The ST2 gene encodes receptor-like molecules that are very similar to the type I interleukin-1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA-1. The main promoter used for the expression of the ST2 gene in UT-7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT-7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT-7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.Keywords: immunoglobulin superfamily; interleukin-1 receptor-related protein; orphan receptor; promoter usage; ST2 gene.The ST2 gene, also designated as T1, Fit-1 or DER4, was cloned as one of the primary response genes in the G 0 /G 1 transitional state of BALB/c-3T3 cells [1], a H-ras oncogeneresponsive gene [2], a Fos-responsive gene [3], and a delayed early serum response gene [4]. Subsequently, ST2L cDNA, encoding a membrane-bound protein the extracellular domain of which is almost identical to the ST2 protein, was cloned [5]. The mRNAs of ST2 and ST2L were produced by alternative 3 H splicing of the primary transcript of the ST2 gene [6]. Based on this discovery of the ST2 gene and its expression in fibroblastic cell lines, our previous studies focused on the function of the ST2 gene in growth control.On the other hand, structural analysis of the ST2 cDNA revealed that the ST2 protein was remarkably similar to the members of the immunoglobulin superfamily, especially to the extracellular portion of the mouse interleukin-1 receptor (IL-1R) [1], and the ST2L protein showed a striking overall similarity to the mouse type I interleukin (IL)-1 receptor (IL-1RI) [5]. Furthermore, the genes encoding ST2 and the two IL-1 receptors, IL-1RI and IL-1RII, were tightly linked on mouse chromosome 1 [7] and human chromosome 2 [8]. The human ST2 gene was assigned to chromosome 2q11.2 [9]. However, IL-1a, b, and receptor antagonist did not bind to the ST2L protein, suggesti...

We recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO K1 (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade (-)A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade (-)A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G(1)/S transition.

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade ؊ A cells (an ATasedeficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.Purine nucleotides are synthesized both via the de novo pathway and via the salvage pathway and are vital for cell functions and cell proliferation through DNA and RNA syntheses and ATP energy supply. Amidophosphoribosyltransferase (ATase) 1 is the rate-limiting enzyme in the de novo pathway of purine ribonucleotide synthesis (1) and is regulated by feedback inhibition by AMP and GMP. Hypoxanthine (Hx) and hypoxanthine guanine phosphoribosyltransferase (HPRT) are the most important substrate and enzyme, respectively, of the salvage pathway (1). The Lesch-Nyhan syndrome is caused by a complete deficiency of HPRT. Although patients with this syndrome show hyperuricemia with accelerated de novo purine synthesis, the mechanism of activation of the de novo pathway is not fully understood.In a previous study, we demonstrated that the expression level of ATase limits the growth rate of cultured fibroblasts, and purine salvage strongly inhibits purine de novo synthesis (1). Two mechanisms by which the salvage pathway inhibits the de novo pathway were presumed. 1) Consumption of 5-phosphoribosyl 1-pyrophosphate (PRPP) by the salvage pathway decreases the activity of the de novo pathway because PRPP is the common source of both pathways. 2) Purin...