Transactivation and growth suppression by the gut-enriched Krüppel-like factor (Krüppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction.

Gut-enriched Krüppel-like factor (GKLF or KLF4) is a pleiotropic (activating and repressive) transcription factor. This study characterizes the mechanisms of transactivation by GKLF. Using a GAL4 fusion assay, the activating domain of murine GKLF was localized to the 109 amino acid residues in the N-terminus. Site-directed mutagenesis showed that two adjacent clusters of acidic residues within this region are responsible for the activating effect. Transactivation by GKLF involves intermolecular interactions as demonstrated by the ability of wild-type, but not mutated, GKLF to compete with the N-terminal activation domain. In addition, wild-type adenovirus E1A, but not a mutated E1A that failed to bind p300/CBP, inhibited transactivation by the N-terminal 109 amino acids of GKLF, suggesting that p300/CBP are GKLF's interacting partners. A physical interaction between GKLF and CBP was demonstrated by glutathione- S -transferase pull-down and by in vivo co-immuno-precipitation experiments. We also showed that the two acidic amino acid clusters are essential for this interaction, since GKLF with mutations in these residues failed to co-immunoprecipitate with CBP. Importantly, the same mutations abrogated the ability of GKLF to suppress cell growth as determined by a colony suppression assay. These studies therefore provide plausible evidence for a structural and functional correlation between the transactivating and growth-suppressing effects of GKLF.

Receptor for epidermal growth factor retains normal structure and function in aging cells.

Cellular responsiveness to epidermal growth factor (EGF) and the structure of the receptor for epidermal growth factor (EGF-R) were compared in young and senescent human fibroblast (HF) cells. Biosynthetic labeling of HF cells with [35S] methionine followed by immunoprecipitation with EGF-R antibody revealed the presence of Mr 170 000 EGF-R in cells from both stages. Autophosphorylation of EGF-R in response to EGF was identical in young and senescent cells. Phosphoamino acid analysis of the autophosphorylated EGF-R indicated that tyrosine residues were phosphorylated in each preparation. Two-dimensional peptide mapping of [125I]EGF-R from young and senescent cells showed essentially the same pattern, indicating that EGF-R does not apparently undergo detectable changes in senescent human fibroblasts. The responsiveness of aging HF cells to EGF for the induction of ornithine decarboxylase activity and for the production of secretory proteins was measured. Young and senescent HF cells showed about a three-fold induction of collagenase activity upon addition of EGF. Ornithine decarboxylase activity was also stimulated by EGF to a comparable level in young and senescent cells. Our results indicate that the responsiveness of HF cells to EGF for these two biochemical parameters does not decline with the loss of proliferative activity.

Tissue prostanoids as biomarkers for chemoprevention of colorectal neoplasia: correlation between prostanoid synthesis and clinical response in familial adenomatous polyposis.

Recent studies indicate that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), lowers mucosal prostanoid levels and regresses colorectal adenomas in patients with familial adenomatous polyposis (FAP). To determine whether they are biomarkers for sulindac-mediated chemoprevention of colorectal adenomas, levels of 5 prostanoids [prostaglandin (PG) D2, PGE2, PGF2alpha, thromboxane B2, and 6-keto-PGF1alpha] in the normal-appearing rectal mucosa from 7 FAP patients with a history of subtotal colectomy and ileorectal anastomosis and 4 FAP patients without surgery, were measured in the absence or presence of exogenously added arachidonic acid before the initiation and at the end of 3 months of sulindac treatment. The addition of arachidonic acid resulted in a uniform increase in the levels of all 5 prostanoids although this increase was selectively attenuated in patients with ileorectal anastomosis who took sulindac. In the latter patients, arachidonic acid also augmented the inhibition of prostanoid synthesis by sulindac. In contrast, sulindac failed to attenuate the increase in prostanoid levels resulting from arachidonic acid in patients without previous surgery. Importantly, when measured in the presence of arachidonic acid, the reduction in the levels of all 5 prostanoids due to sulindac was statistically correlated with a reduction in the size and number of adenomas in the two groups of patients combined. These results suggest that tissue prostanoids measured in the presence of arachidonic acid may serve as sensitive and reliable biomarkers in monitoring the clinical responsiveness of FAP patients undergoing chemoprevention for colorectal neoplasia with NSAIDs.

