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Regulation of alpha 2-macroglobulin gene expression by interleukin-6 (BSF-2/HSF)
T Andus1, T Geiger, J Klapproth
1Biochemisches Institut, Universität Freiburg, F.R.G.
Abstract:
In rat hepatocyte primary cultures recombinant human interleukin-6 (rhIL-6) induced alpha 2-macroglobulin (alpha 2M) synthesis 54-fold. Half-maximal induction was achieved at a rhIL-6 concentration of 30 pM. RhIL-1 beta led only to a 2-fold increase in alpha 2M synthesis, but strongly impaired the action of IL-6. Intraperitoneal injection of rhIL-6 into male rats resulted in a 19.7-fold increase of alpha 2M mRNA already after 4h. In contrast, alpha 2M mRNA levels (50-fold increase) were reached between 16 and 24 h after intramuscular injection of turpentine. Whereas turpentine-induced inflammation resulted in an increased alpha 2M synthesis in male and female rats, rhIL-6 injection had no effect in female rats. The increases after rhIL-6 administration in mRNA concentrations were followed by corresponding changes in alpha 2M levels in serum. By Northern analysis it was demonstrated that LPS-stimulated human monocytes synthesize IL-6 mRNA. The 5'-end of the rat alpha 2M gene has been isolated and the first 3 exons and 166 base pairs of the 5'-flanking region were identified by a combination of oligonucleotide hybridization and DNA sequencing. The transcriptional start site was determined by RNase protection as well as by primer extension experiments. 5'-CATAAAG-3' and 5'-TCAAAA-3' were found as TATA- and CAAT-box equivalent sequences, respectively. Furthermore, a potential glucocorticoid binding site (5'-TGTTCT-3') was localized on the antisense strand of the alpha 2M gene.
Insights
Recombinant human interleukin-6 (rhIL-6) significantly boosts alpha-2-macroglobulin (alpha 2M) synthesis in rats, with gene sequencing revealing regulatory elements. However, rhIL-6
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Interleukin-6 (IL-6) is a key cytokine involved in inflammation and immune responses.
- Alpha-2-macroglobulin (alpha 2M) is a major plasma proteinase inhibitor with diverse biological functions.
- Understanding the regulation of alpha 2M synthesis is crucial for comprehending inflammatory processes.
Purpose of the Study:
- To investigate the effect of recombinant human interleukin-6 (rhIL-6) on alpha 2-macroglobulin (alpha 2M) synthesis in vitro and in vivo.
- To compare the induction of alpha 2M by rhIL-6 with that induced by turpentine inflammation.
- To elucidate the molecular mechanisms underlying rhIL-6-mediated alpha 2M gene regulation.
Main Methods:
- Primary rat hepatocyte cultures treated with rhIL-6 and recombinant human interleukin-1 beta (rhIL-1 beta).
- In vivo studies involving intraperitoneal and intramuscular injections of rhIL-6 and turpentine in male and female rats.
- Northern blot analysis for alpha 2M mRNA quantification, and gene sequencing to identify regulatory elements of the alpha 2M gene.
Main Results:
- rhIL-6 potently induced alpha 2M synthesis in rat hepatocytes, with maximal effect at 30 pM.
- rhIL-6 administration in male rats led to rapid increases in alpha 2M mRNA and serum levels.
- Turpentine-induced inflammation increased alpha 2M synthesis in both male and female rats, unlike rhIL-6, which had no effect in females.
Conclusions:
- rhIL-6 is a potent inducer of alpha 2M synthesis in male rats, acting via transcriptional regulation.
- The alpha 2M gene possesses regulatory elements, including TATA and CAAT boxes, and a potential glucocorticoid binding site.
- Sex-specific differences exist in the response to rhIL-6-induced alpha 2M synthesis, suggesting complex regulatory pathways.