Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice.

Leptin, a hormone secreted by adipocytes, regulates the size of the adipose tissue mass through effects on satiety and energy metabolism. Leptin's precise sites of action are not known. The leptin receptor (Ob-R) is found in many tissues in several alternatively spliced forms raising the possibility that leptin exerts effects on many tissues including the hypothalamus. Ob-R is a member of the gp130 family of cytokine receptors which are known to stimulate gene transcription via activation of cytosolic STAT proteins. In order to identify the sites of leptin action in vivo, we assayed for activation of STAT proteins in mice treated with leptin. The STAT proteins bind to phosphotyrosine residues in the cytoplasmic domain of the ligand-activated receptor where they are phosphorylated. The activated STAT proteins dimerize and translocate to the nucleus where they bind DNA and activate transcription. The activation of STAT proteins in response to leptin was assayed in a variety of mouse tissues known to express Ob-R. Leptin injection activated Stat3 but no other STAT protein in the hypothalamus of ob/ob and wild-type mice but not db/db mice, mutants that lack an isoform of the leptin receptor. Leptin did not induce STAT activation in any of the other tissues tested. Activation of Stat3 by leptin was dose dependent and first observed after 15 minutes and maximal at 30 minutes. Our data indicate the hypothalamus is a direct target of leptin action and that this activation is critically dependent on the gp-130-like leptin receptor isoform missing in C57BLKS/J db/db mice. This is the first in vivo demonstration of leptin signal transduction.

The state of the STATs: recent developments in the study of signal transduction to the nucleus.

The STATs (signal transducers and activators of transcription) are latent cytoplasmic proteins that, upon activation by cell surface bound polypeptide ligands, move to the nucleus to direct transcription. A variety of protein-protein interactions that affect the function of STATs has been recently recognized. It has become clear that the STATs are functional mosaics, or mixtures of signal transduction and transcription modules.

Activation of monocyte effector genes and STAT family transcription factors by inflammatory synovial fluid is independent of interferon gamma.

Activated monocytes play an important role in the pathogenesis of inflammatory arthritis. Blood monocytes which enter the inflamed joint become activated upon adherence to extracellular matrix and exposure to a complex inflammatory environment. We have analyzed the mechanism of monocyte activation by soluble factors present in inflammatory synovial fluid (SF). Greater than 75% of inflammatory SFs tested (a total of 22 fluids to date) increased cell surface expression and dramatically increased mRNA levels of monocyte activation markers Fc gamma RI, Fc gamma RIII, and HLA-DRA. This induction was not triggered by adherence, a known activating stimulus, and several lines of evidence showed that induction was not dependent upon interferon gamma (IFN-gamma). Induction was not prevented by neutralizing anti-IFN-gamma antibodies and IFN-gamma was not detected in the SFs using a sensitive enzyme-linked immunosorbent assay. The SFs also were not able to activate the IFN-gamma-activated transcription factor Stat1, thus providing further support for the absence of IFN-gamma. SFs did activate a related signal transducer and activator of transcription (STAT) family factor, termed Stat-SF, which bound specifically to the IFN-gamma response region (GRR), a well-characterized transcription element in the Fc gamma RI promoter. Based upon DNA-binding specificity and mobilities in gel shift assays, and reactivity with specific antisera, Stat-SF likely contains Stat3, or a closely related STAT family member. Neutralization of interleukin 6, a cytokine present in SFs which is known to activate Stat3, abolished the activation of Stat-SF and inhibited the induction of Fc gamma RI expression by SFs. These results demonstrate the activation of monocytes by inflammatory SF and suggest that monocyte activation at an inflammatory site may occur in the absence of IFN-gamma through the triggering of signal transduction pathways that activate STAT transcription factors.

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

A comparison between heterogeneous nuclear RNA and polysomal messenger RNA in HeLa cells by RNA-DNA hybridization.

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 microg of HeLa cell DNA binds 0.05-0.10 microg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.

A comparison of the heterogeneous nuclear RNA of HeLa cells in different periods of the cell growth cycle.

HeLa cells synthesize heterogeneous nuclear RNA (HnRNA) in the G(1), S, and G(2) portions of the cell cycle. HnRNA prepared from these various periods was compared by RNA-DNA hybridization experiments. The results indicated that some of the HnRNA molecules were equivalent at all times in the cell cycle, but limitations in the sensitivity of the hydridization reactions, as well as in the spectrum of hybridizing molecules, restrict the conclusions that can be drawn from these comparisons.

