IL-1 receptor accessory protein is an essential component of the IL-1 receptor.

The recently described IL-1R accessory protein (IL-1R AcP) interacts with IL-1beta and the IL-1 type-IR (IL-1RI), but an essential requirement for IL-1R AcP in IL-1 signaling in vitro has not been established and its role in vivo has not been examined. In this study, IL-1R AcP-deficient mice and fibroblasts were produced and characterized. All IL-1 agonists bound to IL-1R AcP-deficient cells through the type I IL-1R, but failed to activate gene expression through either the nuclear factor-kappaB or AP-1-dependent signaling pathways. Absence of IL-1R AcP differentially affected the affinity for IL-1 ligands. IL-1R AcP-deficient fibroblasts bound murine IL-1alpha and human IL-1R antagonist protein (IL-1Ra) with only moderately reduced affinity when compared with wild-type cells, whereas murine IL-1beta affinity was reduced by 70-fold. IL-1 also failed to produce a biologic response in vivo in IL-1R AcP-deficient mice. These data demonstrate that a type I IL-1R/IL-1R AcP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1beta. Finally, IL-1R AcP is an essential signal transducing component of the functional IL-1R and should represent a novel target for blocking IL-1 function in human disease.

Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.

IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases. Two IL-1R, the type I and type II receptors, have been identified. This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI). IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection. These data also demonstrate that IL-1 function is not required for normal development or homeostasis.

A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP-1-like subfamily with highly conserved ATPase and protease domains.

We have used a rat pachytene spermatocyte cDNA expression library to clone TBP-1 (for tat-binding protein-1; designated rat testis TBP-1 [rtTBP-1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP-1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP-1 protein. rtTBP-1 protein contains a heptad repeat of six leucine-type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP-1, consists of a protein triplet with a molecular mass range of 52-48 kDa and acidic pI (5.0-5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin-labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP-1 immunoreactive sites colocalize with alpha-tubulin-decorated manchettes of elongating spermatids. In addition, rtTBP-1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP-1 is a member of the highly evolutionary conserved TBP-1-like subfamily of putative ATPases, sharing regions of identity-including ATP-binding sites-with several subunits of the 26S proteasome, known to be involved in the ATP-dependent degradation of ubiquitin-conjugated proteins.

Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse.

mTEF-1 is the prototype of a family of mouse transcription factors that share the same TEA DNA binding domain (mTEAD genes) and are widely expressed in adult tissues. At least one member of this family is expressed at the beginning of mouse development, because mTEAD transcription factor activity was not detected in oocytes, but first appeared at the 2-cell stage in development, concomitant with the onset of zygotic gene expression. Since embryos survive until day 11 in the absence of mTEAD-1 (TEF-1), another family member likely accounts for this activity. Screening an EC cell cDNA library yielded mTEAD-1, 2 and 3 genes. RT-PCR detected RNA from all three of these genes in oocytes, but upon fertilization, mTEAD-1 and 3 mRNAs disappeared. mTEAD-2 mRNA, initially present at approx. 5,000 copies per egg, decreased to approx. 2,000 copies in 2-cell embryos before accumulating to approx. 100,000 copies in blastocysts, consistent with degradation of maternal mTEAD mRNAs followed by selective transcription of mTEAD-2 from the zygotic genome. In situ hybridization did not detect mTEAD RNA in oocytes, and only mTEAD-2 was detected in day-7 embryos. Northern analysis detected all three RNAs at varying levels in day-9 embryos and in various adult tissues. A fourth mTEAD gene, recently cloned from a myotube cDNA library, was not detected by RT-PCR in either oocytes or preimplantation embryos. Together, these results reveal that mTEAD-2 is selectively expressed for the first 7 days of embryonic development, and is therefore most likely responsible for the mTEAD transcription factor activity that appears upon zygotic gene activation.

Preimplantation development of the mammalian embryo and its regulation by growth factors.

