The pancreatic ,8-cell-specific expression of the insulin gene is mediated, at least in part, by the interaction of unique trans-acting ,8-cell factors with a cis-acting DNA element found within the insulin enhancer (5'-GC CATCIM-3'; referred to as the insulin control element [ICE]) present in the rat insulin II Pancreatic ,B-cell-specific expression is controlled at the transcriptional level by 5'-flanking insulin gene enhancer sequences (12,15,18,20,37,41). Cell-specific enhancer expression is mediated predominantly by the insulin control element (ICE) (22-24, 40, 41), whose core sequence motif, 5'-GCCATCTG-3', is found within the transcription unit of all the insulin genes characterized so far (9). This element is regulated by both positive-and negative-acting cellular transcription factors (41). Recent studies have demonstrated that positive activation, mediated by the ICE, is regulated through the binding of factors unique to ,B cells (31,40).The ICE contains the core sequence CANNTG, found in heavy-chain immunoglobulin and muscle creatine kinase enhancer elements (5,17,27,28). A common amino acid sequence motif is present in the proteins that bind to and positively regulate the expression of the heavy-chain immunoglobulin (i.e., E12 and E47) and the muscle creatine kinase (i.e., MyoD) enhancers (29). This motif consists of a helixloop-helix domain (HLH), which is composed of two segments capable of forming amphipathic ot helices connected by a nonconserved loop region that is important in proteinprotein interactions, and a basic region which is involved in DNA-protein interactions and is amino terminal to the HLH domain. The combined motif is referred to as the B-HLH region (13,29,30). This motif is common to a number of transcription factors involved in cell type determination, including the muscle determination proteins MyoD (14, 26, 34), Myf-5 (3), and myogenin (4, 42), proteins of the Drosophila aschaete-scute complex important in neural determination (1,6,36), and genes essential for cell type determination in Drosophila cells, such as daughterless (7,8,11) (Fig. 1, lower diagram)

Lipopolysaccharide (LPS), a maijor envelope component of Gram-negative bacteria, is the most frequent causative agent of septic shock and disseminated intravascular coagulation. LPS activates both CD14-positive (monocytes, macrophages, polymorphonuclear leukocytes) and CD14-negative (B-cell lines, endothelial cells) cells. CD14, a 55-kDa glycosyl-phosphatidylinositol-anchored membrane protein present on mature myeloid cells, serves as a receptor for LPS in complex with a soluble (serum-derived) LPS-binding protein (LBP). In this report, we show that human umbilical vein endothelial cells (HUVEC), which do not express measurable CD14 protein, become 3000-fold more sensitive to LPS-induced activation in the presence of serum, as measured by activation of the transcription factor NF-KB and expression of mRNA encoding tissue factor, a procoagulant molecule. This enhanced responsiveness of HUVEC is specifically mediated by the cell-free pool of CD14 (soluble CD14, sCD14) found in serum. The role of sCD14 in HUVEC activation by LPS was established by .(l) the blocking effect of monoclonal anti-CD14 antibodies which discriminate between cell-bound and sCD14,(ii) the lack of the serum-enhancing effect after immunodepletion of sCD14, and (iiW) establishing a reconstituted system in which recombinant sCD14 was sufficient to enhance the effects of LPS in the absence of serum and without a requirement for LBP. Thus, this mechanism of endothelial cell activation by LPS involves a cell-free pool of sCD14 most likely shed from CD14-positive cells of the monocytic lineage.

The insulin gene is expressed almost exclusively in pancreatic Is-cells. Previous work in our laboratory has shown that pancreatic n-cell-specific expression of the rat insulin II gene is controlled by a number of positive and negative cis-acting DNA elements within the enhancer. We have shown that one element within the enhancer, located between nucleotides -100 and -91 (GCCATCTGCT; referred to as the insulin control element [ICE]) relative to the transcription start site, is controlled by both positive-and negative-acting cellular transcription factors. The positive-acting factor appears to be uniquely active in i-cells. To identify the nucleotides within the ICE that mediate positive cell-type-specific regulation, point mutations within this element were generated and assayed for their effects on expression. Base pairs -97, -94, -93, and -92 were found to be crucial for the activator function of this region, while mutations at base pairs -100, -96, and -91 had little or no effect on activity. The gel mobility shift assay was used to determine whether specific cellular factors associated directly with the ICE. Several specific protein-DNA complexes were detected in extracts prepared from insulin-producing and non-insulin-producing cells, including a complex unique to f-cell extracts. The ability of unlabeled wild-type and point mutant versions of the ICE to compete for binding to these cellular factors demonstrated that the ,-cell-specific complex appears to contain the insulin gene activator protein(s). Interestingly, the adenovirus type 2 major late promoter upstream element (USE; GCCACGTGAC) also competed in the gel mobility shift assay for binding of cellular proteins to the ICE. These results suggested that the cellular factor that binds to the USE (i.e., USF) also interacts with the ICE. This was directly demonstrated by showing that ICE and USE sequences competed for the USF required for adenovirus type 2 major late promoter transcription in vitro and by showing that reticulocyte lysate-translated human USF products bound to the ICE. However, the USE sequences were unable to stimulate fl-cell-type-specific activity in vivo. We discuss the possible relationship of these observations to positive and negative control mediated by the ICE.In a multicellular organism, many genes are under developmental control and are transcribed in specific differentiated cell types. The mechanisms by which tissue-specific transcriptional control is achieved is currently the subject of extensive investigation. Cell-type-and tissue-specific regulation of eucaryotic gene transcription appears to be mediated by binding of regulatory proteins (trans-acting factors) to specific DNA sequences (cis-acting elements) usually, but not always, located in the 5'-flanking region of the gene (promoters-enhancers; for a review, see reference 27). Both enhancers and promoters consist of multiple short DNAbinding sites which are bound by trants-acting factors. There is increasing evidence that sequence-specific DNA-binding proteins confer tr...

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Pancreatic i-cell-type-specific transcription of the insulin gene is principally regulated by a single cis-acting DNA sequence element, termed the insulin control element (ICE), which is found within the 5'-flanking region of the gene. The ICE activator is a heteromeric complex composed of an islet at/-cell-specific factor associated with the ubiquitously distributed E2A-encoded proteins (E12, E47, and E2-5 showed that p-cell-specific transcription was regulated by 5'-flanking insulin DNA sequences (21). These studies, which were conducted with the rat insulin II gene, demonstrated that residues which lie between bp -695 and + 1 relative to the site of initiation are capable of directing 3-cell-type-specific expression. Rodents have two nonallelic insulin genes (I and II), which differ in their number of introns and chromosomal locations (45). The equivalent region of the human insulin gene has also been shown to direct cell-specific expression within transgenic mice (9,17,39).Experiments conducted with insulin-and non-insulin-producing cell lines have demonstrated that the 5'-flanking sequences of the insulin gene that are essential for pancreatic ,3-cell-type-specific expression reside between bp -340 and -91 relative to the transcription start site (for a review, see reference 44). This region exhibits enhancer-like properties and is regulated by both positively and negatively acting transcription factors (12,13,15,31,55). Detailed characterization of the insulin gene enhancer indicates that transcription mediated by these sequences is predominantly regulated by a single element (13, 25), whose core motif, 5'-GCCATCTG-3', is found within the transcription unit of all characterized insulin genes (45). This element, which we refer to as the insulin control element (ICE), is a site of both positive and * Corresponding author. Phone: (615) 322-7236. negative transcriptional control (24,26,55). The ICE is also important in homeostatic control of the insulin gene in glucose-treated a cells (19,40).The ICE activator appears to be restricted to islet ao and L cells (32,38). Experiments conducted with antisera to the expressed E2A gene products, E12, E47, or ITF-1 (or antigenically related proteins), have demonstrated that these ubiquitously distributed proteins are present in the ICE activator complex (11,18,42). (ITF-1 is a generally distributed E47-like factor [23].) Mutagenesis studies have shown that the nucleotides (shown underlined) that define the binding motif for proteins in the basic helix-loop-helix (bHLH) family, CANNTQ, are essential for trans activation (54). In addition, ICE activation is inhibited in ,B cells by a negative regulator of bHLH-mediated activation, Id (11). This factor appears to suppress bHLH activation by sequestering the E2A gene products into transcriptionally nonfunctional complexes (5). These results are all consistent with the proposal that the ICE activator is composed, at least in part, of E2A proteins. The restricted distribution of this ICE activator binding activity sugge...