different extents during development and in pathological situations. A further level of regulation is brought about by the fact that the levels of the HIF-3alpha transcripts themselves are regulated by hypoxia and by changes in HIF-1 levels.

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice.

Collagens carry hydroxylysine residues that act as attachment sites for carbohydrate units and are important for the stability of crosslinks but have been regarded as nonessential for vertebrate survival. We generated mice with targeted inactivation of the gene for one of the three lysyl hydroxylase isoenzymes, LH3. The null embryos developed seemingly normally until embryonic day 8.5, but development was then retarded, with death around embryonic day 9.5. Electron microscopy (EM) revealed fragmentation of basement membranes (BMs), and immuno-EM detected type IV collagen within the dilated endoplasmic reticulum and in extracellular aggregates, but the typical BM staining was absent. Amorphous intracellular and extracellular particles were also seen by collagen IV immunofluorescence. SDS/PAGE analysis demonstrated increased mobilities of the type IV collagen chains, consistent with the absence of hydroxylysine residues and carbohydrates linked to them. These results demonstrate that LH3 is indispensable for biosynthesis of type IV collagen and for BM stability during early development and that loss of LH3's functions leads to embryonic lethality. We propose that the premature aggregation of collagen IV is due to the absence of the hydroxylysine-linked carbohydrates, which thus play an essential role in its supramolecular assembly.

Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80-85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K(m) values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.