Detection of an Mr 200,000 glycoprotein in the culture medium of skin fibroblasts from patients with Huntington disease.

Huntington disease is a progressive neurological disorder with an autosomal dominant mode of inheritance. Using high resolution two-dimensional gel electrophoresis, we analyzed the intracellular and the released protein patterns of skin fibroblasts from HD patients and compared them to cells from apparently normal individuals matched for age and sex. No consistent differences were found in the pattern of total cellular proteins. In contrast, the culture medium from HD patients (12 of 19) contained an Mr 200,000 glycoprotein not found in twelve control cultures. The relation of this protein to the HD gene is unknown.

Induction of collagenase secretion in human fibroblast cultures by growth promoting factors.

In human foreskin fibroblast cultures, two proteins with Mr 60,000 and 55,000 were found to be induced about 3.5-fold by epidermal growth factor (EGF), platelet-derived growth factor, and beta-transforming growth factor. The induced proteins were identified as procollagenases by immunoprecipitation of induced medium with antibodies to purified human fibroblast collagenase. Collagenase enzyme activity in the medium from EGF-treated cultures was also induced at least 3-fold compared to control cultures. Induction of collagenase was dependent upon de novo protein and RNA synthesis and was observed in the medium 10 h after addition of EGF. Although these growth-promoting factors interact with separate membrane receptors, each induced the secretion of a common protein, suggesting that collagenase may be important in some aspect of mitogenesis, cell mobilization, and migration.

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus.

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nucleotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.

Induction and suppression of type I collagenase in cultured human cells.

A number of peptide growth factors have been shown to induce the secretion of type I collagenase into the medium of human fibroblast cultures (Chua et al., 1985). In this study the ability of eye-derived growth factor, lectin and tumor-promoting agents on collagenase induction in human fibroblast cells were examined. These agents were found to be able to induce collagenase production to a similar extent as epidermal growth factor. Dexamethasone at 10-100 nM was found to suppress collagenase induction in human fibroblast cells. The cell-type specificity of this enzyme induction by growth factors was studied by using a human epidermoid carcinoma cell line, A-431. An Mr 55,000 band appeared in the medium of A-431 cells upon 22 h exposure to EGF. Two-dimensional peptide pattern of the Mr 55,000 band in A-431 cells was identical to the counterpart in HF cells. Our results indicated that the induction of collagenase was not unique to human fibroblast cultures.

Characterization of the structure and regulation of the murine gene encoding gut-enriched Krüppel-like factor (Krüppel-like factor 4).

Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor whose expression is associated with growth arrest. In order to understand the mechanisms regulating expression of the gene encoding GKLF, we isolated a genomic clone containing murine GKLF. The gene spans 5.3 kb and contains four exons. A major start site of transcription was mapped to an adenine residue 601 nt 5' of the translation initiation codon. An additional 1 kb of the 5'-flanking region was sequenced and found to contain multiple cis -elements homologous to the binding sites of several established transcription factors including Sp1, AP-1, Cdx, GATA, and USF. In particular, three closely spaced GC-boxes 5' of the TATA box resemble the established binding site for GKLF. DNase I protection and electrophoretic mobility shift assays verified that recombinant GKLF bound to each of the three GC-boxes. In co-transfection experiments, GKLF transactivated a reporter gene linked to the GKLF 1 kb 5'-flanking region, as did Sp1, Sp3 and Cdx-2. Mutations of one or both of the first and second GC-boxes in the promoter resulted in diminished transactivation by GKLF. These results demonstrate that the 5'-flanking sequence of the mouse GKLF gene functions as a promoter and is subject to autoregulation by its own gene product.

Krüppel-like factor 4 (gut-enriched Krüppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle.

Krüppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G(1)/S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G(1)/S boundary of the cell cycle.

The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter.

An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene. We show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21(WAF1/Cip1) due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/Cip1) through a specific Sp1-like cis-element in the p21(WAF1/Cip1) proximal promoter. The same element is also required by p53 to activate the p21(WAF1/Cip1) promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21(WAF1/Cip1) promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21(WAF1/Cip1) promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21(WAF1/Cip1) is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21(WAF1/Cip1) in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21(WAF1/Cip1) and may be part of a novel pathway by which cellular responses to stress are modulated.

Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.

Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.