Journal of Cellular Biochemistry 93:1231–1241 (2004) Carboxy-Terminal Fragment of Osteogenic Growth Peptide Regulates Myeloid Differentiation Through RhoA Letizia Mattii, 1 Rita Fazzi, 2 Stefania Moscato, 1 Cristina Segnani, 1 Simone Pacini, 2 Sara Galimberti, 2 Delfo D’Alessandro, 1 Nunzia Bernardini, 1 and Mario Petrini 2 * 1 Department of Human Morphology and Applied Biology, Section of Histology and General Embryology, University of Pisa, Via Roma, Pisa, Italy 2 Department of Oncology, Transplant and Advanced Technologies in Medicine, Hematology Division, University of Pisa, Via Roma, Pisa, Italy Abstract The carboxy-terminal fragment of osteogenic growth peptide, OGP(10 – 14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10–14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10–14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10 – 14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10 – 14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out. J. Cell. Biochem. 93: 1231–1241, 2004. ß 2004 Wiley-Liss, Inc. Key words: OGP(10–14); GM-CSF; RhoA; HL60; cell differentiation Osteogenic growth peptide (OGP) is a 14-mer peptide, which exerts regulatory effects on bone and bone marrow. This highly-conserved, H4 histone-related peptide was first isolated about 10 years ago in blood during osteogenic remo- deling of post-ablation marrow regeneration and it is found in great abundance in the blood, usually conjugated to a binding protein (OGPBP) [Bab et al., 1992]. OGP administration in vivo enhances bone formation and increases trabecular bone mass. In vitro, OGP stimulates proliferation and alkaline phosphatase activity in osteogenic cell lines and exerts mitogenic effects on fibroblasts [Greenberg et al., 1993, 1995]. Moreover, OGP is able to induce in vivo a balanced increase in white blood cell (WBC) count and overall bone marrow cellularity in mice [Gurevitch et al., 1996]. OGP-derived C-terminal pentapeptide, OGP(10–14), is generated by proteolytic cleav- age of the full-length OGP upon dissociation from OGPBP [Bab et al., 1999]. Synthetic OGP(10–14) retains the OGP effect on cell proliferation, thus suggesting a specific role of the C-terminal region in binding to the putative OGP receptor [Greenberg et al., 1993]. The mechanism of action of the pentapeptide is not fully known; however, it has been recently shown that OGP(10–14) activates the mito- genic Gi protein MAP kinase-signaling cascade in osteogenic cells. This indirectly suggests the presence of a membrane receptor [Gabarin et al., 2001]. We have previously shown that OGP(10–14) is able to enhance bone marrow recovery after cyclophosphamide administration in mice [Fazzi et al., 2002a]. Moreover, OGP(10–14) ß 2004 Wiley-Liss, Inc. *Correspondence to: Mario Petrini, Dipartimento di Onco- logia, dei Trapianti e delle Nuove Tecnologie in Medicina, Sezione di Ematologia, Facolta ` di Medicina e Chirurgia, Universita ` degli Studi di Pisa, Via Roma 67, 56126 Pisa, Italy. E-mail: m.petrini@do.med.unipi.it Received 3 May 2004; Accepted 25 June 2004 DOI 10.1002/jcb.20248