Wayne Petko

Enhancement of beta-amyloid precursor protein transcription and expression by the soluble interleukin-6 receptor/interleukin-6 complex

We investigated a potential role for the soluble interleukin-6 receptor (sIL-6R) in modulating interleukin-6 (IL-6) function in the central nervous system by assessing IL-6 and sIL-6R effects on beta-amyloid precursor protein (beta-APP) transcription and expression in cells of human neuronal origin. Cells transfected with a luciferase reporter plasmid containing a 3.8 kb DNA fragment of the beta-APP promoter were shown to have inducible promoter activity when treated with phorbol ester or basic fibroblast growth factor, but not when treated with lipopolysaccharide or Il-6. PCR amplification analysis revealed the presence of mRNA encoding the signaling subunit of the Il-6 receptor complex, the gp130 subunit, at levels approximating that found in human cortical tissue. The mRNA encoding the IL-6 receptor, however, was poorly expressed and was detectable only at high amplification cycles. When purified sIL-6R protein was added together with IL-6, there was a rapid induction of promoter activity within 2 h of stimulation followed by elevations in protein levels of both cell-associated and secreted beta-APP. Analysis of mRNA transcripts from human cortical brain tissue and cell cultures derived from fetal human brain demonstrated the presence of an alternatively spliced secreted form of the IL-6 receptor mRNA, suggesting that cells of the central nervous system may themselves be a source of sIL-6R protein. The capacity for sIL-6R to enhance IL-6 function and broaden the IL-6 target cell population in the brain has implications for the regulation of beta-APP expression in disease states such as Alzheimers disease where elevations in brain IL-6 levels have been reported.

ADDITIVE EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR AND PHORBOL ESTER ON β-AMYLOID PRECURSOR PROTEIN EXPRESSION AND SECRETION

Expression of the beta-amyloid precursor protein (beta-APP), a proteoglycan whose proteolytically derived fragments have been implicated in the neuropathology observed in Alzheimers disease, is regulated by a variety of stimuli including cytokines, phorbol esters, and growth factors. In this study we report the effects of basic fibroblast growth factor (bFGF) and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on beta-APP expression and secretion in SKNMC human neuroblastoma cells. Treatment of the cells with bFGF for 24 h increased APP promoter activity 200%, cell-associated full-length protein 189%, and secreted amino-terminal fragments 192% compared to basal levels. Treatment of the cells with PMA for 24 h also up-regulated APP expression and secretion with increases of 170, 112, and 161% being observed for promoter activity, cell-associated full-length protein, and secreted amino-terminal fragments, respectively. The effects of bFGF and PMA on the expression and secretion of beta-APP were additive and distinct in that: (a) co-treatment of the cells with maximally stimulating doses of bFGF and PMA had an additive effect on both induced full-length protein expression (242%) and secretion of amino-terminal fragments (311%) compared to basal levels; (b) net levels of full-length protein expression and secretion induced by bFGF and PMA differed significantly from each other; and (c) down-regulation of phorbol ester-stimulated protein kinase C by pre-treatment of the cells for 24 h with 1 microM PMA failed to attenuate bFGF-induced transcription or induced secretion of beta-APP.