Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B

Abstract

Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) not only localizes MM cells in the marrow microenvironment, but also triggers interleukin-6 (IL-6) secretion by BMSCs and related MM cell proliferation. In the present study, we characterized the regulation of IL-6 gene expression in BMSCs during MM cell adhesion. Adhesion of ARH-77, HS-Sultan, IM-9, and U266 MM cell lines to BMSCs and BMSC lines (LP 101 and AA 101) triggered 5-through 15-fold and 2- through 4-fold increases in IL-6 secretion, respectively. IL-6 mRNA transcripts were undetectable by Northern blotting in IM-9 MM cells or LP 101 BMSCs cultured alone; however, adherence of IM-9 cells to LP 101 cells induced a transient increase in IL-6 transcripts at 6 hours, followed by peak IL-6 secretion at 24 hours. To confirm increased IL-6 transcription and characterize its regulation, LP101 BMSCs were transiently transfected with full length and deletion fragments of the IL-6 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of LP101 BMSCs with plasmid containing an intact NF-kappa B site showed a 6.8 +/- 0.4-fold increase in CAT activity triggered by IM-9 MM cell adhesion (n = 3, P < .05). Transfection of LP 101 cells with plasmid containing a single base pair deletion from the NF-kapp B binding motif abolished the MM adhesion- induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF-kappa B sequence resulted in an 8.1 +/- 0.7-fold increase in CAT activity related to MM adhesion (n = 3, P < .05). These data suggest that the NF-kappa B site is one of the essential regulatory elements for MM cell adhesion-induced IL-6 transcription in BMSCs. Electrophoretic mobility shift assays confirmed the involvement of NF-kappa B activation in regulating MM adhesion- induced IL-6 transcription in BMSCs. Further characterization of the upstream events in the signalling cascade regulating IL-6 may not only delineate mechanisms of IL-6 regulation during paracrine MM cell growth, but also provide new therapeutic strategies based on interruption of IL-6 mediated tumor cell growth.