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.

UV-Induced stabilization of c-fos and other short-lived mRNAs.

Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

Activation of the c-fos gene by UV and phorbol ester: different signal transduction pathways converge to the same enhancer element.

In NIH3T3 cells stably transfected with the human c-fos gene, serum, platelet derived growth factor (PDGF), phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA), ultraviolet irradiation (UV) and 3'-5'-cyclic adenosine monophosphate (cAMP) cause a transient and rapid activation of both the endogenous and the transfected c-fos genes. While serum, TPA, UV and PDGF dependent activation of the gene is severely impaired, when the serum responsive element from position -319 to -300 (SRE, Treisman, 1985) is destroyed, a full response to cAMP is retained. Insertion of a synthetic oligonucleotide corresponding to the SRE element upstream of position -96 restores the responses to TPA and serum, and large parts of the responses to UV and PDGF. The signal transduction chains elicited by UV and TPA are blocked by an inhibitor of protein kinase. Only TPA, however, causes the translocation of protein kinase C to the membrane. UV and TPA treated cells become refractory to a second stimulation by the same agent at 3 or 24 hours after the first treatment. Alternating the agents, however, leads to full responses. In addition, saturating doses of UV and TPA are at least additive. Ca-ionophores severely reduce only UV induced c-fos expression. These data indicate, that different signal transduction pathways elicited by growth promoting agents and by UV induced stress converge onto the same enhancer element.

The IFN-gamma response of the murine invariant chain gene is mediated by a complex enhancer that includes several MHC class II consensus elements.

IFN-gamma induces the expression of the MHC class II-associated invariant chain (IN) protein in a variety of cells of nonlymphoid origin. Here we analyze the transcription from the murine invariant chain gene and delimit the cis-acting sequences which confer IFN-gamma responsiveness in human fibroblasts. The major start of transcription of the gene is located 29 bp 3' of a TATA box and 85 bp 5' of the single ATG codon which opens the reading frame. To identify the regulatory elements of the murine IN promoter which respond to IFN-gamma, the 5' flanking region of the gene including its capsite and 85 bp of coding region have been cloned in front of the bacterial chloramphenicol acetyl transferase (CAT) gene. Examination of this construct and various 5' and 3' deletion mutants for IFN-gamma inducibility in transient transfection assays revealed that DNA sequences between -261 and -189 were essential and sufficient for the induction. Removal of sequences between -215 and -189 reduced inducibility of the IN-promoter and abolished the capacity of the element to transmit inducibility to a heterologous promoter. Single or multiple base changes in other parts of the element also abolished inducibility. Cotransfection of a 350 molar excess of the IFN-gamma response element with an inducible IN-CAT chimeric construct blocked inducibility, suggesting positive regulation. A protein binding to the central part of the IFN-gamma response element was detectable in gel retardation experiments; it was active only in extracts from IFN-gamma-treated cells.

Ultraviolet irradiation, although it activates the transcription factor AP-1 in F9 teratocarcinoma stem cells, does not induce the full complement of differentiation-associated genes.

Induction of differentiation of F9 teratocarcinoma stem cells by retinoic acid and cAMP has been shown to involve the activation of the transcription factor AP-1 (a heterodimer of the proto-oncogene products c-Fos and c-Jun); moreover, stable expression of either Fos or Jun drives F9 cells into differentiation. Phorbol ester tumor promoters and short-wave-length ultraviolet (uv) irradiation are efficient inducers of AP-1 activity in various differentiated cells, but it has been shown that phorbol esters do not induce AP-1 activity in undifferentiated F9 cells. We examine here whether uv irradiation induces AP-1 activity in these cells and drives F9 cells into differentiation. We show that uv induces, in contrast to phorbol esters, the formation of active AP-1 by activating transcription from the c-jun gene. Ultraviolet-induced AP-1 drives transcription from AP-1-dependent promoters coding for differentiation-associated proteins (such as urokinase and keratin 18). However, in uv-treated cells, these genes are activated earlier and to a greater extent than in cells treated with retinoic acid and cAMP. More importantly, uv, in contrast to retinoic acid and cAMP, does not induce the accumulation of collagen alpha 1 (IV) and laminin B1 RNA. Our data suggest that the c-jun gene in F9 cells is accessible to immediate activation, but that uv-induced AP-1 activation does not suffice to induce the full program of F9 cell differentiation.