The effect of puromycin on intranuclear steps in ribosome biosynthesis.

Inhibition of protein synthesis by puromycin (100 gamma/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S --> 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.

Persistence of liver-specific messenger RNA in cultured hepatocytes: different regulatory events for different genes.

Normal adult rat hepatocytes plated on rat tail collagen-coated dishes and fed a chemically defined medium supplemented with epidermal growth factor and dimethylsulfoxide (DMSO) were examined over a 40-d culture period for (a) the amount of albumin secreted; (b) steady-state albumin mRNA levels; (c) steady-state mRNA levels for six other liver-specific genes and three common genes; and (d)transcription of several liver-specific and common genes using isolated nuclei. DMSO-treated hepatocytes in culture for 40 d expressed albumin mRNA at 45% the level of normal liver and five other liver-specific genes at levels ranging from 21% to 72% of those in normal liver. The rate of synthesis of ligandin RNA using nuclei from 40-d hepatocytes in a nascent chain extension assay was 130% of the value obtained for normal liver, indicating that liverlike transcriptional activity for ligandin was maintained in this in vitro culture system. In contrast, the rates of synthesis of albumin and phosphoenolpyruvate carboxykinase (PepCK) mRNAs using nuclei from 40-d hepatocytes were 8% and less than 1%, respectively, and, therefore, were at levels that were much lower than was expected given the steady-state mRNA levels for these two genes. The discrepancy between the steady-state mRNA levels and rates of synthesis of RNA was analyzed, and the results suggest that the albumin and PepCK mRNAs from hepatocytes in culture may be more stable than those from liver. A plateau period for secretion of albumin, expression of albumin, alpha 1-antitrypsin, ligandin, phenylalanine hydroxylase, and PepCK mRNAs, and synthesis of albumin RNA using isolated nuclei was observed from days 6 to 40. The usefulness at a biological and molecular level of a hepatocyte culture system in which liver-specific genes are expressed over a long plateau period is discussed.

The turnover of nuclear DNA-like RNA in HeLa cells.

The subcellular distribution of various types of RNA in HeLa cells is described. In addition, the relative rate of synthesis of the major classes of nuclear RNA has been determined. From these experiments it can be deduced that the heterogeneous nuclear RNA fraction is rapidly synthesized and degraded within the cell nucleus.

Structural genes of the mouse major urinary protein are on chromosome 4.

The major urinary proteins (MUPs) of mouse are a family of at least three major proteins which are synthesized in the liver of all strains of mice. The relative levels of synthesis of these proteins with respect to each other in the presence of testosterone is regulated by the Mup-a locus located on chromosome 4. In an effort to determine the mechanism of this regulation in molecular terms, a cDNA clone containing most of the coding region of a MUP protein has been isolated and identified by partial DNA sequence analysis. Using a combination of hybridization analysis and somatic cell genetics, the structural gene family has been unambiguously mapped to mouse chromosome 4. These data suggest that Mup-a regulation operates in a cis fashion and that models proposing trans regulation of MUP protein synthesis are unlikely.

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).

Acquisition of antigens characteristic of adult pericentral hepatocytes by differentiating fetal hepatoblasts in vitro.

Antigens specific to pericentral hepatocytes have been studied in adult mouse liver, during fetal development, and in cultured fetal hepatoblasts. Antibody reactive with glutamine synthetase stained all fetal liver cells but almost all cells lost this antigen after birth; only a single layer of pericentral cells retained it in adulthood. In contrast, monoclonal antibodies to major urinary protein (MUP) did not detect the antigen until approximately 3 wk after birth, after which time the cells within 6-10 cell diameters of the central veins were positive. Cultured fetal liver cells from embryos at 13 +/- 1 d of gestation were capable of differentiating in vitro to mimic events that would occur had the cells remained in the animal. About 10-20% of the explanted cells grew into clusters of hepatocyte-like cells, all of which stained with albumin antibodies. MUP monoclonals were reactive with one-half of the differentiated fetal hepatocytes. Glutamine synthetase was present in all hepatocytes after several days in culture and gradually decreased and remained in only occasional cells, all of which also contained the MUP antigen. These findings suggest that a sequence of gene controls characterizes expression of specific genes in developing liver, and that differentiating fetal hepatoblasts are capable of undergoing similar patterns of gene activity in culture.

