The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.

Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression.

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha‐ras and c‐myc, in transfected Rat1 fibroblasts. While c‐myc overexpression did not modify clusterin gene activity, the Ha‐ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down‐regulation was also observed in the presence of c‐myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c‐myc transformed cell lines, and a weaker induction in the Ha‐ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors. J Cell Physiol 177:593–605, 1998. © 1998 Wiley‐Liss, Inc.

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Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell-cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.

The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papillomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell type-specific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragments with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.

The complete cell biological analysis of human guanylin, a recently discovered regulatory peptide, is offered in this investigation: (i) the nucleotide sequence of the gene, (ii) the isolation and characterization of its circulating molecular form, and (iii) its localization in enterochromaffin cells of the gut. As determined by molecular cloning, DNA sequencing, and comparison with the known cDNA sequence, the approximately 2.6-kbp large gene consists of three exons interrupted by two introns. The putative promoter region contains a TTTAAAA sequence motif and several potential binding sites for transcription factors such as AP-1, AP-2, Sp 1, and glucocorticoid receptors. The isolated hormonal form of guanylin is a 94-amino acid peptide with a molecular mass of 10.3 kDa. Western blot analysis of RP-HPLC fractions from blood plasma confirms this molecular form. Thus, guanylin is synthesized by gut enterochromaffin cells as a prohormone of 115 amino acids and is processed to the molecular form of 94 amino acids circulating in the blood.

The human papillomavirus type 16 is associated with anogenital cancer. Transcription of the viral transforming genes E6 and E7 is under the control of an epithelial cell type-specific enhancer. In the enhancer core, we have identified a regulatory element that is recognized by a novel nuclear factor named MSPF (methylation-sensitive papillomavirus transcription factor). Mutating the MSPF binding site strongly affects the enhancer activity. The MSPF recognition sequence 5'-ATGCGNNNNCGCCT-3' contains two CpG dinucleotides, potential targets for 5-cytidine methylation. DNA recognition by MSPF is strictly methylation-sensitive, since introduction of 5-methylcytidine into either CpG abolishes complex formation. Moreover, CpG methylation of the MSPF binding site suppresses the activity of the enhancer and of the MSPF enhanson subfragment in vivo. In the cervical carcinoma cell line CaSki, which has integrated multiple transcriptionally inactive human papilloma virus 16 genomes, a few of the viral genomes are methylated at the MSPF binding site. These findings suggest that viral transcription can be suppressed by methylation of the regulatory region, an event that prevents binding of the cellular transcription factor MSPF.

The promoter P97 of human papillomavirus-16 (HPV-16) gives rise to transcripts that encode the principal transforming genes of the virus, E6 and E7. The activity of P97 is regulated by a cell-type-specific enhancer, as well as by glucocorticoids and progesterone. We show here, that in CaSki cells, which contain HPV-16 genomes, P97 is also inducible by phorbol esters. Functional analysis of restriction fragments and oligonucleotides of the viral enhancer localizes two phorbol ester response elements on two transcription factor binding sites termed fp4e and fp9e. Sequence comparison, footprint analysis and bandshift competition of the cloned motifs suggest that both fp4e and fp9e are bound by the transcription factor AP1. These AP1 binding sites in HPV-16 and other papillomaviruses may provide a link between cellular oncogenes like jun, fos and possibly ras, whose transcription stimulating activity may lead to an elevated expression of the viral transforming genes E6 and E7.

