TY - JOUR
T1 - Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-κB
AU - Chauhan, Dharminder
AU - Uchiyama, Hiroshi
AU - Akbarali, Yasmin
AU - Urashima, Mitsuyoshi
AU - Yamamoto, Ken Ichi
AU - Libermann, Towia A.
AU - Anderson, Kenneth C.
PY - 1996/2/1
Y1 - 1996/2/1
N2 - 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, LP 101 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 LP 101 BMSCs with plasmid containing an intact NF-κB 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-κB binding motif abolished the MM adhesion-induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF- κB 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-κB 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-κB 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.
AB - 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, LP 101 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 LP 101 BMSCs with plasmid containing an intact NF-κB 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-κB binding motif abolished the MM adhesion-induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF- κB 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-κB 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-κB 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.
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U2 - 10.1182/blood.v87.3.1104.bloodjournal8731104
DO - 10.1182/blood.v87.3.1104.bloodjournal8731104
M3 - Article
C2 - 8562936
AN - SCOPUS:0030041147
VL - 87
SP - 1104
EP - 1112
JO - Blood
JF - Blood
SN - 0006-4971
IS - 3
ER -