Erythropoietin gene expression is suppressed after lipopolysaccharide or interleukin-1 beta injections in rats.

Proinflammatory cytokines play an important role in the pathogenesis of anemia in inflammatory diseases. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been reported to inhibit the synthesis of erythropoietin (EPO) in vitro. To evaluate the in vivo significance of this observation, we have investigated effects of the administration of bacterial lipopolysaccharide (LPS) and IL-1 beta on renal EPO production in rats. Measurements by competitive reverse-transcription polymerase chain reaction showed that EPO mRNA levels were significantly reduced in the kidneys of normoxic rats 6 h after the injection of LPS (0.1 or 1 mg/kg). In addition, LPS and IL-1 beta (1 microgram/kg) inhibited the increase in EPO mRNA and plasma EPO levels when administered to rats before hypoxia exposure (8% O2 in the inspiratory gas). Evidence for an inflammatory reaction in the kidneys of LPS-treated rats was provided by measurements of greatly elevated renal TNF-alpha mRNA levels. Furthermore, kidneys isolated from LPS-created rats produced less immunoreactive EPO when perfused hypoxically in vitro for 2 h. Thus mediators of the immune response inhibit renal EPO gene expression in vivo, which is relevant with respect to the impaired synthesis of EPO in inflammatory diseases in humans.

Effects of antioxidant vitamins on renal and hepatic erythropoietin production.

An important role in O2 sensing has been assigned to microsomal and membrane-bound b-type cytochromes which generate regulatory reactive O2 species (ROS). Recently, ROS have been shown to suppress the in vitro synthesis of erythropoietin (Epo). We investigated the potential of the antioxidant vitamins A, E and C to enhance renal and hepatic Epo production. Renal effects were studied in isolated serum-free perfused rat kidneys. In control experiments without antioxidant vitamins, Epo secretion amounted to 441 +/- 23 mU/g kidney (mean +/- SEM, N = 5) during the three hour period of hypoxic perfusion (arterial pO2 35 mm Hg). Epo secretion significantly increased to 674 +/- 92 mU/g kidney (N = 7) when vitamins A (0.5 microgram/ml), E (0.5 microgram/ml) and C (10 micrograms/ml) in combination were added to the perfusion medium. The effects of the single vitamins were studied in Epo-producing hepatoma cell cultures (lines HepG2 and Hep3B). Vitamin A induced a dose-dependent increase (half-maximal stimulation at 0.2 microgram/ml) in the production of immunoreactive Epo during 24 hours of incubation (such as 680 +/- 51 U Epo/g cell protein in HepG2 cultures with 3 micrograms/ml retinol acetate compared to 261 +/- 15 U/g in untreated controls; N = 4). In contrast, vitamin E (tested from 0.05 to 500 micrograms/ml) and vitamin C (tested from 2 to 200 micrograms/ml) did not increase Epo production in hepatoma cell cultures. Thus, while vitamins E and C may have the potential to protect cells from oxidative damage, vitamin A exerts a specific stimulation of Epo production. Preliminary evidence suggests that this effect of vitamin A involves increased mRNA levels of hypoxia-inducible factor 1 alpha (HIF-1 alpha).

A transcript family from a long-range repeat cluster of the house mouse.

A family of closely related genes is a component of the polymorphic long-range repeat cluster D1Lub1 of the house mouse. Members of the gene family have diverged from one another by rearrangements and point mutations. D1Lub1 cluster have low (approximately 50) or high (> or = 500) copy numbers. In mice with high-copy clusters five or six poly(A)+ RNAs are found, while in mice with low-copy clusters only a single member of the RNA family is detected. The RNA family is synthesized in a tissue-independent manner. Each member of the RNA family is defined by a set of DNA probes. Cross hybridization with the probes reveals common 5' regions and variable remaining parts. The RNA variants are probably transcribed from different gene copies.

A member of the mouse LRR transcript family with homology to the human Sp100 gene.

A previously isolated cDNA sequence with homology to the long-range repeat (LRR) cluster in chromosome 1 of the house mouse, Mus musculus, was identified as derived from a 1.3 kb polyadenylated RNA. This transcript belongs to a family of polyadenylated RNAs which are synthesized from a multicopy gene included in the LRR copies. The representation of the 1.3 kb transcript in genomic DNA was studied in lambda and cosmid clones from the LRR cluster. Two different types of LRRs were detected with respect to the arrangement of coding regions. In the type-1 arrangement, the sequence is split into five exons, and in the type-2 arrangement, into six exons. The respective exons with their flanking regions were sequenced. The analysis of splice signals revealed that LRR copies with a type-1 arrangement are presumably the source of the 1.3 kb transcript. The 1.3 kb transcript has sequence homology to a human gene encoding Sp100, a nuclear antigen recognized by autoantibodies from patients suffering from some autoimmune diseases including primary biliary cirrhosis. Mouse exons II and III exhibit 71% homology at the nucleotide level and 56% homology at the amino acid level to the human Sp100 cDNA. We mapped the human Sp100 gene to chromosome 2. This location corroborates the assumption that the human Sp100 gene and the mouse LRR gene are homologous, as the human chromosome 2 contains the segment which is homologous to the mouse LRR region.

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus.

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3' end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, an estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1-4 million years ago.