Hans J. Rahmsdorf

Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor

The promoter regions of several phorbol diester-(TPA-) inducible genes (collagenase, stromelysin, hMT IIA, and SV40) share a conserved 9 bp motif. Synthetic copies of these closely related sequences conferred TPA inducibility upon heterologous promoters. Footprinting analysis indicated that these TPA-responsive elements (TREs) are recognized by a common cellular protein: the previously described transcription factor AP-1. A point mutation that eliminated the basal and induced activity of the TRE also interfered with its ability to bind AP-1. Treatment of cultured cells with TPA led to a rapid 3- to 4-fold increase in TRE binding activity, by a posttranslational mechanism. These results strongly suggest that AP-1 is at the receiving end of a complex pathway responsible for transmitting the effects of phorbol ester tumor promoters from the plasma membrane to the transcriptional machinery.

Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone

Glucocorticoid hormones counteract inflammation and phorbol ester tumor promotion and drastically decrease the expression of several extracellular proteases, including collagenase I. Glucocorticoid hormone inhibits basal and induced transcription of collagenase by interfering with AP-1, the major enhancer factor of the collagenase promoter. The mechanism of interference is novel in that it does not require protein synthesis, it depends on the hormone receptor but not its binding to DNA, it occurs at hormone doses one order of magnitude below those required for gene activation, and it involves down-modulation of the trans-activating function of preexisting unbound and DNA-bound AP-1. Coprecipitation experiments suggest direct AP-1-hormone receptor interaction, which also possibly explains the reverse experiment: overexpression of Fos or Jun inhibits the expression of hormone-dependent genes.

A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1.

Steroid receptors activate and repress genes. An important class of genes that they repress is controlled by the transcription factor AP‐1. The activity of AP‐1 is inhibited by the receptor, a mechanism exploited for the therapy of various forms of pathological hyperproliferation in humans. We show here by point mutations in the DNA binding domain and by the choice of steroid ligands that repression of AP‐1 activity and transactivation functions of the glucocorticoid receptor (GR) are separable entities. While DNA binding and activation of glucocorticoid‐regulated promoters require GR dimerization, we present data that suggest that repression is a function of GR monomers.

Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents.

Several non‐physiologic agents such as radiation, oxidants and alkylating agents induce ligand‐independent activation of numerous receptor tyrosine kinases (RTKs) and of protein tyrosine kinases at the inner side of the plasma membrane (e.g. Dévary et al., 1992; Sachsenmaier et al., 1994; Schieven et al., 1994; Coffer et al., 1995). Here we show additional evidence for the activation of epidermal growth factor receptor (EGFR), and we show activation of v‐ErbB, ErbB2 and platelet‐derived growth factor receptor. As a common principle of action the inducing agents such as UVC, UVB, UVA, hydrogen peroxide and iodoacetamide inhibit receptor tyrosine dephosphorylation in a thiol‐sensitive and, with the exception of the SH‐alkylating agent, reversible manner. EGFR dephosphorylation can also be modulated by these non‐physiologic agents in isolated plasma membranes in the presence of Triton X‐100. Further, substrate (EGFR) and phosphatase have been separated: a membrane preparation of cells that have been treated with epidermal growth factor (EGF) and whose dephosphorylating enzymes have been permanently destroyed by iodoacetamide can be mixed with a membrane preparation from untreated cells which re‐establishes EGFR dephosphorylation. This dephosphorylation can be modulated in vitro by UV and thiol agents. We conclude that RTKs exhibit significant spontaneous protein kinase activity; several adverse agents target (an) essential SH‐group(s) carried by (a) membrane‐bound protein tyrosine phosphatase(s).

UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein

UV irradiation of human and murine cells enhances the transcription of several genes. Here we report on the primary target of relevant UV absorption, on pathways leading to gene activation, and on the elements receiving the UV-induced signal in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, in the gene coding for collagenase, and in the cellular oncogene fos. In order to induce the expression of genes. UV radiation needs to be absorbed by DNA and to cause DNA damage of the kind that cannot be repaired by cells from patients with xeroderma pigmentosum group A. UV-induced activation of the three genes is mediated by the major enhancer elements (located between nucleotide positions -105 and -79 of HIV-1, between positions -72 and -65 of the collagenase gene, and between positions -320 and -299 of fos). These elements share no apparent sequence motif and bind different trans-acting proteins; a member of the NF kappa B family binds to the HIV-1 enhancer, the heterodimer of Jun and Fos (AP-1) binds to the collagenase enhancer, and the serum response factors p67 and p62 bind to fos. DNA-binding activities of the factors recognizing the HIV-1 and collagenase enhancers are augmented in extracts from UV-treated cells. The increase in activity is due to posttranslational modification. While AP-1 resides in the nucleus and must be modulated there, NF kappa B is activated in the cytoplasm, indicating the existence of a cytoplasmic signal transduction pathway triggered by UV-induced DNA damage. In addition to activation, new synthesis of AP-1 is induced by UV radiation.

