Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.

Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense.

The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction of cell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1beta-converting enzyme. Actin cleaved by interleukin 1beta-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin. These findings suggest a mechanism for the coordination of the proteolytic, endonucleolytic, and morphogenetic aspects of apoptosis.

Amyloid beta peptide potentiates cytokine secretion by interleukin-1 beta-activated human astrocytoma cells.

Neurodegenerative processes in Alzheimer disease (AD) are thought to be driven in part by the deposition of amyloid beta (A beta), a 39- to 43-amino acid peptide product resulting from an alternative cleavage of amyloid precursor protein. Recent descriptions of in vitro neurotoxic effects of A beta support this hypothesis and suggest toxicity might be mediated by A beta-induced neuronal calcium disregulation. In addition, it has been reported that "aging" A beta results in increased toxic potency due to peptide aggregation and formation of a beta-sheet secondary structure. In addition, A beta might also promote neuropathology indirectly by activating immune/inflammatory pathways in affected areas of the brain (e.g., cortex and hippocampus). Here we report that A beta can modulate cytokine secretion [interleukins 6 and 8 (IL-6 and IL-8)] from human astrocytoma cells (U-373 MG). Freshly prepared and aged A beta modestly stimulated IL-6 and IL-8 secretion from U-373 MG cells. However, in the presence of interleukin-1 beta (IL-1 beta), aged, but not fresh, A beta markedly potentiated (3- to 8-fold) cytokine release. In contrast, aged A beta did not potentiate substance P (NK-1)- or histamine (H1)-stimulated cytokine production. Further studies showed that IL-1 beta-induced cytokine release was potentiated by A beta-(25-35), while A beta-(1-16) was inactive. Calcium disregulation may be responsible for the effects of A beta on cytokine production, since the calcium ionophore A23187 similarly potentiated IL-1 beta-induced cytokine secretion and EGTA treatment blocked either A beta or A23187 activity. Thus, chronic neurodegeneration in AD-affected brain regions may be mediated in part by the ability of A beta to exacerbate inflammatory pathways in a conformation-dependent manner.

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.

Interleukin 1 beta up-regulates the expression of sulfoglucuronosyl paragloboside, a ligand for L-selectin, in brain microvascular endothelial cells.

Treatment of cultured bovine brain microvascular endothelial cells (BMECs) with interleukin 1 beta (IL-1 beta), an inflammatory cytokine, was shown to induce the accumulation of sulfoglucuronosyl paragloboside (SGPG), a glycolipid bearing the HNK-1 epitope. This resulted in the attachment of a greater number of human lymphocytes to the treated than to the untreated BMEC monolayers. Attachment of human lymphocytes to the IL-1 beta-activated BMEC cells could be blocked either by incubation of the human lymphocytes with an anti-L-selectin antibody or by application of an anti-SGPG antibody to the BMECs. These results suggest that SGPG may act as an important ligand for L-selectin for the regulation of the attachment of activated lymphocytes and their subsequent invasion into the nervous system parenchyma in inflammatory disorders of the central and peripheral nervous systems.

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.