Cooperation Between IL-7 and TNF-Alpha Results in Suppression of Runx2/Cbfa1 Expression and Inhibition of Osteoblast Differentiation in Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy in which almost all patients develop osteolytic lesions characterized by enhanced osteoclast (OCL) activity and reduced bone formation. Although much is known about the mechanisms responsible for the increased OCLs activity, the mechanisms responsible for osteoblast (OBL) inhibition are still not clearly understood. However, it is now clear that OBL suppression represents an important feature of the disease. Factors reported to inhibit OBL differentiation in MM include Dickkopf-1 (DKK-1), secreted Frizzle-Related Protein-2 (sFRP2), which are inhibitors of the Wingless (Wnt) signaling pathway that is critical for the OBL differentiation, IL-3, IL-7 and tumor necrosis factor-alpha (TNF-alpha). To further define the mechanisms responsible for OBL suppression in MM, we co-cultured MC4 cells, a MC-3T3-derived mouse marrow OBL precursor cell line, with 5TGM1 cells, an aggressive murine MM cell line known to reproduce MM bone disease in vivo, and measured the expression levels of OBL differentiation markers by Real-time PCR. Type I collagen, bone sialoprotein and osteocalcin mRNA expression were downregulated by 10-fold after the co-culture for 48 hours, and mineralization was inhibited with longer co-culture. Further, expression of mRNAs for two major transcription factors that regulate OBL differentiation, Runx2 and osterix were suppressed 10-fold, while activating transcription factor-4 (ATF4) was unaffected. Time-course experiments demonstrated that Runx2 mRNA downregulation was detectable within 2 hours of co-culture. To identify the promoter region responsible for the suppression of Runx2, MC4 cells were then transfected with Runx2-promoter luciferase expression vectors carrying different deletions in the Runx2 promoter and co-cultured with 5TGM1 cells. 5TGM1 cells strongly downregulated Runx2 promoter activity and a 900 bp region was shown to be the region required for Runx2 suppression. Within this promoter region, several potential transcription factor sites were identified including GFI-1 that can suppress Runx2 expression. However, GFI-1 mRNA expression was only increased after 48 hours of co-culture, suggesting another mechanism was involved. To determine if OBL suppression was mediated by cell-cell interaction or by soluble factors, we then co-cultured MC4 and 5TGM1 cells in a transwell system and found that 5TGM1 cells were still capable of suppressing Runx2 expression, indicating a soluble inhibitor was responsible for suppression of OBL differentiation. Importantly, DKK-1, sFRP2 or IL-3 expression was not detectable in 5TGM1 cells, but the cells did produce IL-7 and TNF-alpha. Using blocking antibodies for IL-7 and TNF-alpha in the co-culture system, Runx2 expression increased to 90% of normal levels with either anti-TNF or anti-IL-7 and to 100% when both antibodies were added. These results suggest that IL-7 and TNF-alpha cooperate to suppress Runx2 expression. Moreover, when MC4 cells were treated with low dose (0.1ng/ml) of TNF-alpha or IL-7, no effect on Runx2 expression was detected. However, when low concentrations of IL-7, and TNF-alpha were both added, Runx2 was downregulated almost 80% within 2 hours. These results demonstrate that TNF-alpha and IL-7 can cooperate to rapidly suppress OBL differentiation in MM.