Preimplantation development in mammals involves both the development of the embryo and the preparation of the uterus in anticipation of blastocysts implantation. Preparation of the uterus for implantation is primarily under the control of the ovarian sex steroids, estrogen and progesterone. Increasing evidence is revealing that their effects on cell proliferation and differentiation in the uterus are medicated by locally produced growth factors and cytokines. In contrast, preimplantation development of the embryo to the blastocyst stage appears to be independent of exogenous growth factors. Implantation, the point at which the blastocyst forms a more intimate association with the maternal tissues, is regulated by the uterine expression of leukemia inhibitory factor (LIF). LIF is required both to promote embryo attachment and for decidualization of the uterus. In the absence of LIF, neither of these events occur. Uterine expression of LIF at the time of implantation has been described in many species, suggesting that LIF may be of general significance in regulating embryo implantation in mammals.

Reproduction in mice lacking a functional type 1 IL-1 receptor.

Growth factors and cytokines have been identified in having critical roles at mediating maternal-fetal interactions during pregnancy, with interleukin-1 being a recently implicated factor. Previous experiments indicated that repeated intraperitoneal injections of the Il-1 receptor antagonist (Il-1Ra), which inhibits binding of interleukin-1 (Il-1) to the type 1 Il-1 receptor (Il-1Rt1) blocks blastocyst implantation in superovulated mice. To gain a greater insight into the role of Il-1 receptor in implantation, we analyzed the reproduction of mice deficient for the Il-1Rt1. Our results show that mice lacking this receptor do not exhibit any profound alterations in their reproduction, apart from a slight reduction in mean litter size. Furthermore, repeated intraperitoneal injections of either IL-1Ra or the monoclonal antibody 35F5, which also blocks ligand binding to the Il-1Rt1, did not affect embryo implantation in either wild type and Il-1 receptor deficient mice.

Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation.

The uterine expression of leukemia inhibitory factor (LIF) is essential for embryo implantation in the mouse. Here, we describe the expression of LIF, related members of this group of cytokines, oncostatin M and ciliary neurotrophic factor, and the LIF receptor beta and glycoprotein gp130 in normal human tissues and in the endometrium of fertile women. Our results show that LIF is the only one of these factors expressed at detectable levels in the endometrium of women of proven fertility. LIF expression is restricted to the endometrial glands during the secretory/postovulatory phase but is not present in the endometrium during the proliferative/preovulatory phase. The LIF receptor beta is expressed during the proliferative and secretory phases of the cycle and is restricted to the luminal epithelium. The associated signal-transducing component of the LIF receptor, gp130, is also expressed in both the luminal and glandular epithelium throughout the cycle. These results suggest that uterine expression of LIF in humans, like mice, may have a role in regulating embryo implantation, possibly through an autocrine/paracrine interaction between LIF and its receptor at the luminal epithelium.

[Regulation by maternally derived cytokines of pre-implantation development and uterine receptiveness]. / Régulation par les cytokines d'origine maternelle du développement pré-implantatoire et de la réceptivité utérine.

Increasing evidence is revealing that cytokines, under the control of steroid hormones, are important in regulating fetal-maternal interactions during pregnancy. Mice carrying mutated genes coding for these factors or their receptors, have been instrumental in clarifying the in vivo roles of these factors in reproduction. Lack of many of the cytokines expressed in the male and female reproductive tracts, such as TNF-alpha, Inf-gamma, the majority of the interleukins and the transforming growth factors TGF-alpha and beta do not result in any gross impairment in reproductive efficiency. However, some cytokines do have a critical role, in particular Leukemia Inhibitory Factor (LIF), Colony stimulating Factor-1 (CSF-1), Epidermal Growth Factor (EGF) and possibly interleukin-1 (IL-1). The recent advances relating to the reproductive roles of these factors up to the time of embryo implantation will be discussed.

Extrachromosomal chromatin: novel target for bleomycin cleavage in cells and solid tumors.

