Wolfgang Doppler

Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway.

Nitric oxide (NO) produced from L‐arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post‐transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non‐macrophage cell lines activates RNA binding by iron regulatory factor (IRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO‐induced binding of IRF to iron‐responsive elements (IRE) specifically represses the translation of transfected IRE‐containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-λ, ϵ and ζ

The implication of protein kinase C (PKC) isoforms cPKC‐α, nPKC‐ϵ, aPKC‐λ and aPKC‐ζ in the transcriptional activation of a c‐fos promoter‐driven CAT‐reporter construct by transforming Ha‐Ras has been investigated. This was achieved by employing antisense constructs encoding RNA directed against isoform‐specific 5′ sequences of the corresponding mRNA, and expression of PKC mutants representing either kinase‐defective, dominant negative, or constitutively active forms of the PKC isoforms. The data indicate that in HC11 mouse mammary epithelial cells, transforming Ha‐Ras requires the activities of the three PKC isozymes: aPKC‐λ, nPKC‐ϵ and aPKC‐ζ, not, however, of cPKC‐α, for the transcriptional activation of c‐fos. Co‐expression of oncogenic Ha‐Ras with combinations of kinase‐defective, dominant negative and constitutively active mutants of the various PKC isozymes are in agreement with a tentative model suggesting that, in the signaling pathway from Ha‐Ras to the c‐fos promoter, aPKC‐λ acts upstream whereas aPKC‐ζ functions downstream of nPKC‐ϵ.

MECHANISM OF INTERACTION BETWEEN THE GLUCOCORTICOID RECEPTOR AND STAT5 : ROLE OF DNA-BINDING

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of beta-cascin gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were nonclassical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

The Elf Group of Ets-Related Transcription Factors

Expression of mammary-specific genes is under the control of both hormone dependent and independent mechanisms, neither of which may be totally mammary-specific. It seems that a complex interplay of transcription factors may be responsible for mammary expression. Our challenge is to determine whether ELF3 anhion of mammary-specific genes in vivo, and whether they cooperate with other stimuli, such as prolactin. Because the mammary gland is not a static tissue, but can continually undergo cycles of growth, differentiation and function, it will be interesting to examine whether ELF3 and ELF5 operate cooperatively or differentially during these different phases.

Synergistic and Antagonistic Interactions of Transcription Factors in the Regulation of Milk Protein Gene Expression

The stage and tissue specific expression of milk protein genes in the mammary gland is controlled by modular response regions with multiple binding sites for distinct classes of transcription factors, which either co-operate or are antagonistic. In addition, the activity of some of these factors is individually control-led by diverse extracellular signals. A well studied paradigm for a synergistic co-operation is the activation of beta-casein gene transcription by prolactin and glucocorticoids mediated by the signal transducer and activator of transcription STAT5 and the glucocorticoid receptor (GR). As an example for an antagonistic interaction we can demonstrate inhibition of prolactin signalling by TNF-alpha, which is mediated by NF-kappa B. In both cases, the interactions occur at several levels: For GR and STAT5, the synergy is discussed to be promoted by protein-protein interactions. Furthermore, we can demonstrate a co-operation between GR and STAT5 in DNA binding by a mechanism, which is dependent on the integrity of the DNA binding domain of the GR and on the existence of half-palindromic GR binding sites in the hormone response region. Indirect effects of glucocorticoids by modulation of the expression of secondary genes are also important. They might account for the observed enhancement of prolactin induced tyrosine phosphorylation of STAT5 by glucocorticoids. For NF-kappa B and STAT5, one component of the antagonism is the inhibition of STAT5 tyrosine phosphorylation by activation of NF-kappa B. Another potential mechanism is the inhibition of DNA binding of STAT5 due to overlapping binding sites for STAT5 and NF-kappa B in the beta-casein gene promoter. Thus, synergistic and antagonistic interactions between GR, NF-kappa B, and STAT5 involve (a) cross-talk mechanisms influencing the activation of STAT5 and (b) promoter-dependent interactions modulating the DNA binding activity of the transcription factors.

Plasma membrane as target of alkylating agents

N mustard resistant Walker cells exhibit the same frequency of DNA interstrand cross-links and the same rate of cross-link removal as the sensitive parental line. Employing cytostatically active concentrations of chlorambucil covalently bound to polyethyleneimine, the extent of DNA cross-linking is reduced to levels observed in the presence of nontoxic concentrations of free chlorambucil. It is concluded, therefore, that DNA cross-links alone are not sufficient to explain the inhibition of cell multiplication by alkylating agents and that additional mechanisms have to be considered. Evidence for an interference of alkylating agents with several enzymes of the plasma membrane is presented. An inhibition by N mustard of the furosemide-sensitive Na+/K+/Cl- -cotransport and the Na+/H+-antiport is described in greater detail. Considering the fact that the enzymes which are affected by alkylating agents are controlled by growth factors it was investigated whether a synergism between inhibitors of early growth-factor-controlled reactions and alkylating agents is to be seen. It is demonstrated that mepacrine, an inhibitor of phospholipase C, and the calmodulin binding drugs, chlorpromazine and flunarizine, amplify the action of N mustard.

