Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

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.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

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.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.

Human intestinal epithelial cells express functional cytokine receptors sharing the common gamma c chain of the interleukin 2 receptor.

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.

Cloning and characterization of a human type II receptor for bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily. Several members of this family have been shown to transduce their signals through binding to type I and type II serine-(threonine) kinase receptors. Here we report the cDNA cloning and characterization of a human type II receptor for BMPs (BMPR-II), which is distantly related to DAF-4, a BMP type II receptor from Caenorhabditis elegans. In transfected COS-1 cells, osteogenic protein (OP)-1/BMP-7, and less efficiently BMP-4, bound to BMPR-II. BMPR-II bound ligands only weakly alone, but the binding was facilitated by the presence of previously identified type I receptors for BMPs. Binding of OP-1/BMP-7 to BMPR-II was also observed in nontransfected cell lines. Moreover, a transcriptional activation signal was transduced by BMPR-II in the presence of type I receptors after stimulation by OP-1/BMP-7.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.