Identification and characterization of the gene encoding human cytoplasmic polyadenylation element binding protein.

The maturation of human oocytes occurs in the absence of gene transcription. In model organisms, such as Drosophila, Xenopus, and the mouse, oocyte maturation and early pattern formation is mediated through the regulated translation of maternally derived mRNAs. The maturation-dependent stimulation of maternal mRNA translation is correlated with increases in poly(A) tail length, controlled through a process termed cytoplasmic polyadenylation. However, this mechanism of mRNA translational control has not been characterized in humans. In this study we report the cloning of a human cytoplasmic polyadenylation element binding (hCPEB) protein with sequence-specific RNA binding activity. Our data demonstrate that alternative splicing generates hCPEB mRNAs that encode proteins with a conserved C-terminal RNA binding domain but with different N-terminal regulatory domains. The hCPEB mRNA is expressed in the brain and heart as well as in immature oocytes, consistent with the hypothesis that cytoplasmic polyadenylation may regulate the translation of human mRNAs in both oocytes and somatic cells.

Ultraviolet light attenuates heat-inducible gene expression.

Ultraviolet light (UV) induces a stress response mediated through transcription factors such as NF-kB and AP-1, yet little is known about its effect on other transactivators of stress gene expression such as heat shock factor (HSF1). Analysis of UV-treated HeLa cells unexpectedly revealed uncoupling of the heat shock response. UV weakly induced HSF1 into its DNA bound state and markedly attenuated heat-inducible gene expression. HSF1 was further analyzed as a potential target for the uncharacteristic uncoupling of the thermal stress response by another type of stress. Heat-inducible multimerization and nuclear translocation of HSF1 were found to be intact in UV-treated cells; however, the monomeric rather than the multimeric form of HSF1 become hyperphosphorylated by UV. This effect could be partially abolished by the antioxidant N-acetyl cysteine with partial reconstitution of hs gene expression. The reported role of a MAP kinase blockade of HSF1 transactivating properties could not be confirmed by an inhibitor of the MAP kinase pathway. Fibroblasts defective in SAP kinase activity also did not exhibit resistance to UV-inducible phosphorylation of HSF1. Two-dimensional phosphopeptide mapping of HSF1 revealed a single tryptic peptide to be affected by UV, but no new pattern of phosphorylation was evident relative to tryptic phosphopeptide profile observed in control cells. These data suggest that UV uncoupling of the hs response possibly involves steps in addition to those associated with phosphorylation the monomeric form of HSF1.

Attenuated stress responses in young and old human lymphocytes.

Aging generally is understood to be a period defined by altered responses to physiological stress. At the molecular level, several stress responses involving specific gene expression have been revealed, and thermal stress has been tightly linked to induction of the heat shock gene family (D.A. Jurivich. In E. Bittar (ed.), Principles of Medical Biology, Vol. 4, JAI press, San Diego, 1996, pp. 411-462). Perturbations in heat shock gene transcription consistently have been noted in senescent cells from all species examined thus far. Because heat shock proteins serve several vital functions in the immune system, changes in the thermal stress response could potentially contribute to immunosenescence. Inadequate promoter priming by the transactivator or heat shock genes, heat shock factor 1 (HSF1), is thought to account for age-dependent diminution in expression of these genes, although the exact mechanism for this loss is not clearly understood. We have found that human lymphocytes exhibit an age-dependent loss in HSF1-DNA binding, although a range of binding has been observed in both young and old donor cells. This report characterizes a subset of young and old human donor lymphocytes that are non-responders to thermal stress defined by the absence of HSF1-DNA binding after a 42 degrees C heat shock. Whole cell extracts from these donor cells have the capacity to inhibit HSF1-DNA binding when mixed with pre-activated HSF1 from HeLa cells. This inhibitory activity is lost upon heat denaturation and does not appear to be protease mediated. Serial passage of lymphoblasts recapitulates loss of heat inducible HSF1-DNA observed in old donor lymphocytes, thus suggesting that loss of replicative potential and aging lead to altered stress responses. Uncoupling of the thermal response and its potential relevance to apoptosis and aging are discussed.

Phospholipase A2 triggers the first phase of the thermal stress response and exhibits cell-type specificity.

To understand the relationship of inflammatory and cellular stress responses, phospholipase A2 (PLA2) was examined for its role in the first phase of the transcriptional response to cellular stress. Electromobility shift analysis revealed heat shock transcription factor (HSF1)-DNA binding when HeLa S3 and Jurkat cells were exposed to exogenous PLA2. Although PLA2-inducible HSF1-DNA binding was comparable to thermal stress, it did not induce maximal heat shock gene expression. PLA2-induced HSF1 was not hyperphosphorylated relative to the heat-inducible form, thus suggesting that exogenous PLA2 affects the signal for HSF1 multimerization but not its phosphorylation. Because inflammation often involves elevated temperatures, the effect of PLA2 on thermal regulation of HSF1-DNA binding activity was examined. PLA2 exposure altered the thermal threshold for HSF1 activation, and pore-gradient gel analysis indicated that either conformational changes or other modifications of HSF1 are being induced when cells are treated by PLA2, thus creating a synergistic environment for HSF1 activation into its DNA-bound state. Surprisingly, the monocyte-like cell line, U-937, was insensitive to the action of exogenous PLA2. Neither HSF1-DNA binding or lowering of the temperature threshold for HSF1 activation was observed in PLA2-treated U-937 cells. These data suggest that inflammatory mediators such as PLA2 partially affect transcriptional switches mediating thermal stress in some cell types but not others. The purpose of HSF1 activation during inflammation and its differential induction are discussed relative to these observations.

Salicylate triggers heat shock factor differently than heat.

Sodium salicylate has the unusual property of partially inducing the human heat shock response (Jurivich, D. A., Sistonen, L., Kroes, R., and Morimoto, R. I. (1992) Science 255, 1243-1245). Salicylate induces the DNA binding state of the human heat shock transcription factor (HSF), but this is insufficient to elevate heat shock gene expression. Because it is not known how HSF enhances heat shock gene expression, further analysis of the transcriptionally inert, salicylate-induced HSF was undertaken to potentially identify components of the heat shock response that are necessary for full transcriptional induction. Like thermal stress, exposure of HeLa cells to salicylate led to the induction of HSF1 into a DNA-bound state. Despite continued exposure of cells to salicylate, HSF1.DNA binding attenuated much more rapidly than a continuous heat shock. Western blot analysis revealed that the salicylate-induced form of HSF1 was not hyperphosphorylated like the heat-induced form. Furthermore, supershifts of the HSF1 bound to an heat shock element (HSE) oligonucleotide by monoclonal antibodies to phosphoamino acids revealed that salicylate induced threonine phosphorylation of HSF1, whereas heat led to a predominance of HSF1 serine phosphorylation. These data suggest that salicylate-independent signals are necessary to convert HSF1 into a transactivator of heat shock gene expression and that brief acquisition of DNA binding by this factor is insufficient to maximally enhance transcription.