T1-deficient and T1-Fc-transgenic mice develop a normal protective Th2-type immune response following infection with Nippostrongylus brasiliensis.

The IL-1 receptor-related protein T1 is expressed on the surface of Th2, but not Th1 cells. Studies with anti-T1 monoclonal antibodies have suggested that T1 is critical for development of normal Th2-type responses. To elucidate the role of T1 in vivo, we generated T1-deficient mice and a T1-transgenic strain which secretes soluble T1-Fc fusion protein into the serum. These were analyzed for the Th2 immune response induced by infection with the parasitic nematode Nippostrongylus brasiliensis. Although Th2 cytokine production by lymph node cells was similar in all groups of N. brasiliensis-infected mice, a decrease in IL-5 production by lung lymphocytes was detected in both T1-deficient and T1-Fc-transgenic mice compared to control littermates. This difference in IL-5 production did not influence blood eosinophilia, but recruitment of eosinophils into lung tissue, especially in T1-Fc-transgenic mice was slightly decreased. However, induction of all other immune parameters was normal and both T1-deficient and T1-Fc-transgenic mice were able to clear the parasite infection within 12 days with kinetics similar to those in control mice. Therefore, in contrast to previous suggestions, we conclude that the T1 protein is not obligatory for normal development of Th2 immune responses.

PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells.

The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

Effects of ras transformation on the induction of the IL-1 receptor related T1 gene in response to mitogens, anisomycin, IL-1 and TNFalpha.

The T1 gene gives rise to two transcripts encoding a 62 kDa membrane-bound and a 37 kDa secreted protein with similarity to the type I IL-1 receptor. It is weakly expressed in proliferating but not in resting fibroblasts and is strongly induced during the entry of quiescent cells into the cell cycle. Here we show that the T1 gene is also transcriptionally activated in response to the treatment of fibroblasts with cycloheximide and anisomycin. These protein synthesis inhibitors are known to stimulate the JNK and p38/RK signal transduction pathways. We provide evidence that anisomycin triggers T1 gene induction through the stimulation of the p38/RK MAP kinase. This observation is in line with our finding that physiological activators of the p38/RK pathway, the proinflammatory cytokines IL-1 and TNFalpha, stimulate T1 gene expression efficiently. Growth factor mediated T1 gene induction is a delayed early event, requiring ongoing protein synthesis. In contrast, anisomycin induces T1 gene expression at concentrations which block translation completely. Thus, transcriptional induction of the T1 gene via the p38/RK pathway is an immediate early event not requiring de novo protein synthesis. The T1 gene is strongly induced by various mitogens in quiescent NIH3T3 fibroblasts but not in ras transformed NIH3T3 cells. In contrast, all of the three tested agent which activate the p38/RK pathway, IL-1, TNFalpha, and anisomycin led to strong T1 gene expression in normal and ras transformed NIH3T3 cells alike. Thus, the T1 gene can be induced through the activation of at least two MAP kinase pathways: signaling through the ERK pathway can occcur in normal but not in ras transformed NIH3T3 cells, whereas the signaling through the p38/RK pathway is not affected by ras transformation.

Growth factor-mediated induction of the delayed early gene T1 depends on a 12-O-tetradecanoylphorbol 13-acetate-responsive element located 3.6 kb upstream of the transcription initiation site.

The T1 gene is a delayed early serum-responsive gene which encodes a secreted glycoprotein of the immunoglobulin superfamily. We have addressed the question of what promoter elements are needed to allow for growth factor-mediated T1 gene expression. By deletion analysis we have identified a 448-bp DNA region 3.5-4.0 kb upstream of the transcription start site which can confer serum inducibility onto a foreign minimal promoter. Within this sequence there is a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE) which is essential for T1 promoter induction in response to the forced expression of the transcription factor AP-1 in NIH 3T3 fibroblasts and F9 teratocarcinoma cells. This TRE is crucial for growth factor-mediated T1 gene expression. A point mutation within this TRE attenuated serum inducibility. Two E boxes are positioned 6 and 40 bp downstream of the TRE. Point mutations within these sequence motifs reduced basal T1 promoter activity and serum inducibility. Additional, as-yet-unidentified, promoter elements within the 448-bp serum-responsive region are required for T1 gene activation in response to growth stimulation.

