bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells Aikaterini Berdiaki a , Dragana Nikitovic a , Aristeidis Tsatsakis a , Pavlos Katonis b , Nikos K. Karamanos c , George N. Tzanakakis a, ⁎ a Department of Histology, Division of Morphology, School of Medicine, University of Crete, 71110, Heraklion, Greece b Department of Orthopedics, University Hospital of Heraklion, 71003, Heraklion, Greece c Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26110, Patras, Greece abstract article info Article history: Received 30 January 2009 Received in revised form 29 May 2009 Accepted 25 June 2009 Available online 3 July 2009 Keywords: Fibrosarcoma Hyaluronan Basic fibroblast growth factor Hyaluronan synthase Hyaluronidase Background: Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration. Methods: Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized. Results: bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high mole- cular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p b 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed. Conclusions: bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism. HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cancer cells interact with the surrounding extracellular matrix (ECM) in a manner supportive to their growth and metastasis. The ECM acts both as a scaffold to which tumor cells adhere to and migrate in, as well as, a pool of growth factors and cytokines that modulate their malignant behavior [1]. Hyaluronan (HA), a nonsulfated high molecular weight glycosa- minoglycan (GAG) is one of the major ECM components. HA acts as a messenger capable of transmitting ECM-derived stimulus to the cell interior [2], in addition to its well established structural role in tissue homeostasis [3]. This GAG specifically binds to its respective cell surface receptors, CD44 and Receptor for HA-Mediated Motility (RHAMM) to activate distinct downstream signaling cascades [4,5] shown to regulate various biological functions [6–8]. HA is synthesized by the three hyaluronan synthases (HAS1, HAS2 and HAS3), enzymes located at the plasma membrane. Importantly, each HAS produces HA of specific molecular weight, which ranges from 100–2000 kDa [3,9–12]. Hyaluronidases (HYAL1 and 2) are enzymes responsible for the degradation of HA in human tissues [11,13]. It has previously been shown, that the biological effects of HA are highly dependent on its molecular weight [14]. Thus, the differential expres- sion of the HAS and HYAL isoforms, which regulate the balance bet- ween the synthesis and the degradation of HA, defines HA-dependent downstream signaling in both normal and tumor cells [15,16]. Our previous study [17] demonstrated that growth factor secretion in fibrosarcoma cells can potentially modulate specific HAS isoforms expression and HA synthesis. This correlates well with studies on HA metabolism in other tumor cell lines [7,16–19]. Manipulation of the HAS and HYAL genes expression in vitro, has shown that HA may have a crucial role in tumor progression [12,15,16,20–24]. In this study, we investigated the changes in HA metabolism induced by basic fibroblast growth factor (bFGF) in fibrosarcoma cell lines. Fibrosarcoma, a soft tissue sarcoma originating from fibroblasts, is a rare malignant tumor. Different cell lines of fibroblastic origin have Biochimica et Biophysica Acta 1790 (2009) 1258–1265 Abbreviations: HAS, hyaluronan synthase; HYAL, hyaluronidase; HA, hyaluronan; GAG, glycosaminoglycans; ECM, extracellular matrix; bFGF, basic fibroblast growth factor; RHAMM, Receptor for HA-Mediated Motility ⁎ Corresponding author. Tel./fax: +302810394719. E-mail address: tzanakak@med.uoc.gr (G.N. Tzanakakis). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.06.013 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen