Macrophages play a key role in both normal and pathological processes involving immune and inf lammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor ␤ (TGF-␤) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1␤, tumor necrosis factor ␣ (TNF-␣), interleukin 2, and macrophage colony-stimulating factor but not interferon ␥, or lipopolysaccharide (LPS). Its expression is also increased by TGF-␤. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of M r 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-␣ production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinf lammatory cytokines and TGF-␤ may serve to limit the later phases of macrophage activation.

Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-beta receptor II, extracellular signal-regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.

Abstract-Here we identified growth-differentiation factor 15 (GDF15) (also known as MIC-1), a secreted member of the transforming growth factor (TGF)-␤ superfamily, as a novel antihypertrophic regulatory factor in the heart. GDF15 is not expressed in the normal adult heart but is induced in response to conditions that promote hypertrophy and dilated cardiomyopathy. To elucidate the function of GDF15 in the heart, we generated transgenic mice with cardiac-specific overexpression. GDF15 transgenic mice were normal but were partially resistant to pressure overload-induced hypertrophy. Expression of GDF15 in neonatal cardiomyocyte cultures by adenoviral-mediated gene transfer antagonized agonist-induced hypertrophy in vitro. Transient expression of GDF15 outside the heart by intravenous adenoviral delivery, or by direct injection of recombinant GDF15 protein, attenuated ventricular dilation and heart failure in muscle lim protein gene-targeted mice through an endocrine effect. Conversely, examination of Gdf15 gene-targeted mice showed enhanced cardiac hypertrophic growth following pressure overload stimulation. Gdf15 gene-targeted mice also demonstrated a pronounced loss in ventricular performance following only 2 weeks of pressure overload stimulation, whereas wild-type controls maintained function. Mechanistically, GDF15 stimulation promoted activation of SMAD2/3 in cultured neonatal cardiomyocytes. Overexpression of SMAD2 attenuated cardiomyocyte hypertrophy similar to GDF15 treatment, whereas overexpression of the inhibitory SMAD proteins, SMAD6/7, reversed the antihypertrophic effects of GDF15. These results identify GDF15 as a novel autocrine/endocrine factor that antagonizes the hypertrophic response and loss of ventricular performance, possibly through a mechanism involving SMAD proteins. Key Words: cardiac Ⅲ signaling Ⅲ hypertrophy Ⅲ growth factors Ⅲ mouse genetics C ardiac hypertrophy is typically characterized by an enlargement of the heart associated with an increase in cardiomyocyte cell volume that occurs during postnatal development, in response to physiologic stimuli such as exercise and in response to diverse pathophysiological stimuli such as hypertension, ischemic heart disease, valvular insufficiency, infectious agents, or mutations in sarcomeric genes. 1 Pathologic hypertrophic growth of the myocardium is a leading predictor for the development of arrhythmias and sudden death, as well as dilated cardiomyopathy and heart failure. 2,3 In general, the hypertrophic growth of the myocardium is regulated by endocrine, paracrine, and autocrine growth factors that activate membrane-bound receptors resulting in signal transduction that culminates in altered gene transcription and protein accumulation. 4 Both pro-and antihypertrophic growth factors have been characterized. For example, angiotensin II (Ang II) serves as an endocrine and autocrine growth factor underlying pathological cardiac hypertrophy, whereas insulin-like growth factor (IGF)-1 signaling is thought to function, in part, by mediating deve...

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among Ϸ12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as ␣-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide͞bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-␤ superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation͞protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.gene expression ͉ microarray ͉ genome ontology ͉ sequence analysis ͉ immunohistochemistry

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24 -Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.Chloride ion channels control a variety of cellular processes that are central to normal function and disease states (1). The CLIC 1 family is a recently identified class of Cl Ϫ channel proteins that consists of seven members (p64, parchorin, CLIC1-5) (2, 3). A conserved C-terminal CLIC module of ϳ240 amino acids is present in each member of the family with several members containing additional, unrelated Nterminal domains. Most CLICs are localized to intracellular membranes and have been linked to functions including apoptosis, pH, and cell cycle regulation (4 -6). The CLIC ion channels are unusual in that they possess both soluble and integral membrane forms (2). In this regard they are similar to some bacterial toxins and several classes of intracellular proteins including Bcl-x L and the annexins (7). Our understanding of how such dual natured proteins enter the membrane is limited by the dearth of high resolution structures for key states in this process.We have recently determined the crystal structure of a soluble monomeric form of CLIC1 (8) and found that it is a structural homologue of the GST superfamily of proteins (9). This soluble form of CLIC1 consists of two domains, the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all ␣-helical C-domain, which is typical of the GST superfamily. CLIC1 contains an intact glutathione-binding site that was shown to covalently bind glutathione via a conserved CLIC cysteine residue, Cys-24. This led to the suggestion that CLIC1 function may be under redox control, possibly via reactive oxygen or nitrogen species.The structure and stoichiometry of the integral membrane form of the CLIC proteins is still unclear. Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 1...