Effect of Ha-ras on phosphatidylinositol metabolism, Na+/H+-antiporter and mobilization of intracellular calcium

NIH3T3 cells were transfected with activated Ha-ras and the corresponding proto-oncogene was subjected to transcriptional control by recombination in vitro with MMTV-LTR. Induction of p21ras expression in quiescent cells by dexamethasone causes an increased turnover of phosphatidylinositol 4,5-bisphosphate with a concomitant rise in inositol phosphates, and an activation of the Na+/H+-antiporter. Addition of serum growth factors to dexamethasone treated cells does not result in an additional stimulation of phosphatidylinositol metabolism or Na+/H+-exchange. There is also a desensitization to exogenous growth factors of the intracellular Ca2+-mobilizing system, leading to a depression of the transitory increase in cytosolic Ca2+ after addition of serum growth factors. None of these effects are seen after expression of the Ha-ras proto-oncogene. Results are discussed as indicating a constitutive growth factor independent activation of growth factor signal transduction by the activated Ha-ras.

Nitrogen mustard interference with potassium transport systems in Ehrlich ascites tumor cells

SummaryNitrogen mustard (N-mustard) inhibits the ouabain-sensitive and the furosemide-sensitive Rb uptake of Ehrlich ascites tumor cells, whereas the transport, which is resistant to both inhibitors, is not affected by the alkylating agent. At N-mustard concentrations below 10 μM, the reduction in Rb uptake is predominantly due to an interference with the furosemide-sensitive system. The dose response curve for the inhibition by N-mustard of the furosemide-sensitive Rb uptake closely parallels the dose response curve for the anti-tumor activity of the alkylating drug. This is in contrast to the behaviour of the ouabain-sensitive Rb transport.The inhibition of the furosemide-sensitive Rb uptake is expressed much less in cells which are resistant to N-mustard. The recovery of the furosemide-sensitive transport system after a single exposure to N-mustard is relatively slow and characterized by an initial 4 h lag period, whereas the repair of DNA-interstrand cross-links starts immediately after removal of the drug. At mM concentrations furosemide blocks the multiplication of Ehrlich ascites tumor cells. However, lower concentrations of furosemide which cause a 50% reduction in the furosemide-sensitive Rb uptake do not interfere with cell proliferation. This is in contrast to the behaviour of N-mustard which exerts a clear-cut depression of cell growth at concentrations leading to a 50% inhibition of the furosemide-sensitive Rb transport. It is concluded, therefore, that the inhibition of the furosemide-sensitive system alone is not sufficient to explain the anti-tumor activity of the alkylating agent.The effect is discussed as part of a more extended N-mustard-induced membrane alteration which may be important for the growth inhibitory effect of the alkylating agent.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Stimulation of IRE-BP Activity of IRF by Tetrahydrobiopterin and Cytokine Dependent Induction of Nitric Oxide Synthase

Hypoferric anemia is very frequent in patients suffering from chronic inflammatory disorders or malignancies. This type of anemia is characterized by decreased amounts of serum iron and hemoglobin but increased body iron stores reflected by an increase of ferritin concentrations as well as by enhanced amounts of cellular immune activation markers like interferon-gamma and pteridines (Weinberg, 1984; Fuchs et al., 1991 and 1993; Means and Krantz, 1992). It was shown recently that increased intracellular concentrations of low molecular weight iron inhibit the efficiency of the interferon-gamma signal in human monocytic cells as checked by decreased cytokine induced MHC class II antigen expression, tryptophan degaradtion or pteridine formation in the presence of iron (Weiss et al., 1992 and 1993a). Pteridines are pyrazino-pyrimidino compounds which are synthezised in excess by human monocytes/macrophages upon stimualtion with cytokines such as interferon-gamma (Huber et al., 1984). Starting from GTP the first intermediate of the biosynthetic pathway, i.e. 7,8-dihydroneopterintriphosphate, is either converted by two subsequent enzymes to 5,6,7,8-tetrahydrobiopterin known to be an essential cofactor for hydroxylation reacrtions or nitric oxide synthase (Kaufmann, 1963; Tayeh and Marietta 1989; Kwon et al., 1989), or cleaved by omnipresent phosphatases to form 7,8-dihydroneopterin (Nichol et al., 1986; Werner et al., 1990).