Molecular cloning, sequence analysis, expression, and tissue distribution of suppressin, a novel suppressor of cell cycle entry.

Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990) J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5'- and 3'-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at approximately 2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator in Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types.

Interleukin-1 receptors in the brain: characterization by quantitative in situ autoradiography.

Autoradiography was performed on mouse brain cryosections to localize interleukin-1 (IL-1) receptors in the mouse brain and pituitary gland and to identify the cell types expressing these receptors. Interleukin-1 receptor binding sites were mapped in the mouse central nervous system (CNS) with [125I]IL-1 alpha and [125I]IL-1 beta. IL-1 receptors were detected in high density in the dentate gyrus of the hippocampus, choroid plexus, meninges, and anterior pituitary. IL-1 receptors are also expressed in the frontoparietal cortex at very low density. Both neurons and glial cells were shown to express IL-1 receptors. An intrahippocampal injection of colchicine, a selective neurotoxin, induced the concurrent disappearance of neuronal cells and [125I]IL-1 alpha binding in the hippocampus. This treatment established that IL-1 bound to the neurons of the dentate gyrus. IL-1 receptors on glial cells were not detected in situ in the CNS under basal conditions. However, [125I]IL-1 alpha bound to glial cells at the site of astrogliosis induced by a local mechanical injury. These results suggest that activated astrocytes express IL-1 receptors. Furthermore, the results of histoautoradiography of [125I]IL-1 alpha binding on astrocyte and microglial cultures showed that astrocytes express IL-1 receptors in vitro. The biochemical characterization of IL-1 binding in the dentate gyrus was achieved by quantitative in situ autoradiography. In the dentate gyrus IL-1 bound to a single class of receptor with characteristics similar to those of the receptor expressed on immune cells (Kd = 0.3 +/- 0.2 nM, Bmax = 60 +/- 10 fmol/mg protein). Competition experiments with IL-1 alpha and IL-1 beta showed that the neuronal receptor characteristics were similar to those of the type I IL-1 receptor, which binds the two isoforms of IL-1 with the same affinity. Regulation of IL-1 receptor density in the CNS and pituitary was studied after peripheral injection of LPS. Stimulation of IL-1 synthesis by LPS was shown to induce a major decrease in the number of receptors available for IL-1 binding in the CNS. A decrease of 84 +/- 9% was observed in the dentate gyrus and in the choroid plexus, but no change occurred in the pituitary gland.

Suppressin: an endogenous negative regulator of immune cell activation.

We have recently identified a new suppressor molecule we named suppressin (SPN) that has all the characteristics of a global negative regulator of the immune system. SPN is a unique 63-kD monomeric polypeptide with a pI of 8.1 that is produced and secreted under basal conditions by murine splenocytes, human peripheral mononuclear cells, and hormone-secreting pituitary cells. The biological actions of SPN in vitro include the inhibition of mitogen-induced proliferation and immunoglobulin synthesis of lymphocytes and the suppression of interleukin-2-dependent CTLL-2 cell proliferation. In addition, SPN enhances natural killer cell activity by eliciting interferon-alpha and -beta synthesis and secretion. SPN effects are reversible, nontoxic, and require the continuous presence of exogenous SPN. T lymphocytes stimulated with concanavalin A or phytohemagglutinin are more sensitive to SPN (90% inhibition) than are lipopolysaccharide-stimulated B cells (60% inhibition). SPN arrests lymphocytes in the G0/G1 phase of the cell cycle after reduction of their RNA, protein and DNA synthesis, suggesting that SPN inhibits the processes required for G0 transition to G1. SPN is found intracellularly in all unstimulated lymphocyte subsets, monocytes, and in phytohemagglutinin-activated T lymphocytes immunopositive for the low affinity interleukin-2 receptor. These results suggest that SPN may be a major negative regulator of cell proliferation in the immune system. All SPN-producing cell types are also sensitive to SPN. Collectively, the results of these experiments provide the foundations for a model in which SPN regulates lymphocyte proliferation in an autocrine and/or paracrine manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of suppressin-producing cells in the rat pituitary.

Suppressin (SPN) is a novel polypeptide that is synthesized and secreted by normal rat pituitary cells and a rat pituitary tumor cell line, GH3. Specifically, SPN is a negative regulator of growth that inhibits lymphoid and neuroendocrine cell proliferation. The objective of the present study was to identify the cells in the normal rat pituitary that produce SPN. A double immunofluorescence technique, using antibodies to SPN in conjunction with antibodies to the six major adenohypophyseal hormones, was used to colocalize SPN and a specific hormone in a single dispersed pituitary cell. The results of these experiments showed that, on the average, 42% of the cells in the pituitary produce SPN. Suppressin production in the pituitary was restricted to the adenohypophysis. The SPN-producing population in the pituitary was composed of somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs, while gonadotrophs did not produce SPN. Additionally, a PRL reverse hemolytic plaque assay was used to examine SPN production in lactotrophs. The results of these experiments showed that SPN production and the amount of PRL secreted covaried. Specifically, SPN production was observed primarily in non-PRL-secreting lactotrophs or in lactotrophs secreting a high amount of PRL. The results of these experiments suggest a potential regulatory relationship between the synthesis and secretion of SPN and PRL. In summary, this report provides the first identification of SPN-producing cells in the pituitary and shows that SPN production occurs primarily in somatotrophs and lactotrophs.

Interleukin-1 binding sites on astrocytes.

Interleukin-1 has been shown to have regulatory effects on glial cell functions. In this study, we examined the capacity of astroglial cells to specifically bind recombinant iodinated human interleukin-1 alpha. This was performed in mouse brain by both in situ and in vitro autoradiography, on areas of gliosis and on astrocytes and microglia primary and secondary cultures respectively. Specific binding was shown in the brain sections over areas of glial proliferation, and in addition, quantitative autoradiography was performed. Analysis of competition experiments by autoradiography led to EC50 values of 5 x 10(-11) M for human interleukin-1 alpha and approximately 10(-9) M for the interleukin-1 receptor antagonist. In cultures, iodinated human interleukin-1 alpha bound specifically to astrocytes but was unable to bind to microglial cells. Competition binding experiments in astrocyte cultures led to EC50 values of 8 x 10(-11) M and 1 x 10(-10) M for human interleukin-1 alpha and mouse interleukin-1 beta respectively, and an EC50 higher than 10(-9) M for the antagonist. The presence of interleukin-1 receptors on astroglial cells provides biochemical support for the various effects of interleukin-1 in the central nervous system, particularly those concerning the formation of scar tissue, possibly by astroglia proliferation after brain injury.