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Differential effects of oncostatin M and leukaemia inhibitory factor expression in astrocytoma cells.

ABSTRACT: The effects of the production of two closely related cytokines, oncostatin M (OSM) and leukaemia inhibitory factor (LIF), by astrocytoma cells were investigated using the stable cell line human U373-MG, which expressed and secreted both biologically active polypeptides. The expression of LIF by these cells caused resistance to this cytokine due to loss of the LIF receptor (LIFR), from the cell surface, suggesting its retention. In contrast, cells expressing OSM were stimulated by this cytokine, utilizing an autocrine mechanism, and possessed receptors for OSM, but not LIF, on the cell surface. In these cells the continuous up-regulation of OSM-induced gene expression was found even though the Janus kinase-signal transducer and activator of transcription ('JAK/STAT') pathway was almost exhausted due to long-term autocrine stimulation of the cells by OSM. The amount of LIFR was down-regulated in both LIF- and OSM-producing cells and this effect was not found in wild-type U373-MG cells treated with externally added cytokines. To investigate the mechanism of autocrine stimulation by OSM we constructed a stable cell line expressing a form of OSM that is retained in the endoplasmic reticulum (ER). This biologically active cytokine was not secreted, but was localized in the ER. In addition, it did not stimulate the astrocytoma cells in an autocrine manner. We conclude that expression of LIF causes resistance of astrocytoma cells to this cytokine, whereas expression of OSM leads to autocrine stimulation.

SUBMITTER: Kasza A

PROVIDER: S-EPMC1221740 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

Project description:Aquaporin 2 (AQP2), AQP5 and AQP8 participate in adenomyosis (AM). ?owever, the roles of these three molecules in AM have not been fully elucidated. In the present study, Institute of Cancer Research female mice were used to establish a model of AM. Subsequently, the endometrial tissues of the mice were observed by hematoxylin?eosin staining, and AM severity, uterus diameter, uterus index, ovary index and numbers of nodules on the uterine surface were evaluated and counted. In addition, eutopic and ectopic endometrial stromal cells (ESCs) were isolated from eutopic and ectopic endometrial samples derived from patients with AM and were then identified by immunofluorescence. The viability, and migratory and invasive ability of ESCs transfected with small interfering RNA targeting AQP5 (siAQP5) were determined by Cell Counting ?it?8, scratch wound?healing and Transwell assays, respectively. Reverse transcription?quantitative polymerase chain reaction was performed to determine the mRNA expression levels of AQP5, epithelial?mesenchymal transition (EMT)?related genes (E?and N?cadherin), matrix metalloproteinase (MMP)?2 and ?9. Protein expression levels of AQP2, AQP5, AQP8, E?, N?cadherin, MMP?2 and ?9 were detected by western blotting. AM severity and uterus index were higher, and there were a greater number of nodules on the uterine surface in the AM group compared with the sham group. AQP2, AQP5 and AQP8 proteins were highly expressed in eutopic and ectopic endometrium of the AM group, and AQP5 was more highly expressed than AQP2 or AQP8. In addition, the data showed that Vimentin was positively expressed in ESCs, and that siAQP5 suppressed the mRNA expression levels of AQP5, cell viability, migration, invasion, EMT and MMP?2 and ?9 expression in ESCs. In conclusion, AQP2, AQP5 and AQP8 were highly expressed in eutopic and ectopic endometrium. Notably, AQP5 silencing may suppress AM by inhibiting viability, migration, invasion, EMT, and MMP?2 and ?9 expression in ESCs.

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Differential effects of oncostatin M and leukaemia inhibitory factor expression in astrocytoma cells.

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