The preference of bleomycin, a DNA strand scission antitumor agent, to damage extrachromosomal (episomal) DNA was investigated. These episomes contain transcriptional promoters, replication origins, and oncogenes from MMTV, BPV, and v-Ha-ras and confer a neoplastic phenotype to a mouse fibroblast cell line. We found that bleomycin induces dose-dependent single- and double-stranded cleavage of intracellular episomes as measured by topological forms conversion. Bleomycin scission of episomes occurs within 1 min, and upon drug removal, damaged episomes are as rapidly repaired. By expressing the episomal and genomic damage as breaks per nucleotide, bleomycin has a 30-50-fold cleavage preference for episomal chromatin compared to genomic DNA. The episomes have preferred regions of the bleomycin-induced damage, particularly within the MMTV LTR and BPV origin of replication. Also, it is possible to assess bleomycin action on episomes in solid tumors in mice. Single intravenous injections of BLM into tumor-bearing mice result in single- and double-stranded cleavage of episomes that are dose related and occur within 1 min. Specific double-stranded breaks occur in the same regulatory regions of episomes in solid tumors and in cultured cells. Finally, we observe that damage to the episomal drug target occurs at therapeutic doses in mice.

Topoisomerase II-mediated DNA damage of episomes in tumor-bearing mice.

Observations of cells in culture have demonstrated that, for many antitumor agents, topoisomerase II-mediated DNA damage relates to cytotoxicity. However, there is no evidence in tumor-bearing animals to suggest that such agents induce topoisomerase II-mediated damage of DNA in solid tumors or that such damage reflects inhibition of tumor growth. To address this question, a mouse fibroblast cell line neoplastically transformed by an episomal element containing the v-Ha-ras and bovine papillomavirus genes was utilized to measure topoisomerase II-induced DNA damage and growth inhibition of solid tumors derived from this line. Using the topoisomerase II inhibitor amsacrine, the episomal element was found to be a sensitive indicator of topoisomerase II-mediated damage in vivo. The DNA breaks induced by single i.v. injections of amsacrine were protein linked and occurred preferentially in episomal regulatory regions. A strong correlation between suppression of tumor growth and topoisomerase II-mediated damage of the episome was demonstrated.

A study of drug-induced topoisomerase II-mediated DNA lesions on episomal chromatin.

A well defined extrachromosomal DNA element, referred to as an episome (Ostrowski, M., Richard-Foy, H., Wolford, R., Berard, D., and Hager, G. (1983) Mol. Cell. Biol. 3, 2045-2057), was employed as a target for the topoisomerase II inhibitors amsacrine and teniposide. Both drugs have distinct mechanisms of action in cleaving the episome, as defined by topological forms conversion assays. The concentration ranges required to measure episomal cleavage are similar. The onset of damage induced by amsacrine begins within 1 min and is maintained at that level for at least 1 h. Teniposide induces damage that peaks between 30 and 60 min. The amsacrine-induced damage is only partially reversible, whereas teniposide-induced damage is almost completely reversible. Sites of specific cleavage are quite dissimilar. Multiple cleavage sites are formed in the episomal regulatory regions after amsacrine treatment, whereas a single cleavage in the regulatory region and one outside this region are found after teniposide treatment. Transcriptional activation using dexamethasone does not change the amount or site preference of episomal cleavage induced by either agent. Damage to the episome was quantitatively compared with damage produced in genomic DNA between 500 and 24,000 rad equivalents. The study showed that amsacrine has a significant (33-38-fold) preference for episomal DNA over genomic DNA.

Neocarzinostatin and auromomycin preferentially cleave simian virus 40 DNA and chromosomes at a number of discrete locations.

Neocarzinostatin and auromomycin were shown to cleave simian virus 40 (SV40) DNA with preference for distinct regions of the viral genome. The positions cut by neocarzinostatin and auromomycin were similar, while micrococcal nuclease cleaved at positions other than those recognized by neocarzinostatin and auromomycin. Breaks were distributed throughout the viral genome and were not associated with any single type of genetic element. The limited number of locations in SV40 DNA that were preferentially cut by neocarzinostatin and auromomycin suggests that drug attack is directed by DNA structures other than the known trinucleotide sequence specificity of the drugs. Neocarzinostatin and auromomycin cut purified, cell-free, nuclear and intracellular chromosomal SV40 DNA at similar regions. The data indicate that there are regions in DNA that are hypersensitive to the drugs; the hypersensitivity may be determined by the microstructure of the DNA. The conformational change associated with the packing of the DNA into nucleosomes did not affect the microstructure of the sensitive region, nor did the shielding effect of nuclear proteins affect the drug's access to it. In addition, intracellular drug metabolism or other cellular factors did not alter the ability of drugs to interact at these sensitive regions.