Induction of differentiation in F9 cells and activation of peroxisome proliferator-activated receptor delta by valproic acid and its teratogenic derivatives.

The antiepileptic drug valproic acid (VPA) is teratogenic, because it induces birth defects in some children of mothers treated for epilepsy. Cellular and molecular actions associated with teratogenicity were identified by testing differentiation of F9 embryocarcinoma cells. VPA altered cell morphology and delayed proliferation. Specific differentiation markers (e.g., c-fos and keratin 18 mRNA and particularly the activating protein-2 transcription factor protein) were induced. This pattern differs from the pattern induced by other teratogens or F9 cell-differentiating agents. Induction of differentiation correlated with teratogenicity because teratogenic derivatives of VPA, such as (S)-4-yn-VPA, induced differentiation, whereas closely related nonteratogenic compounds, such as (R)-4-yn-VPA, 2-en-VPA, and 4-methyl-VPA, did not. In the cellular signaling network, the peroxisome proliferator-activated receptor delta (PPARdelta) was activated selectively by VPA and teratogenic derivatives. Depletion of PPARdelta by antisense RNA expression precluded the response of F9 cells to VPA. In conclusion, our data show that VPA and its teratogenic derivatives induce a specific type of F9 cell differentiation and that PPARdelta is a limiting factor in the control of differentiation.

Posttranscriptional regulation of c-fos mRNA expression.

The transient induction of c-fos mRNA and protein suggests that regulation occurs not only by transcriptional activation but also at the level of turnover of the gene product. Here we present evidence for the rapid turnover of c-fos mRNA and some of the requirements for its specific degradation. The half life of induced mature cytoplasmic c-fos mRNA is 9 min in both serum-starved and growing primary human fibroblasts and in NIH 3T3 cells. A structure present at the 3' end of the c-fos mRNA molecule is involved in its low stability since the substitution or the removal of the untranslated 3' portion prolongues the RNA life time. The rapid turnover of fos mRNA requires, in addition, continued protein synthesis. Treatment of cells with cycloheximide stabilizes c-fos mRNA. Washing out cycloheximide reestablishes the rapid turnover. Both changes occur with lag periods of less than 17 minutes.

Interferon-gamma, mitomycin C, and cycloheximide as regulatory agents of MHC class II-associated invariant chain expression.

The murine and human major histocompatibility complex class II-associated invariant chain genes are expressed in mature B cells and in antigen-presenting cells. Several pre-B cell lines and fibroblasts do not naturally contain invariant chain mRNA. Expression is inducible, however, by interferons and other agents interfering with proliferation. Mitomycin C induces the transcription of the gene in pre-B cells, but not in fibroblasts. Interferon-gamma acts in both types of cells. Cycloheximide inhibits the induction of the invariant chain mRNA by interferon-gamma, suggesting that protein synthesis is required. In fact, cycloheximide itself increases the transcriptional rate at the invariant chain gene, suggesting the existence of a labile repressor or an indirect action through cycloheximide arrest of the cell cycle. Lipopolysaccharide (LPS) activation of B lymphocytes causes a rapid decrease of the invariant chain mRNA level and of the amount of invariant chain protein due to rapid turnover. Also class II alpha and beta mRNA expression decreases after LPS treatment. The decrease of invariant chain protein is accompanied by increased surface expression of alpha and beta. The murine invariant chain gene transfected into human fibroblasts is regulated by the same agents and the same dose of agents as is the endogenous gene. The differentiation marker invariant chain thus seems to be transcribed from a gene that is accessible to regulation even in nonlymphoid cells and the expression of which is linked to states of nonproliferation. The sequence responsible for these responses is contained within the cloned genomic fragment and is conserved between mouse and man.