ORP-3 - a novel gene with a potential role in the regulation of oxysterol-induced apoptosis of hemopoietic progenitors

We have compared gene expression between purified CD34+ and CD34+ cells from bone marrow and cord blood using Differential Display RT-PCR. One of the differentially expressed genes identified was identical to an EST with homology to Oxysterol Binding Protein. This EST has been identified by others as one of a family of sequences bearing the OSBP family signature, and has been termed OSBP-Related Protein 3, or ORP-3. We have determined the full coding and genomic sequence of ORP-3, which shows it to be a protein of 887 amino acids coded by 23 exons spanning 185 kb of chromosome 7pl5-21. Using Real Time PCR we have examined expression of ORP-3 in haemopoietic cells. It is > 3 fold more highly expressed in CD34+ than CD34 cells, and is more highly expressed in CD34+CD38 than CD34+CD38+ cells. It is also 1.5 fold more highly expressed in CD34+ cells derived from adult bone marrow than in equivalent cells from cord blood. In addition, ORP-3 is down regulated in CD34+ cells after one week culture in media containing SCF, IL3, IL6 and GM-CSF. In view of the homology between ORP-3 and OSBP, a known mediator of oxysterol (OS) actions, we investigated the effects of OS on haemopoietic progenitors and cell lines. OS are a group of hydroxylated derivatives of cholesterol with a wide range of known functions, both in vitro and in vivo, including the ability to induce apoptosis of a number of cell types. Growth of both CFU-GM and of the myeloid cell line HL60 is inhibited by the OS 25 hydroxy cholesterol (25-OH-C), 7κ cholesterol and 7β cholesterol at levels at or near the upper end of physiological levels measured in serum. 25-OH-C is the most potent, causing significant inhibition at levels of 600 nmol/L (cf 100-200 nmol/L in serum, 300400 nmol/L in bone marrow aspirates), and causing > 90% inhibition of growth at concentrations above 2000 nmol/L. Growth inhibition is caused by initiation of apoptosis, as measured by Annexin V/PI flow cytometry. Apoptosis of HL60 is partially abrogated in a subclone which constitutively over expresses BCL-2. These results suggest that OS are capable of causing apoptosis of haemopoietic progenitors at concentrations that may be encountered in a physiological setting. These observations, together with the identification of a differentially expressed gene bearing the OS binding sequence motif, suggest a possible role for ORP-3 and OS in the regulation of apoptosis in haemopoietic cells.