Breast cancer speci®c gene 1 (BCSG1), also referred as synuclein g, is the third member of a neuronal protein family synuclein. BCSG1 is not expressed in normal breast tissues but highly expressed in advanced in®ltrat-ing breast carcinomas. When over expressed, BCSG1 signi®cantly stimulates breast cancer metastasis. To elucidate the molecular mechanisms underlying the abnormal transcription of BCSG1 in breast cancer cells, in this study, we isolated a 2195 base pair fragment of human BCSG1 gene. This fragment includes 1 kb 5'-anking region, exon 1, and intron 1. By analysing the promoter activity and the methylation status of the exon 1 region, we show that (1) Intron 1 plays critical roles in the control of BCSG1 gene transcription through cisregulatory sequences that a ect BCSG1 transcription in cell type-speci®c and cell type-nonspeci®c manners. (2) The activator protein-1 (AP-1) is functionally involved in BCSG1 transcription in breast cancer cells through its binding to an AP-1 motif located in the intron 1. (3) The exon 1 region of BCSG1 gene contains a CpG island that is unmethylated in BCSG1-positive SKBR-3 and T47D cells but densely methylated in BCSG1-negative MCF-7 cells. (4) Treating MCF-7 cells with a demethylating agent 5-Aza-2'-deoxycytidine speci®cally activated BCSG1 transcription. Thus, our results suggest that while the cellular content of transcription activators and repressors that interact with the cis-regulatory sequences present in the intron 1 contribute prominently to the tissue-speci®c expression of BCSG1, demethylation of exon 1 is an important factor responsible for the aberrant expression of BCSG1 in breast carcinomas. Oncogene (2001) 20, 5173 ± 5185.
The cytokine oncostatin M (OM) activates human low density lipoprotein receptor (LDLR) gene transcription through a sterol-independent mechanism. Previous studies conducted in our laboratory have narrowed the OM-responsive element to promoter region ؊52 to ؉13, which contains the repeat 3 and two TATA-like sequences. We now identify LDLR promoter region ؊17 to ؊1 as a sterol-independent regulatory element (SIRE) that is critically involved in OM-, transcription factor CCAAT/enhancer-binding protein (C/EBP)-, and second messenger cAMP-mediated activation of LDLR transcription. The SIRE sequence overlaps the previously described TATA-like element and consists of an active C/EBP-binding site (؊17 to ؊9) and a functional cAMPresponsive element (CRE) (؊8 to ؊1). We demonstrate that (a) mutations within either the C/EBP or CRE site have no impact on basal or cholesterol-mediated repression of LDLR transcription, but they completely abolish OM-mediated activation of LDLR transcription; (b) replacing the repeat 3 sequence that contains the Sp1-binding site with a yeast transcription factor GAL4-binding site in the LDLR promoter construct does not affect OM inducibility, thereby demonstrating that OM induction is mediated through the SIRE sequence in conjunction with a strong activator bound to the repeat 3 sequence; (c) electrophoretic mobility shift and supershift assays confirm the specific binding of transcription factors C/EBP and cAMP-responsive element-binding protein to the SIRE; (d) cotransfection of a human C/EBP expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234 increases LDLR promoter activity; and (e) OM and dibutyryl cAMP synergistically activate LDLR transcription through this regulatory element. This study identifies, for the first time, a cis-acting regulatory element in the LDLR promoter that is responsible for sterol-independent regulation of LDLR transcription.Previous studies of the human low density lipoprotein receptor (LDLR) 1 promoter identified a 177-base pair fragment of the 5Ј-flanking DNA from Ϫ142 to ϩ35, relative to the major transcription start site, that is sufficient for controlling basal transcription as well as negative feedback regulation by cholesterol and its derivatives (1-3). The positive regulatory elements within this region were identified as three GC-rich imperfect 16-base pair direct repeats and two TA-rich TATA-like sequences of 7 base pairs each. Repeats 1 and 3 contain transcription factor Sp1-binding sites. Interference in Sp1 binding to either repeat severely decreases basal transcriptional activity. Sterols regulate LDLR transcription through a 10-base pair sequence within repeat 2 designated as sterol regulatory element-1 (SRE-1) (4, 5). When intracellular cholesterol is low, SRE-binding protein-1 and -2 bind to the SRE-1 sequence and interact with Sp1 in repeat 3, thereby activating LDLR transcription (6 -9). To date, no studies have been conducted to investigate further how the TATA-like sequences affect LDLR transcription, and the trans-acting ...
