Riboflavin and photoproducts in MC3T3-E1 differentiation Antonio Hernandes Chaves Neto a, * , Claudia Lumy Yano a , Edgar Julian Paredes-Gamero b , Daisy Machado a , Giselle Zenker Justo b , Maikel P. Peppelenbosch c , Carmen Veríssima Ferreira a a Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP6109, 13083-970 Campinas, SP, Brazil b Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil c Department of Cell Biology, University Medical Center Groningen, University of Groningen, A. Deusinglaan 1, NL-9713 AV, Groningen, The Netherlands article info Article history: Received 24 May 2010 Accepted 29 July 2010 Available online 3 August 2010 Keywords: Ascorbic acid Differentiation b-Glycerophosphate Osteoblast Riboflavin Signaling abstract Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascor- bic acid and b-glycerophosphate. These compounds decreased the osteoblast proliferation, increased the alkaline phosphatase activity, promoted a reduction in matrix metalloproteinase-2 activity and the decreased in the OPG/RANKL ratio. The effects of flavins on osteoblasts were unrelated to the antioxidant activity of these compounds. The biological activity of osteogenic medium containing riboflavin and its photoderivatives involved the activation of different signaling pathways (AKT, FAK, CaMKII), caspases- 3, -8 and -9, and up-regulation of the expression and/or stabilization of osteoblastic transcription factors (Runx2 and b-catenin). These findings suggest a potential use of flavins as adjuvants to improve bone metabolism. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Osteoporosis is a systemic skeletal disease characterized by low bone mass and the microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (Anonymous, 1993). Since osteoporosis is influenced by genetic and environmental factors such as diet and lifestyle it is important to develop strategies and recommendations for both the treatment and prevention of osteoporosis. In recent years, nutrients have received considerable interest because of their po- tential to prevent or minimize the risk of developing osteoporosis (Weber, 1999; Cashman, 2007; Trzeciakiewicz et al., 2009). Riboflavin is the precursor for essential flavo-coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FAD and FMN are prosthetic groups of numerous enzymes that catalyse electron transfer reactions during energy production, biosynthesis, detoxification and electron scavenging (McCormick, 1999). Besides being an important component of coenzymes, other biological properties have been ascribed to riboflavin. Dietary B vitamins can influence circulating homocysteine levels and several studies that have examined the relationship between methylenetetrahy- drofolate reductase (MTHFR) polymorphisms and B complex vita- mins have suggested that riboflavin intake and MTHFR genotype may interact to regulate bone mineral density (Macdonald et al., 2004; Yazdanpanah et al., 2007, 2008). We have recently shown that photoderivatives of riboflavin provide strong protection against haematological malignancy (de Souza et al., 2006) and are cytotoxic to human prostate cancer cells (de Souza Queiroz et al., 2007). Since the chemical structure of compounds is related to their biological activity, in this work we investigated the possible additive or synergistic effect of a combi- nation of riboflavin (Rb) or its photoproducts (irradiated Rb – IRb) and classic osteogenic inducers on bone composition during the differentiation of preosteoblastic MC3T3-E1 cells; this cell line is a widely accepted model of osteogenesis in vitro. The differentia- tion of MC3T3-E1 cells induced by ascorbic acid and b-glycero- phosphate is characterized by the induction of specific genes associated with the osteoblastic phenotype, including type I colla- gen, alkaline phosphatase, osteopontin and osteocalcin (Sudo et al., 1983; Quarles et al., 1992; Franceschi and Iyer, 1992; Fratzl-Zel- man et al., 1998). Our results indicate that riboflavin and its photoproducts had an additive effect on ascorbate and b-glycerophosphate-induced osteoblast differentiation of MC3T3-E1 cells. This effect was characterized by enhanced alkaline phosphatase activity, attenu- ated proliferation and the activation of multiple signaling path- ways. These findings provide clues to alternative therapeutic approaches for treating osteoporosis and indicate that the thera- peutic usefulness of riboflavin photoproducts deserves further investigation. 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.07.026 * Corresponding author. Tel.: +55 19 3521 6659; fax: +55 19 3521 6129. E-mail address: ahcnfoa@yahoo.com.br (A.H. Chaves Neto). Toxicology in Vitro 24 (2010) 1911–1919 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit