Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner Jiyuan Zhao a , Shinsuke Ohba b , Masashige Shinkai a , Ung-il Chung c,d , Teruyuki Nagamune a,c,d, * a Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan b Center for Disease Biology and Integrated Medicine, School of Medicine, University of Tokyo, Tokyo 113-0033, Japan c Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan d Center for NanoBio Integration, University of Tokyo, Tokyo 113-8656, Japan Received 28 January 2008 Available online 22 February 2008 Abstract To effectively treat bone diseases using bone regenerative medicine, there is an urgent need to develop safe cheap drugs that can potently induce bone formation. Here, we demonstrate the osteogenic effect of icariin, the main active compound of Epimedium pubes- cens. Icariin induced osteogenic differentiation in pre-osteoblastic MC3T3-E1 cells and mouse primary osteoblasts. Icariin upregulated the mRNA expression levels of the osteoblast marker genes runt-related transcription factor 2 (Runx2) and inhibitor of DNA-binding 1 (Id-1). The osteogenic effect was inhibited by the introduction of Smad6 or dominant-negative Runx2, as well as Noggin treatment. Fur- thermore, icariin induced the mRNA expression of bone morphogenetic protein (BMP)-4. These data suggest that icariin exerts its potent osteogenic effect through induction of Runx2 expression, production of BMP-4 and activation of BMP signaling. The extremely low cost of icariin and its high abundance make it appealing for bone regenerative medicine. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Bone regeneration; Icariin; Osteogenic differentiation; Osteoblast Bone disorders, especially osteoporosis, are becoming increasingly prevalent as the aging population grows, and bone fractures also occur frequently. The treatment of serious bone defects remains a great challenge [1]. Local transplantation of autologous multipotent cells has been widely used for bone regeneration [2]. For enhancement of the therapeutic effects, growth factors and cytokines are usually introduced into the system by direct protein delivery or viral gene delivery [3–5]. Although the use of bone morphogenetic proteins (BMPs) has been extensively studied for bone regeneration, large amounts of these BMPs are required and BMP-containing devices tend to fail in a certain percentage of cases, thereby raising concerns over costs and safety [6–8]. The high cost and rapid degradation of BMPs [9] limit their clinical use. Therefore, there is an urgent need to develop alternative methods with higher efficacies and lower costs than the existing methods. Despite recent successes with drugs that inhibit bone resorption, there is only a limited number of reports on anabolic agents that effectively increase bone formation, such as statins [10], isoflavone derivatives [11,12] and TAK-778 [13]. The herb Epimedium pubescens is recorded in the Chinese pharmacopoeia as the traditional Chinese medicine ‘‘yinyanghuo” and was reportedly used to cure bone diseases in ancient China. Icariin is a flavonoid iso- lated from this herb and is its main active compound. An E. pubescens extract was recently reported to have thera- peutic effect on a rat model of osteoporosis induced by 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.054 * Corresponding author. Address: Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Fax: +81 3 5841 8657. E-mail address: nagamune@bio.t.u-tokyo.ac.jp (T. Nagamune). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 369 (2008) 444–448