Bernd J. Thiele

The diversity of the lipoxygenase family: Many sequence data but little information on biological significance

Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian lipoxygenase isoforms and critically reviews the concepts for their biological importance.

Translational Control of Collagen Prolyl 4-Hydroxylase-α(I) Gene Expression under Hypoxia

Hypoxia is a pro-fibrotic stimulus, which is associated with enhanced collagen synthesis, as well as with augmented collagen prolyl 4-hydroxylase (C-P4H) activity. C-P4H activity is controlled mainly by regulated expression of the α C-P4H subunit. In this study we demonstrate that the increased synthesis of C-P4H-α(I) protein in human HT1080 fibroblasts under long term hypoxia (36 h, 1% oxygen) is controlled at the translational level. This is mediated by an interaction of RNA-binding protein nucleolin (∼64 kDa form) at the 5′- and 3′-untranslated regions (UTR) of the mRNA. The 5′/3′-UTR-dependent mechanism elevates the C-P4H-α(I) expression rate 2.3-fold, and participates in a 5.3-fold increased protein level under long term hypoxia. The interaction of nucleolin at the 5′-UTR occurs directly and depends on the existence of an AU-rich element. Statistical evaluation of the ∼64-kDa nucleolin/RNA interaction studies revealed a core binding sequence, corresponding to UAAAUC or AAAUCU. At the 3′-UTR, nucleolin assembles indirectly via protein/protein interaction, with the help of another 3′-UTR-binding protein, presumably annexin A2. The increased protein level of the ∼64-kDa nucleolin under hypoxia can be attributed to an autocatalytic cleavage of a high molecular weight nucleolin form, without alterations in nucleolin mRNA concentration. Thus, the alteration of translational efficiency by nucleolin, which occurs through a hypoxia inducible factor independent pathway, is an important step in C-P4H-α(I) regulation under hypoxia.

Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) α-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-α (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-α (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)16 element located in the 3′-untranslated region of C-P4H-α (I) mRNA. Luciferase reporter gene assays depending on C-P4H-α (I) 3′-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)16 element is necessary and sufficient for posttranscriptional control of C-P4H-α (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-α (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-α (I) mRNAs.

Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).

Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes

Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous [1-14C]-arachidonic acid to 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE). Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase. Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is also present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrence of an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradation of mitochondria.

Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene

15‐Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH‐GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH‐GPx is less well investigated. In this study we cloned the PH‐GPx cDNA from a mouse fibroblast cDNA library and the PH‐GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5′‐flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.