Details

Autor(en) / Beteiligte

• Alloza Moral, Iraide

Titel

Analysis of the folding and secretion of the heterodimeric pro-inflammatory cytokine interleukin-12 in a recombinant HEK cell system

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2003

Link zum Volltext

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Interleukin-12 (IL-12) is a pro-inflammatory cytokine produced by antigen presenting cells (APC), principally macrophages and dendritic cells, and to a lower extent by B lymphocytes. IL-12 is composed of two disulfide-linked glycoproteins, i.e. α-chain or p35 (35 kD) and β-chain or p40 (40 kD). A body of literature points to different regulatory mechanisms for the α- and β-chains production. We have developed tightly controlled, ponasterone A inducible HEK-EcR cell lines expressing C-terminally hexahistidine tagged α, β, β/β and α/β chains. We have used these cell lines as means to analyse regulation of dimerization, folding and secretion of IL-12. In order to identify proteins that regulate these processes, i.e. chaperones, we performed immunoprecipitation with Ni2+-NTA. This method allowed us to purify the 6x histidine tagged-α- and β-chains and simultaneously, co-capture chaperones in a complex with the α- and β-chains. Thus, we detected the chaperone calreticulin (CRT) in association with both α- and β-chains and the GRP94 chaperone in a complex with the β-chain. These observations imply a role for CRT and GRP94 in the folding of the α- and β-chains. In addition, these cell lines were treated with pharmacological agents that disturb the function of the endoplasmic reticulum (ER). We found that the production of the α/β heterodimer and the β/β homodimer can be prevented by blocking N-glycosylation with tunicamycin. Castanospermine, in the other hand, produced an obvious increase in the secretion of α-chain but not the α/β or β/β forms. The effects of these two inhibitors in the secretion of the α-chain and the dimeric forms of IL-12 support the previous results in which CRT was shown to be involved in the folding of α- and β-chains. Moreover, treatment with ER Ca 2+-ATPase inhibitors (i.e. thapsigargin and celecoxib) and geldanamycin (inhibitor of GRP94) appeared to inhibit homodimer formation, a finding suggestive for a role of Ca2+ and Ca2+ binding proteins (e.g. GRP94) in regulation of the folding and secretion of the IL-12. Celecoxib was also found to be a strong inhibitor of IL-12 heterodimer production, and we present evidence that it acts by inhibiting secretion but not intracellular formation of dimeric of IL-12. This study provides evidence that celecoxib may find use as a method of treatment of conditions in which the dimeric forms of IL-12 play a disease-promoting role.