0000000000020678
AUTHOR
Peter C. Heinrich
Different protein turnover of interleukin-6-type cytokine signalling components.
Interleukin (IL)-6 and IL-6-type cytokines signal through the gp130/Jak/STAT signal transduction pathway. The key components involved are the signal transducing receptor subunit gp130, the Janus kinases Jak1, Jak2 and Tyk2, STAT1 and STAT3 of the family of signal transducers and activators of transcription, the protein tyrosine phosphatase SHP2 and the suppressors of cytokine signalling SOCS1, SOCS2 and SOCS3. Whereas considerable information has been accumulated concerning the time-course of activation for the individual signalling molecules, data on the availability of the proteins involved in IL-6-type cytokine signal transduction are scarce. Nevertheless, availability of these molecules…
Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.
Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize a2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized a2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and fluorography. a2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5 - 11 days after the isolation of the cells, increasing amounts of a2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled a2-macroglob…
The membrane distal half of gp130 is responsible for the formation of a ternary complex with IL-6 and the IL-6 receptor
AbstractGp130 is the signal transducing subunit of the interleukin-6 receptor. Signaling is initiated by the complex formation of gp130 with IL-6 bound to the IL-6 receptor (IL-6R). We have subdivided the extracellular domain of gp130 in two parts and expressed the mutant proteins as soluble IgG fusion proteins in COS-7 cells. By studying the formation of the ternary complex we show that the membrane distal half of gp130 which contains a cytokine receptor domain is responsible for the interaction with the IL-6/IL-6R complex. Interestingly this is the same region which is believed to be involved in specific recognition of the related cytokines LIF, OM, and probably also of CNTF and IL-11.
Tissue inhibitor of metalloproteinases-2 (TIMP-2) in rat liver cells is increased by lipopolysaccharide and prostaglandin E2
AbstractTo explore the functional role of TIMP-2 in liver, we determined TIMP-2 mRNA levels in primary rat hepatocytes and in total rat liver. Rat hepatocytes constitutively express TIMP-2 mRNA at a low level. Incubation with dexamethasone, prostaglandin E2 and a combination of inflammatory cytokines leads to an up-regulation of TIMP-2 mRNA. In rats in vivo we found a dramatic increase of TIMP-2 expression after intraperitoneal injection of lipopolysaccharide. Compared to our previous findings on TIMP-1 we conclude that TIMP-2 mRNA expression is regulated in a distinct and partially opposite manner. Over-production of TIMP-2 could inhibit the activity of metalloproteinases and thus lead to …
Cytoplasmic STAT proteins associate prior to activation
The commonly accepted model of STAT factor activation at the cytoplasmic part of the receptor assumes that signal transducers and activators of transcription (STATs) are recruited from a cytoplasmic pool of monomeric STAT proteins. Based on a previous observation that non-phosphorylated STAT3-Src homology 2 domains dimerize in vitro, we investigated whether the observed dimerization is of physiological relevance within the cellular context. We show that STAT1 and STAT3 are pre-associated in non-stimulated cells. Apparently, these complexes are not able to translocate into the nucleus. We provide evidence that the event of STAT activation is more complex than previously assumed.
Purification and Characterization of the Soluble Interleukin-6 Receptor from Human Plasma and Identification of An Isoform Generated through Alternative Splicing
The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein α1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglyc…
TIMP expression in toxic and cholestatic liver injury in rat.
Abstract Background/Aims: Hepatic fibrosis is a dynamic pathological process with a net accumulation of extracellular matrix proteins. Recent evidence suggests that besides their increased synthesis, inhibition of matrix degradation plays a significant role. ECM degradation occurs via metalloproteinases which are inhibited in situ by specific tissue inhibitors of metalloproteinases (TIMPs). The aim of our studies was to determine the expression of TIMPs during toxic liver injury and cholestatic liver injury leading to fibrosis. Methods: We examined the expression of TIMP-1, -2 and -3 in two different rat models for liver injury (intraperitoneal CCl 4 injection and bile duct ligation) by Nor…
Participation of Two Ser-Ser-Phe-Tyr Repeats in Interleukin-6 (IL-6)-Binding Sites of the Human IL-6 Receptor
The alpha-subunit of interleukin-6 (IL-6) receptor is a member of the hematopoietin receptor family. The alignment of its amino acid sequence with those of other members of this family (human somatotropin receptor/murine IL-3 receptor beta and human IL-2 receptor beta) has suggested that amino acids included in two SSFY repeats found in each of its hematopoietin receptor domains, contribute to the binding of the ligand. The involvement of these amino acids in IL-6 binding and signal transduction was studied by site-directed mutagenesis and molecular modelling. We present a computer-derived three-dimensional model of the IL-6/IL-6 receptor complex based on the structure of the human somatotr…
Gene Therapy of Human Melanoma — from Animal Experiments to the Clinical Trial
Low immunogenecity of tumour cells is one of the reason that specific immune response is insufficient to destroy malignant cells. In an attempt to augment weakly immunogenic B78H1 mouse melanoma cell line, these cells were transfected with genomic DNA from a line of human melanoma cells expressing a 96kD melanoma associated antigen (MAA) that is intercellular adhesion molecule 1 (ICAM 1). The transfected cells expressed fivefold higher quantities of the melanoma associated antigen from which the DNA was obtained. Human ICAM 1 expressed by mouse melanoma cells appeared to be highly immunogenic leading to the rejection of the modified mouse melanoma cells. The transfected cells appeared to be…