Upregulation of HARP during in vitro myogenesis and rat soleus muscle regeneration DANIE ` LE CARUELLE, ZOHRA MAZOUZI, IRENE HUSMANN, JEAN DELBE ´ , ARLETTE DUCHESNAY, JEAN GAUTRON, ISABELLE MARTELLY and JOSE ´ COURTY* Laboratoire de Recherche sur la Croissance Cellulaire, la Re´paration et la Re´ge´ne´ration Tissulaires FRE CNRS 2412, Universite´ Paris Val de Marne, Avenue du Ge´ne´ral de Gaulle, 94010 Cre´teil, France Received 10 March 2003; accepted in revised form 21 October 2003 Abstract Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation. Introduction In response to injury, adult skeletal muscle has the ability to regenerate. This regeneration depends on the activation of muscle precursor cells named satellite cells. These cells, located between the muscle fiber plasma- lemma and its sheath of basement membrane, either fuse directly to form multinucleated young muscle fibers (myotubes) or fuse with damage muscle fibers (Carlson, 1973). Several growth factors including fibroblast growth factors, platelet derived growth factor BB, insulin-like growth factors, transforming growth factor and more recently hepatocyte growth factor/scatter factor (Tatsumi et al., 1998) have been shown to be involved in this process (Husmann et al., 1996). In addition to these well-known molecules, several results indicate that more recently discovered, growth factor, named heparin affin regulatory peptide (HARP), might play a role in muscle regeneration. HARP is a secreted heparin-binding protein that displayed mitogenic (Courty et al., 1991) and neurite outgrowth-promoting activity (Rauvala, 1989). Recently, we showed that the biological activity of HARP is modulated by glycosaminoglycans (Vacherot et al., 1999) and that the C-terminal part of HARP is involved in the mitogenic activity (Bernard-Pierrot et al., 2001). During development, the expression of this protein, also known as heparin-binding growth associated molecule (HB- GAM); (Merenmies and Rauvala, 1990) or pleiotrophin (PTN) ; (Li et al., 1990) is tighly regulated with strong expression in the nervous system just after birth (Meren- mies and Rauvala, 1990). More recent studies suggest that HARP is involved in stroma-epithelium interactions (Vanderwinden et al., 1992; Mitsiadis et al., 1995). Despite numerous studies, very little is really known about the precise physiological role of this molecule. However, several reports would support the idea that HARP plays a role in muscle regeneration. Previous investigations have demonstrated that HARP displayed neurotrophic activity (Li et al., 1992a; Raulo et al., 1992; Rauvala et al., 1994; Kinnunen et al., 1996), and induces clustering of acetylcholine receptors (AchR) at neuromuscular junctions (Peng et al., 1995). During embryogenesis, immunocytological analysis indicated that HARP was expressed in close proximity to AchR clusters on the muscle cell surface at E16 during rat development (Szabat and Rauvala, 1996). Since skeletal muscle regeneration recapitulates events in muscle development, we postulated that HARP would also be involved in muscle fiber reconstruction following myol- ysis in regenerating muscle. Therefore, in this study, we investigated the mRNA and protein expression of HARP during muscle regen- eration using a crush-induced model of muscle regener- ation in rat and rat satellite cells in primary cultures. The experiments reported herein demonstrated that HARP is expressed in myogenic precursor cells and is up regulated after rat muscle injury during muscle fiber reconstruction. *To whom correspondence should be addressed: Tel.: +33-1-45-17- 17-97; Fax: +33-1-45-17-18-16; E-mail: courty@univ-paris12.fr Journal of Muscle Research and Cell Motility 25: 45–53, 2004. 45 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.