E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma via promoting ubiquitination-mediated degradation of RARα

All-trans retinoic acid (ATRA) is a widely used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA-induced osteogenic differentiation of osteosarcoma cells. We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to downregulate retinoic acid receptor α (RARα), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RARα and promotes its ubiquitination-mediated degradation; as a consequence, RARα-mediated differentiation is inhibited in osteosarcoma. Therefore, our studies present E2F1 as a potent biomarker, as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients.     

All-trans retinoic-acid inhibits heterodimeric bone morphogenetic protein 2/7-stimulated osteoclastogenesis,and resorption activity

Background

Bone regenerative heterodimeric bone morphogenetic protein 2/7 (BMP2/7) enhances but all-trans retinoic acid (ATRA) inhibits osteoclastogenesis. However, the effect of ATRA on physiological and/or BMP2/7-induced osteoclastogenesis in still unclear. In this study, we aimed to test the effect of combined treatment of BMP2/7 and ATRA on osteoclastogenesis, and resorption activity.

Results

All-trans retinoic acid (1 µM)?±?BMP2/7 (5 or 50 ng/ml) was added in murine pre-osteoclasts cell line RAW264.7 or mouse bone marrow derived macrophages (BMM) cultures. Osteoclast marker gene expression, osteoclastogenesis, and resorption activity were analyzed. BMP2/7 robustly enhanced osteoclast maker gene expression, osteoclastogenesis, and resorption activity. Interestingly, ATRA completely inhibited osteoclast formation in presence or absence of BMP2/7. Pan-antagonist of retinoic acid receptors (RARs) and antagonist of RARα, β or γ failed to reverse the inhibitory effect of ATRA on osteoclastogenesis. ATRA strongly inhibited Rank and Nfatc1 expression.

Conclusions

All-trans retinoic acid inhibits BMP2/7-induced osteoclastogenesis, and resorption activity possibly via RANKL–RANK pathway. Our findings from previous and current study suggest that combination of ATRA and BMP2/7 could be a novel approach to treat hyperactive osteoclast-induced bone loss such as in inflammation-induced severe osteoporosis and bone loss caused by cancer metastasis to bone.

     

胃癌细胞中AP-1活性与RARα介导的相关性

为确定全反式视黄酸 ( ATRA)抑制胃癌细胞 AP- 1活性过程中 RARα介导的作用机理 ,利用 Northern印迹和 Western印迹测定 RARα基因和 c Jun、c Fos蛋白表达水平 ;氯霉素乙酰转移酶活性 ( CAT)分析 AP- 1活性 ;以及 MTT法检测细胞生长速率 .结果表明 ,ATRA能够诱导 RARα表达 ,抑制 c Jun和 c Fos蛋白表达和 AP- 1活性 ,由此导致胃癌细胞生长抑制 .结果证实 ,ATRA抑制胃癌细胞 AP- 1活性是抑制细胞生长的重要途径之一 ,并与 RARα介导密切相关 .