Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-��B and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor���2

TNF-induced activation of the transcription factor NF-��B and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-��B-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-��B. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved ���WKI��� motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-��B activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-��B activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-��B and JNK, respectively.

Glucocorticoid-mediated repression of nuclear factor-��Bdependent transcription involves direct interference with���transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-��B. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory I��B molecule, which in turn would bind and remove activated, DNA-bound NF-��B complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-��B-dependent transcription, without changing the expression level of I��B. Neither the mRNA of I��B nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-��B also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-��B. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-��B p65 subunit.

Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Epidermal growth factor-induced nuclear factor ��B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (���) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER��� and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER��� and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER��� cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER��� human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF���EGFR interaction is mediated via activation of nuclear factor ��B (NF-��B). The basal level of active NF-��B in ER��� cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-��B activation factor. NF-��B transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER��� cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294���002, blocked this event, suggesting a role of the former in the activation of NF-��B by EGF. Go6976, a well-characterized NF-��B inhibitor, blocked EGF-induced NF-��B activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER��� cells. Thus Go6976 and similar NF-��B inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER��� breast cancer patients.

Induction of inhibitory factor ��B�� mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the���rat

In this study we investigate the mRNA expression of inhibitory factor ��B�� (I��B��) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). I��B controls the activity of nuclear factor ��B, which regulates the transcription of many immune signal molecules. The detection of I��B induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of I��B�� mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of I��B�� mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1���2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, I��B�� mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood���brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood���brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein���1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein���protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth���hormone

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-��-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2���/��� T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2���/��� T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3���-kinase in NIH 3T3���cells

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3���-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

Parasite-mediated nuclear factor ��B regulation in lymphoproliferation caused by Theileria parva���infection

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor ��B (NF��B) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NF��B activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the I��B molecules which normally sequester NF��B in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of I��B��. However, I��B�� reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NF��B-mediated positive feedback loop which restores cytoplasmic I��B��. In contrast, T. parva mediated continuous degradation of I��B�� resulting in persistently low cytoplasmic I��B�� levels. Normal I��B�� levels were only restored following T. parva killing, indicating that viable parasites are required for I��B�� degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both I��B degradation and consequent enhanced expression of NF��B dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either I��B levels or NF��B activation, indicating that the parasite subverts the normal I��B regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

Many of the protein���protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-��, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-�� was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain���containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from���Xenopus���oocytes

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl��� currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5���11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl��� current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-��B) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK���/��� mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1���/��� (recombinase activating gene 1) mice, indicating that RANK���/��� mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK���/��� mice without inducing hypercalcemia, although tumor necrosis factor �� treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK���/��� mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.