CD is also known as choline transporter like
CD92 is also known as choline transporter-like protein 1 (CTL1) and its main function is to transport choline, which is then incorporated into phosphatidylcholine (PC), across the cell membrane (Michel and Bakovic, 2009; Nakamura et al., 2010). In vivo, PC is found in the lipid fraction in the calcification front during both intramembranous and endochondral bone formation and the addition of PC to bone graft material has been shown to increase the osteoinductivity of the material and ALP activity in the surrounding tissue (Han et al., 2003). Albeit speculative, it is therefore possible that the strong upregulation of CD92 in osteogenically differentiated cells, demonstrated in the present study, is related to the increased synthesis of PC during osteogenic differentiation. Furthermore, the gene expression of CD92 is upregulated in response to dexamethasone in the context of choline uptake in human lung adenocarcinoma (Nakamura et al., 2010). This is in line with the present results, as dexamethasone was the glucocorticoid steroid used in this study to induce differentiation. The present results extend previous observations, demonstrating the upregulation of CD92 in osteogenically differentiated hBMSCs at protein level. A significant increase in CD92 expression was also seen in adipogenically differentiated hBMSCs compared with undifferentiated hBMSCs, a difference that was most pronounced during the initial stages of differentiation. There appears to be a lack of knowledge in the literature regarding the expression of CD92 in relation to both osteogenically and adipogenically differentiated win 55 212-2 and further studies of the mechanism via which CD92 affects the differentiation process are needed.
In addition to the identified membrane-bound proteins, the subcellular fractionation further isolated proteins that were not annotated as membrane bound. The intracellular protein, CRYaB, is a small heat shock protein belonging to the alpha family, which is composed of two gene products, alpha-A (acidic) and alpha-B (basic) proteins. Alpha-A is associated with the vertebrate eye lens, while alpha-B is expressed in many tissues and organs. The gene expression of CRYaB has been shown to be significantly regulated in the early stages of the chondrogenic differentiation of the ATDC-5 chondroprogenitor cell line (Chen et al., 2005). Further, Lambrecht et al. (2009) have reported a reduction in CRYaB expression in dedifferentiating chondrocytes, thus indicating that CRYaB perhaps plays a role in the chondrogenic differentiation process. However, the present results indicate an increase in the protein expression of CRYaB during the osteogenic differentiation of hBMSCs. The data indicate an osteogenic lineage specificity of this marker, since the present results did not reveal any upregulation of CRYaB during early adipogenic and chondrogenic differentiation. The elevated expression of CRYaB has previously been associated with many neurological diseases (Fort and Lampi, 2011; Klemenz et al., 1991). However, relatively little is known about the role of CRYaB in osteogenic differentiation. Furushima et al. (2002) previously reported that CRYaB gene expression is associated with bone metabolism, which has also been demonstrated in a gene microarray study of hBMSCs during differentiation into osteoblasts (Kulterer et al., 2007). Apart from this, no specific role for CRYaB in osteogenic differentiation is known. The role and the underlying mechanisms of this protein in the osteogenic differentiation process therefore require further studies.
Acknowledgments The support of the Swedish Research Council (VR grants K2009-52X-09495-22-3 and 621-2011-6037), the BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy and the Västra Götaland Region, the Inga-Britt and Arne Lundberg Research Foundation and Handlanden Hjalmar Svensson Forskningsfond is gratefully acknowledged. The authors are grateful to Katarina Junevik for assistance during flow cytometry.