We have previously identified in the long control region of the genome of human papillomavirus type 16 (HPV-16) a DNA segment which functions as a cell-type-specific enhancer as well as mediating glucocorticoid response. It contains multiple transcription-factor-binding sites, including several for nuclear factor I and one for the glucocorticoid receptor, which binds to the partially palindromic sequence TGTACANNNTGTCAT. We report here that this sequence element, when separated from the surrounding transcription-factor-binding sites and placed as an oligonucleotide into a test vector, retains its function as a glucocorticoid response element (GRE) in HeLa cells. In T47D cells, which express the progesterone receptor, the HPV-16 enhancer fragment mediates progesterone responsiveness. A point mutant in this fragment and the response of the oligonucleotide clone to both steroids prove the identity of the progesterone response element (PRE) with the GRE. The antiprogesterone and antiglucocorticoid RU486 interferes with both hormonal responses. In SiHa cells, the HPV-16 GRE mediates an increase in transcripts encoding E6 and E7 proteins, which are involved in transformation by HPV-16. Hormonal regulation is not restricted to HPV-16: DNA segments containing the cell-type-specific enhancers of HPV-11 and HPV-18 also mediate glucocorticoid and progesterone response. We identified sequence elements in the long control regions of HPV-11 and HPV-18 which function as GRE/PREs when tested as oligonucleotides. These findings suggest that GRE/PREs are an integral part of gene expression regulation in genital HPVs.

ALTHOUGH glucocorticoids and progestins control vastly different physiological processes, the receptors mediating the effects of these hormones interact with the same DNA sequences1–4. Transfer experiments involving synthetic genes5 and in vitro binding studies6 have shown that progesterone and glucocorticoid receptors both recognize the same 15-base pair DNA element (TGTACAGGATGTTCT), raising the question of how the two steroids affect gene expression selectively. We considered the possibility that their selectivity arises from either the differential expression of the receptors in target cells or the differential dependence of receptor function on additional transcription factors. To test these alternatives we introduced a progesterone-receptor expression pi asm id into the rat hepatoma cell line Fto2B-3 which contains glucocorticoid receptor but is devoid of progesterone receptor. We report that expression of the progesterone receptor in Fto2B-3 cells renders endogenous glucocorticoid-regulated genes inducible by progestins. Our data show that the responsive-ness of a cell to external stimuli can be reprogrammed by the expression of a single transcription factor and that differential expression of hormone receptors is at least one mechanism by which steroid-specific gene activation is achieved.

Control of gene expression by steroid hormones is exerted mainly by modulation of the transcriptional activity of the regulated genes. The response to the various steroid hormones is highly specific. It is mediated by binding of the hormone to its specific receptor which, after binding, attains the capacity to recognize a specific DNA region on regulated genes. This receptor-DNA interaction eventually leads in a still-unknown way to an increase of the rate of transcription of the target gene (Yamamoto, 1985).

The 7905 bp genomic DNA of the human papilloma virus type 16 (HPV-16), which is associated with invasive cervical neoplasia, contains a 832 bp upstream regulatory region (URR) supposed to contain most of its cis-acting promoter elements. A search for DNA sequences known to bind transcription factors has resulted in the recognition of numerous consensus sequences. A functional analysis of a segment of this URR containing some of these consensus sequences has resulted in the recognition of an enhancer element, that is functional only in human HeLa, but not in human MCF7 cells. The same region contains an element that confers regulatibility by glucocorticoid hormones, but not by sex steroids, to a linked promoter.

Steroids control gene expression by mainly modulating the rate of transcription. The effect of the hormone in target cells is mediated by specific receptors that bind the steroid with high affinity and selectivity. Binding of the hormone alters the properties of the receptor, allowing interaction of the hormone-receptor complex with specific recognition sequences in the vicinity of regulated genes. As a consequence of this interaction, the expression of a specific set of genes is increased or decreased, resulting in altered levels of mRNAs for specific proteins (Ringold 1985; Yamamoto 1985).

The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.

To define the recognition sequence of the glucocorticoid receptor and its relationship with that of the progesterone receptor, oligonucleotides derived from the glucocorticoid response element of the tyrosine aminotransferase gene were tested upstream of a heterologous promoter for their capacity to mediate effects of these two steroids. We show that a 15-base-pair sequence with partial symmetry is sufficient to confer glucocorticoid inducibility on the promoter of the herpes simplex virus thymidine kinase gene. The same 15-base-pair sequence mediates induction by progesterone. Point mutations in the recognition sequence affect inducibility by glucocorticoids and progesterone similarly. Together with the strong conservation of the sequence of the DNA-binding domain of the two receptors, these data suggest that both proteins recognize a sequence that is similar, if not the same.