Expression of alpha B-crystallin in human brain tumors.

We have previously shown that alpha B-crystallin is a heat-shock protein which specifically accumulates in response to the expression of c-Ha-ras and v-mos oncogenes in mouse NIH 3T3 fibroblasts. Elevated levels of alpha B-crystallin mRNA or protein were shown to be associated with pathological conditions of the brain. Therefore, we have examined the expression of alpha B-crystallin in normal human brains and brain tumors by Western blot analysis. alpha B-crystallin is moderately expressed in adult but not fetal brain. Elevated levels of alpha B-crystallin expression are observed in glial tumors such as astrocytoma, glioblastoma multiforme, and oligodendroglioma. alpha B-crystallin in these tumors is predominately unphosphorylated. High amounts of accumulated alpha B-crystallin in astrocytic tumors are preferentially found in the more aggressive stages. Glioblastoma multiforme is exceptional in that high alpha B-crystallin expression is observed in only one half of the analyzed samples whereas no alpha B-crystallin could be detected in the other. These results indicate that alpha B-crystallin may be a useful biochemical marker for studying the pathogenesis of various human brain tumors.

Cloning of the mouse hsp25 gene and an extremely conserved hsp25 pseudogene.

A genomic clone of the murine gene encoding the small heat-shock protein, Hsp25, was isolated. The coding region is interrupted by two introns of 128 bp and approximately 600 bp at identical positions as the human hsp27 gene. The 5' flanking regions of the mouse and human genes are very strongly conserved and contain several sequence motives for the transcription factors, HSF and Sp1. In the same screen we also isolated a hsp25 pseudogene. The sequence conservation between this pseudogene and hsp25 cDNA is very high (99%) indicating that this pseudogene emerged very recently.

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.

Expression of the murine small heat shock proteins hsp 25 and alpha B crystallin in the absence of stress.

Stress induces the synthesis of several large and small heat shock proteins (hsp's). Two related small hsp's, hsp25 and alpha B crystallin exist in mice. alpha B crystallin is an abundant protein in several tissues even in the absence of stress. Particularly high amounts accumulate in the eye lens. Here we show that hsp25 is likewise constitutively expressed in many normal adult tissues. In the absence of stress the protein is most abundant in the eye lens, heart, stomach, colon, lung, and bladder. The stress-independent expression pattern of the two small hsp's is distinct. In several tissues the amount of hsp25 exceeds that accumulating in NIH 3T3 fibroblasts in response to heat stress. hsp25, like alpha B crystallin, exists in a highly aggregated form in the eye lens. The expression of hsp25 and alpha B crystallin in normal tissues suggests an essential, but distinct function of the two related proteins under standard physiological conditions.

Alpha B-crystallin is a small heat shock protein.

Sequence similarity between alpha B-crystallin and small heat shock proteins (HSPs) has prompted us to investigate whether alpha B-crystallin expression is induced by heat shock. Indeed, accumulation of alpha B-crystallin was detected immunologically in NIH 3T3 cells after incubation at elevated temperatures and after addition of Cd2+ or sodium arsenite to these cells. Two-dimensional gel electrophoresis revealed identity between alpha B-crystallin from eye lenses and from heat-treated fibroblasts. The promoter of the alpha B-crystallin gene was fused to the bacterial chloramphenicol acetyltransferase gene and was shown to confer heat inducibility on this reporter gene in transient transfection assays. A perfect heat shock element within the promoter region is likely to mediate this response. Small HSPs and alpha B-crystallin were shown to share the following two physical properties: (i) they form supramolecular structures with sedimentation values around 17 S and (ii) they are associated with the nucleus at high temperatures and are localized in the cytoplasm under normal conditions. We conclude that alpha B-crystallin has to be considered a member of the class of small HSPs.

Alpha B crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.