In this study, we showed that Osx down-regulates IL-1α expression in osteosarcoma through repression of the IL-1α promoter. This down-regulation was specific as Osx had no effect on the transcription of OPG or the expression of IL-6, IL-11, and prostaglandin E2. Although Osx
is an essential gene for osteoblast differentiation, its role in bone tumors is poorly understood. We showed in previous studies that bone tumors induced after intratibial injection of K7M2 osteosarcoma cells were osteolytic. Osx expression was absent in these cells. The reexpression of Osx by stable gene transfection altered the osteolytic phenotype (5
). However, the mechanism of reduced osteolysis following Osx
gene transfection was unclear and did not involve the suppression of RANKL by Osx (5
). Cytokines, such as IL-1α, IL-6, IL-11, and prostaglandin E2, have been shown to participate in the bone remodeling processes by inducing RANKL and osteoclast formation. Therefore, changes in the levels of these cytokines could affect bone lysis. Here, we showed for the first time that expression and production of IL-1α, a cytokine that stimulates osteoclastogenesis through the production of RANKL by marrow stromal cells and osteoblasts (13
), was down-regulated at the transcriptional level by Osx. The secretion of IL-1α by tumor cells will have an effect on the expression of RANKL on the stromal cells and osteoblasts. This increased RANKL on stromal cells and osteoblasts increases osteoclast activity. In our previous report, we showed that reexpression of Osx in K7M2 cells had no effect on the RANKL expression in tumor cells.
Our data also showed that the effect of Osx on IL-1α expression was not restricted to one osteosarcoma cell line. The suppression of Osx expression by siRNA in two other osteosarcoma cell lines, Dunn and DLM8, resulted in the up-regulation of both IL-1
α promoter activity and IL-1α protein expression. The osteoblastic phenotype of these two cell lines was also altered to a lytic phenotype following Osx suppression. The possible autocrine effect of IL-1α on tumor cell lines has been ruled out because K7M2, Dunn, and DLM8 do not express IL-1α receptors (data not shown). To determine whether Osx acts through the Sp1-binding site on the IL-1
α promoter, we used a site-directed mutagenesis reporter vector to show that Osx was unable to repress the IL-1
α promoter activity in osteosarcoma cells after mutating the Sp1-binding site. These data indicate that this Sp1-binding site is important for Osx-mediated repression of the IL-1
α promoter. The ChIP assay was used to confirm the occupancy of the IL-1α promoter by Osx. These data suggest that Osx represses the activity of IL-1α promoter via binding to the −1,532 to −1,056 and −272 to −13 regions of IL-1α promoter. Our present studies are consistent with the fact that Osx belongs to the Sp1 family (4
In summary, this study is the first to link Osx
, a gene involved in bone differentiation, with the repression of the IL-1α promoter. IL-1α participates in bone remodeling, and therefore, alteration in the production of IL-1α by tumor cells may in turn affect the phenotype of the tumor cells when they metastasize to the bone. Indeed, we have shown that the lytic phenotype of prostate, breast, and renal cell carcinoma cells inversely correlated with the expression of Osx (19
). Other cytokines and proteins may also contribute to the lytic activity of tumor cells. It is not our contention that IL-1α is the only cytokine involved. However, the data presented here indicate a strong correlation between the presence or absence of Osx, the production of IL-1α by tumor cells, and tumor-induced bone lysis. Our findings support the premise that Osx has potential as a therapeutic target for decreasing tumor-induced bone destruction and pathologic bone fractures. We hypothesize that Osx-induced repression of IL-1α expression in osteosarcoma changed the microenvironment, decreasing osteoclastogenesis resulting in the change in bone phenotype. Further studies will focus on identifying the critical pathways involved in the regulation of osteoclastogenesis by Osx.