The biosynthesis of cachectin/TNF is largely regulated at a post-transcriptional level. Bacterial endotoxin, which strongly induces cachectin/TNF production, thus seems to elicit at least some of its effects by altering the macrophage cytoplasmic milieu. It has previously been shown that the 3'-untranslated TTATTTAT element present in numerous cytokines and proto-oncogenes is capable of repressing the translation of mRNA molecules in which it is represented. Using constructs in which the CAT coding sequence is followed by varying segments of the cachectin/TNF 3'-untranslated region, we now demonstrate that downstream sequences present in the cachectin/TNF mRNA are sufficient to mediate greater than 200-fold induction of CAT synthesis in response to activation by endotoxin. Induction of CAT activity is not attributable to a change in cytoplasmic mRNA concentration, but to a marked enhancement of translational efficiency. The response to endotoxin represents "derepression," and is conferred chiefly by the translationally repressive TTATTTAT element, acting in concert with essential flanking sequences.

SummaryThe induction of cachectin/tumor necrosis factor (TNF) synthesis by bacterial endotoxins is a process that entails activation at several levels . Cachectin/TNF gene transcription is accelerated, leading to rapid accumulation ofmP NA within the macrophage cytosol . In addition, translational derepression occurs, leading to far more efficient message utilization . Through the use of posttranscriptional reporter constructs, we now demonstrate that certain agents capable of inhibiting cachectin/TNF biosynthesis operate through different mechanisms. In RAW 264.7 macrophages, pentoxifylline blocks cachectin/TNF mRNA accumulation but has no effect upon the efficiency of reporter mRNA translation. Dexamethasone, on the other hand, has only a modest effect on cachectin/TNF mkNA accumulation, but strongly impedes translational derepression . Combined application of dexamethasone and pentoxifylline to macrophages causes a greater suppression of cachectin/TNF biosynthesis than can be achieved by either agent alone. These findings suggest that the signaling pathway activated by endotoxin is branched, and that selective inhibition of different parts of the pathway may be achieved through the use of distinct agents .

Vascular endothelial cells, serving as a barrier between vessel and blood, are exposed to shear stress in the body. Although endothelial responses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological conditions-e.g., in atherosclerosis and reperfusion injury. We have previously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-1-encoding gene to define the cis element responding to shear stress. The shear stress/luciferase assay on the deletion constructs revealed that a 38-bp segment (-53 to -90 bp relative to the transcription initiation site) containing two divergent phorbol ester "12-O-tetradecanoylphorbol 13-acetate" (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was lost after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in both the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferase gene, shear stress induced the reporter activities by 35-fold and 7-fold in HeLa cells and BAEC, respectively. The application of shear stress on BAEC also induced a rapid and transient phosphorylation of mitogen-activated protein kinases. Pretreatment of BAEC with TPA attenuated the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduction pathway in activating cells. The present study provides a molecular basis for the transient induction of MCP-1 gene by shear stress.

Noting the scarcity of CpG dinucleotide in total genomic DNA derived from higher organisms and the scarcity of TpA dinucleotide in total genomic DNA derived from most life forms, we examined the distribution of these dinucleotides in sequences derived from functionally distinct types of human DNA, including mitochondrial DNA, intergenic DNA, intron DNA, and DNA destined to be represented in the cytoplasm as mRNA, tRNA, or rRNA. While CpG frequency has fallen to its lowest levels in DNA that is transcriptionally silent, TpA is most stringently excluded in DNA destined to be expressed as mRNA in the cytosol. This observation suggests that the selective pressures leading to the removal of CpG and TpA operate at different levels. With respect to TpA, dinucleotide scarcity may reflect a requirement for mRNA stability and may indicate the action of UpA-selective ribonucleases. We propose that, by reason of its instability, UpA must have been very rare in primordial RNA. Therefore, tRNA with the anticodon for this dinucleotide may have failed to evolve, making UpA the primordial doublet "stop" codon. The modern triplet code has faithfully conserved this arrangement in the two universal stop codons, UAA and UAG.In 1961, at a time when the determination of DNA sequences was but a dream, Kornberg and his associates (1, 2) developed the ingenious technique of nearest-neighbor analysis and found that representation of some dinucleotides in DNA deviated significantly from that expected were the mononucleotides associated randomly with each other. Specifically, there seemed to be a marked deficiency in the number of CpG dinucleotides and a slight but significant underrepresentation of TpA dinucleotides in animal and plant cells. With the development of techniques for sequencing large stretches of DNA and with the establishment of computer-accessible data bases, sequence analyses also revealed a scarcity of CpG and TpA dinucleotides (3-5).Underrepresentation of these dinucleotides could have evolved because of constraints operating at the level of DNA, primary messenger, processed messenger, or protein. Although a number of studies comparing the frequency of dinucleotides in different species have been performed (5-7), the distribution of dinucleotides in functionally different types of DNA has received scant attention.We have now studied the occurrence of CpG and TpA dinucleotides in different functional domains of DNA. While CpG frequency is lowest in transcriptionally silent DNA, TpA is most stringently excluded from DNA destined to be expressed as cytosolic mRNA. We show that UpA dinucleotides in RNA are unstable to nucleosidic cleavage and suggest that instability of this dinucleotide may account for the fact that the conserved stop codons start with UpA. MATERIALS AND METHODSData were from the GenBank and the EMBL data libraries supplemented by sequences determined in our own laboratory and made available through the generosity of E. W. Davie (University of Washington, Seattle). Only human genes were examined and...