Leukemia Research 30 (2006) 1241–1248 Expression and functional significance of osteocalcin splicing in disease progression of hematological malignancies Peter Wihlidal a , Franz Varga b , Michael Pfeilst ¨ ocker a,c , Heidrun Karlic a,∗ a Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria b Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Medical Department, Hanusch Hospital, Vienna, Austria c 3rd Medical Department, Hanusch Hospital, Vienna, Austria Received 19 October 2005; received in revised form 19 October 2005; accepted 18 November 2005 Available online 4 January 2006 Abstract The aim of this study was to investigate the expression of osteocalcin (OCN) splicing variants in hematological malignancies. We analysed bone marrow obtained from two patients with chronic myeloid leukemia (CML), seven patients with other myeloproliferative diseases (MPD) and four patients with acute myeloid leukemia (AML). RT-PCR analyses were performed in order to assess and quantify spliced (OCNs) and unspliced (OCNu) mRNA, the associated transcription factors (AML1 and AML3) as well as c-KIT which is a marker for activated stem cells. Our data indicate that OCNs mRNA and OCN protein is expressed in c-KIT positive neoplastic stem cells in hematological malignancies. © 2005 Elsevier Ltd. All rights reserved. Keywords: Osteocalcin, c-KIT; Hematological malignancy; Runt-domain transcription factors 1. Introduction OCN has originally been defined as marker for mature osteoblasts – cells involved in bone formation. The protein itself is a small (MW 6.9 kDa) molecule and represents the most abundant non-collagenous protein in bone. It has - helical domains forming a tightly packed charged molecule that coordinates Ca 2+ at the surface of the hydroxyapatite-like lattice of bone mineral crystals. The fact that no OCN defi- cient persons have been found to date suggests a crucial role of OCN for human life and development, but the dimension of its action has yet to be completely defined. OCN may function as a matrix signal in the recruitment and differentiation of bone-resorbing cells [1] and studies of murine long-term bone marrow cultures with rat bone OCN indicate that OCN promotes osteoclastic differentiation of a ∗ Corresponding author at: Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, H. Collinstr. 30, A-1140 Vienna, Austria. Tel.: +43 69919241457; fax: +431 9143214. E-mail address: heidrun.karlic@chello.at (H. Karlic). stroma-free subpopulation of hematopoietic progenitors in the presence of GM-CSF [1]. These bone-resorbing cells are derived from hematopoietic stem cells, an indication that OCN may influence the differentiation of this stem cell pool as well [2]. Recently, OCN has been shown to enhance bone remodelling, a process depending on the number and activity of both osteoclasts and osteoblasts [3]. In addition to bone-related tissues, OCN is synthesised by vascular smooth muscle cells [4] and its mRNA is also expressed in megakaryocytes and peripheral blood platelets, which possibly contribute to the OCN levels in blood and the regulation of bone turnover [5]. Recent data demonstrated that activated HSC (hematopoi- etic stem cells) from hematologic malignancies, as charac- terised by KIT, also express the osteocalcin protein (OCN) [6]. There is a species dependent difference in the regulation of this protein between human, rats and mice: in human and rats OCN expression is strongly increased by 1,25-dihydroxy- Vitamin D3 while in mice the transcription is attenuated by this vitamin [7]. Thyroid hormones, however, increase the 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.11.008