Oncostatin M (OM) activates the transcription of the human low density lipoprotein receptor (LDLR) inHepG2 cells through a sterol-independent mechanism. Our previous studies showed that mutations within the repeat 3 sequence of the LDLR promoter significantly decreased OM activity on LDLR promoter luciferase reporter constructs that contain the sterol responsive element-1 (repeat 2) and Sp1 binding sites (repeats 1 and 3). In this study, we investigated the signal transduction pathways that are involved in OM-induced LDLR transcription. In HepG2 cells, OM induced a rapid increase in LDLR mRNA expression, with increases detected at 30 min and maximal induction at 1 h. This OM effect was not blocked by protein synthesis inhibitors, inhibitors of p38 kinase, phosphatidylinositol 3-kinase, or c-Jun Nterminal kinase, but OM activity was completely abolished by pretreating cells with inhibitors of the extracellular signal-regulated kinase (ERK) kinase (mitogen/ ERK kinase (MEK)). To investigate whether the repeat 3 sequence of the LDLR promoter is the OM-responsive element that converts ERK activation at the promoter level, three luciferase reporters, pLDLR-TATA containing only the TATA-like elements of the promoter, pLDLR-R3 containing repeat 3 and the TATA-like elements, and pLDLR-234 containing repeats 1, 2, 3 and the TATA-like elements were constructed and transiently transfected into HepG2 cells. OM had no effect on the basal promoter construct pLDLR-TATA; however, including a single copy of repeat 3 sequence in the TATA vector (pLDLR-R3) resulted in a full OM response. The activity of OM on pLDLR-R3 was identical to that of pLDLR-234. Importantly, the ability of OM to increase luciferase activities in both pLDLR-R3-and pLDLR-234-transfected cells was blocked in a dose-dependent manner by inhibition of MEK. These results demonstrate that the mitogen-activated protein kinase MEK/ERK cascade is the essential signaling pathway by which OM activates LDLR gene transcription and provide the first evidence that the repeat 3 element is a new downstream target of ERK activation.
Previously, we identified the low density lipoprotein receptor (LDLR) promoter region ؊ 17 to ؊ 1 as a novel sterol-independent regulatory element (SIRE) that mediates the stimulating effect of oncostatin M (OM). The goal of this study was to identify the OM-induced transcription activator that binds to the SIRE sequence. By conducting a electrophoretic mobility shift assay (EMSA) followed by UV crosslinking and SDS-PAGE, we show that a protein with a molecular mass of 85 kDa was present in the OM-induced SIRE DNA-protein complex. Western blotting and supershift assays reveal that the 85 kDa factor is early growth response gene 1 ( Transcription of the low density lipoprotein receptor (LDLR) is largely controlled by a cholesterol-mediated feedback mechanism through interaction of a sterol regulatory element-1 (SRE-1) and SRE binding proteins (SREBPs) (1-9). In addition to this sterol-dependent pathway that is regulated by intracellular cholesterol levels, cumulative evidence from both in vivo studies and cell culture models suggests the existence of a sterol-independent regulatory pathway for LDLR transcription that is modulated by cytokines, growth factors, hormones, and secondary messengers (10-19). Some of these modulators appear to increase LDLR transcription under cholesterolrepressed conditions, and their activities do not require SRE-1. However, in contrast to the well-characterized mechanism of cholesterol regulation, the molecular and cellular mechanisms underlying sterol-independent regulation have not been clearly defined. This is at least in part due to the lack of understanding at the promoter level of the transcription factors and their interacting cis -acting elements that play critical roles in sterol-independent regulation.Recently, we identified a cis regulatory element in the human LDLR promoter that is responsible for cytokine oncostatin M (OM), CCAAT/enhancer binding protein (c/EBP), and cAMP-stimulated transcription of LDLR (20). This regulatory sequence designated as the sterolindependent regulatory element (SIRE) lies down stream of the SRE-1 and Sp1 sites. It is located in the LDLR promoter region Ϫ 17 to Ϫ 1 that overlaps the previously described TATA-like sequences ( Ϫ 23 to Ϫ 8). The SIRE sequence consists of a putative binding site for c/EBP ( Ϫ 17 to Ϫ 9) and a cAMP-responsive element (CRE; Ϫ 8 to Ϫ 1). Mutations within the SIRE sequence have no effect on cholesterol-mediated suppression and only slightly lower basal promoter activity to levels 60-80% of the wild-type sequence. However, alterations of nucleotides (even a sinAbbreviations: Ap1, activator protein 1; ATF, activating transcription factor; C/EBP, CCAAT/enhancer binding protein; CRE, cAMPresponsive element; CREB, cAMP-responsive element binding protein; Egr1, early growth response gene 1; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal regulated kinase; LDLR, low density lipoprotein receptor; OM, oncostatin M; SIRE, sterol-independent regulatory element; SRE-1, sterol regulatory element-1; S...
Cytokine oncostatin M (OM) has profound effects on proliferation and differentiation of breast cancer cells. OM treated cells show reduced growth rate and differentiated phenotypes. The mechanisms underlying the OM growth-inhibitory activity in breast cancer cells have not been fully elucidated. In this study, we investigated the OM-elicited signaling pathways in breast cancer cell lines MDA-MB231 and MCF-7. We show that OM rapidly activates the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription (STAT) 1 and 3 in both cell lines. Intriguingly, OM-induced growth inhibition and morphological changes in MDA-MB231 cells are completely abolished by inhibitors to ERK upstream kinase MEK (nitrogen/extracellular-regulated protein kinase kinase), but the MEK inhibitors have little effects on OM growth-inhibitory activity in MCF-7 cells. In addition, expressions of the cyclin kinase inhibitors p21 and p27 are strongly induced by OM in MCF-7 cells, but their expression is only slightly increased by OM in MDA-MB231 cells. These data together demonstrate that the growth-inhibitory activity of OM can be mediated by different signaling pathways in a cell line-specific manner. While the MEK/ERK pathway is the predominant signaling pathway that leads to the growth inhibition of MDA-MB231 cells, activation of additional signaling pathways are necessary for OM to exert its growth-inhibitory activity in MCF-7 cells.
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