STATs and gene regulation.

STATs (signal transducers and activators of transcription) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Biochemical and molecular genetic explorations have defined a single tyrosine phosphorylation site and, in a dimeric partner molecule, an Src homology 2 (SH2) phosphotyrosine-binding domain, a DNA interaction domain, and a number of protein-protein interaction domains (with receptors, other transcription factors, the transcription machinery, and perhaps a tyrosine phosphatase). Mouse genetics experiments have defined crucial roles for each known mammalian STAT. The discovery of a STAT in Drosophila, and most recently in Dictyostelium discoideum, implies an ancient evolutionary origin for this dual-function set of proteins.

Implications of RNA-RNA splicing in evolution of eukaryotic cells.

The differences in the biochemistry of messenger RNA formation in eukaryotes compared to prokaryotes are so profound as to suggest that sequential prokaryotic to eukaryotic cell evolution seems unlikely. The recently discovered noncontiguous sequences in eukaryotic DNA that encode messenger RNA may reflect an ancient, rather than a new, distribution of information in DNA and that eukaryotes evolved independently of prokaryotes.

Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6.

The STAT family of proteins carries out a dual function: signal transduction and activation of transcription. A new family member, Stat3, becomes activated through phosphorylation on tyrosine as a DNA binding protein in response to epidermal growth factor (EGF) and interleukin-6 (IL-6) but not interferon gamma (IFN-gamma). It is likely that this phosphoprotein forms homodimers as well as heterodimers with the first described member of the STAT family, Stat91 (renamed Stat1 alpha), which is activated by the IFNs and EGF. Differential activation of different STAT proteins in response to different ligands should help to explain specificity in nuclear signaling from the cell surface.

Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein.

Interferon-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in interferon-stimulated gene factor-3 (ISGF-3), a transcription complex activated by IFN-alpha. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 113-kilodalton ISGF-3 protein that is phosphorylated in response to IFN-alpha was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.

Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor.

The interferon-alpha (IFN-alpha)-stimulated gene factor 3 (ISGF3), a transcriptional activator, contains three proteins, termed ISGF3 alpha proteins, that reside in the cell cytoplasm until they are activated in response to IFN-alpha. Treatment of cells with IFN-alpha caused these three proteins to be phosphorylated on tyrosine and to translocate to the cell nucleus where they stimulate transcription through binding to IFN-alpha-stimulated response elements in DNA. IFN-gamma, which activates transcription through a different receptor and different DNA binding sites, also caused tyrosine phosphorylation of one of these proteins. The ISGF3 alpha proteins may be substrates for one or more kinases activated by ligand binding to the cell surface and may link occupation of a specific polypeptide receptor with activation of transcription of a set of specific genes.

Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.

Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.

Structure of the amino-terminal protein interaction domain of STAT-4.

STATs (signal transducers and activators of transcription) are a family of transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The crystal structure of an NH2-terminal conserved domain (N-domain) comprising the first 123 residues of STAT-4 was determined at 1.45 angstroms. The domain consists of eight helices that are assembled into a hook-like structure. The N-domain has been implicated in several protein-protein interactions affecting transcription, and it enables dimerized STAT molecules to polymerize and to bind DNA cooperatively. The structure shows that N-domains can interact through an extensive interface formed by polar interactions across one face of the hook. Mutagenesis of an invariant tryptophan residue at the heart of this interface abolished cooperative DNA binding by the full-length protein in vitro and reduced the transcriptional response after cytokine stimulation in vivo.

Polyadenylic acid sequences: role in conversion of nuclear RNA into messenger RNA.

Polyadenylic acid [poly(A)] segments containing 150 to 250 nucleotides appear to be covalently linked to heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA) in eucaryotic cells. The poly(A) is synthesized in the nucleus, and is probably linked initially to HnRNA that is ultimately transported as mRNA to the cytoplasm. Studies with inhibitors of RNA or poly(A) synthesis indicate that synthesis of poly(A) segments is independent of transcription. The poly(A) marker may prove useful to elucidate mRNA modification and transport in eucaryotic cells.s