Involvement of growth factor receptors in the mammalian UVC response

Irradiation of HeLa cells with short-wavelength ultraviolet light (UVC) induces the modification and activation of the preexisting transcription factors c-Fos-c-Jun (AP-1) and TCF/Elk-1, as well as the protein synthesis independent transcriptional activation of the c-fos and c-jun genes. This response to UVC is mediated via obligatory cytoplasmic signal transduction, involving Ras and Raf, Src, and MAP kinases. The UVC response is inhibited by prior down-modulation of growth factor receptor signaling upon growth factor prestimulation, by suramin (an inhibitor of receptor activation) or by expression of a dominant negative epidermal growth factor (EGF) receptor mutant. These data suggest the involvement of several growth factor receptors in the UVC response. Indeed, UVC induces the suramin-inhibitable immediate tyrosine phosphorylation of the EGF receptor.

Requirement for fos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters

Transcription from the c-fos promoter and from minimal promoter constructs carrying the phorbol ester-responsive element [12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element (TRE)] corresponding to the sequence in the human collagenase gene is activated by elevated levels of the oncogene products v-src, c-Ha-ras, activated c-Ha-ras, and v-mos, as well as by phorbol ester. Elevated c- or v-fos expression stimulates TRE-dependent transcription but represses the c-fos promoter. Antisense fos sequences abolish basal and induced transcription from TRE constructs and derepress the c-fos promoter. These results establish a key role for fos in signal transduction and implicate the fos protein as a trans-activating and -repressing molecule.

UV-INDUCED SIGNAL TRANSDUCTION

Irradiation of cells with wavelength ultraviolet (UVA, B and C) induces the transcription of many genes. The program overlaps with that induced by oxidants and alkylating agents and has both protective and other functions. Genes transcribed in response to UV irradiation include genes encoding transcription factors, proteases and viral proteins. While the transcription factor encoding genes is initiated in minutes after UV irradiation (immediate response genes) and depends exclusively on performed proteins, the transcription of protease encoding occurs only many hours after UV irradiation. Transcription factors controlling the activity of immediate response genes are activated by protein kinases belonging to the group of proline directed protein kinases immediately after UV irradiation. Experimental evidence suggests that these kinases are activated in UV irradiated cells through pathways which are used by growth factors. In fact, the first cellular reaction detectable in UV irradiated cells is the phosphorylation of several growth factor receptors at tyrosine residues. This phosphorylation does not depend on UV induced DNA damage, but is due to an inhibition of the activity of tyrosine phosphatases. In contrast, for late cellular reactions to UV, an obligatory role of DNA damage in transcribed regions of the genome can be demonstrated. Thus, UV is absorbed by several target molecules relevant for cellular signaling, and it appears that numerous signal transduction pathways are stimulated. The combined action of these pathways establishes the genetic program that determines the fate of UV irradiated cells.

Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo.

Phorbol esters stimulate and glucocorticoid hormones down‐regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP‐1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP‐1 to its cognate DNA binding site in vivo. The DNA‐protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP‐1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos‐Jun heterodimers or of purified AP‐1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP‐1 binding site in vivo. The hormone receptor acts without itself binding to DNA.

Sequential DNA damage‐independent and ‐dependent activation of NF‐κB by UV

NF‐κB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF‐κB from IκBα and degradation of IκBα, targeting for degradation and signaling are different. Early IκBα degradation at 30 min to ∼6 h is not initiated by UV‐induced DNA damage. It does not require IκB kinase (IKK), as shown by introduction of a dominant‐negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IκBα, carrying serines at positions 32 and 36. Induced IκBα degradation requires, however, intact N‐ (positions 1–36) and C‐terminal (positions 277–287) sequences. IκB degradation and NF‐κB activation at late time points, 15–20 h after UV irradiation, is mediated through DNA damage‐induced cleavage of IL‐1α precursor, release of IL‐1α and autocrine/paracrine action of IL‐1α. Late‐induced IκBα requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the ‘alarmone’ IL‐1α may account for some of the systemic effects of sunlight exposure.