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-Å resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.Chloride ion channels, located both within the plasma membrane and other internal cell membranes (1, 2), are involved in diverse physiological processes. They are known to participate in the control of secretion and absorption of salt, regulation of membrane potentials, organellar acidification, and cell volume homeostasis (3). Malfunction in these channels can lead to severe disease states (4).Chloride channels fall into several classes based on their sequence relationships. The three best characterized classes are the ligand-gated receptor channels (␥-aminobutyric acid and glycine receptors), the cystic fibrosis transmembrane conductance regulator family, and the ClC chloride ion channels (1, 2). A new class of chloride ion channel, the "chloride intracellular channels" (CLICs), 1 has recently been characterized at a molecular level. To date, there are seven members of the CLIC family: CLIC1 (NCC27) (5), CLIC2 (6), CLIC3 (7), CLIC4 (8), CLIC5 (9), p64 (10), and parchorin (11). All of these proteins exist as soluble globular proteins that can form ion channels in organellar and plasma membranes (5,7,8,(12)(13)(14)(15). Five of the CLIC proteins are each composed of ϳ240 residues, while the longer p64 and parchorin consist of distinct amino-terminal domains followed by the 240-residue CLIC module. This module has recently been shown to share weak sequence homology with the glutathione S-transferase (GST) superfamily (16).The CLIC proteins are expressed in a wide variety of tissues and appear to have diverse physiological functions. p64 is associated with kidney function (17), while CLIC1 and CLIC4 appear to have a broad tissue distribution (5,8,18,19). Several CLICs interact with protein kinases (7,11,20). CLICs are associated with a variety of intracellular membranes including the nuclear membrane (5), the endoplasmic reticular membrane (8), large dense-core vesicles (19), mitochondria (21), trans-Golgi vesicles (22), and secretory vesicles (23). Parchorin forms the chloride channel in water-secreting cells,...

Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles. However, until recently they were believed not to occur at the nuclear membrane. In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937. NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles. NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.Whereas ion channels have been found on the membranes of various intracellular organelles, it has only been recently that patch clamping studies have suggested their existence at the nuclear membrane. The nuclear pore complexes have been considered the site of communication and exchange between the nucleus and cytoplasm (1, 2). Studies of traffic across the nuclear envelope have in general conformed to the paradigm that ions and small metabolites with diameters of less than 3-4 nm passively diffuse across the nuclear envelope (3). Thus, the concept of nuclear membrane ion channels seems at variance with the generally accepted views of the nuclear envelope, with their function at this location seeming redundant. However, this does not appear to be the case.The first demonstration of ionic conductances in the nuclear membrane were in mouse zygote pronuclei (4). Further evidence then followed with the demonstration of ion-selective channels in avian erythrocytes (5), in mouse oocyte germinal vesicles, in nuclei from two-cell embryos and liver (6), in the nuclei of cardiac myocytes (7), and in rat hepatocyte nuclei (8). Mak and Foskett (9) described the presence of inositol 1,4,5-trisphosphate-dependent receptor channels in isolated nuclei which were activated by inositol 1,4,5-trisphosphate, inhibited by heparin and selective to calcium ions. Similarly, Pasyk and Foskett (10), have shown chloride channel activity in isolated nuclei from CHO 1 cells. More recently, following fractionation and reconstitution of inner and outer nuclear membrane fractions into lipid bilayers, Rousseau et al. (11) have shown the presence of two types of chloride channels. The use of calcium ion imaging techniques has also demonstrated variations in calcium ion concentrations between the nucleus and cytoplasm, again suggesting a selective uptake or retention of these ions by the nucleus (12, 13).With growing electrophysiological data for the existence of nuclear ion channels, it is clear that the cloning and isolation of these proteins will greatly assist in determining their structure and function. This paper describes the molecular cloning and characterization of what we believe to be the first chloride ion channel protein of the nuclear membrane and only the second cloned ion chan...

Macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor-B superfamily cytokine, is involved in tumor pathogenesis, and its measurement can be used as a clinical tool for the diagnosis and management of a wide range of cancers. Although generally considered to be part of the cell's antitumorigenic repertoire, MIC-1 secretion, processing, and latent storage suggest a complex, dynamic variability in MIC-1 bioavailability in the tumor microenvironment, potentially modulating tumor progression and invasiveness.