Cells of bone Within the skeleton there are 4 basic types of bone cells; osteoclasts, osteoblast, bone lining cells and osteocytes. Osteoclasts (Figure 1.2A) are multinucleated cells derived from monocytes that are ultimately from HSCs first discovered in 1873 by Kolliker. Osteoclast maturation requires the presence of receptor activator of nuclear factor κβ ligand (RANKL) and Macrophage colony-stimulating factor (M-CSF) (, , ). Overexpression of osteoprotegerin (OPG), soluble receptor that competes with RANK for RANKL, reduces osteoclastogenesis in mice. On the other hand, lack of OPG has been shown to accelerate osteoclastogenesis and OPG Ko mice (OPG−/− mice) develop severe osteoporosis (). Osteoclasts form at skeletal sites, in which stromal cells and osteoblasts express RANKL and M-CSF and it is the interaction of these ligands with their receptors on monocyte/macrophage cells that induce commitment to the osteoclast phenotype. Osteoclasts have a critical role in the regulation of bone formation and regulation of bone mass, in which mature and active osteoclasts are responsible for resorbing mineralized bone matrix and breaking up the organic bone (90% collagen). Differentiated osteoclasts express αvβ3 integrin, which binds to bone matrix via proteins leading to the formation of sealed pocket for the secretion of cathepsin K, the major protease involved in the degradation of type I collagen (, ). After attachment, osteoclasts exhibit a highly polarized cytoplasmic organization. The
Zhang et al (2003) used immunohistochemistry technology to identify the HSC niche. It was found that LT-HSCs reside in the osteoblastic niche (Figure 1.5) and were only attached to the N-cadherin+ osteoblasts (). Xie et al (2009) developed new ex vivo imaging technology. Green fluorescent protein expressing (GFP+) HSCs homed through the vascular niche to the osteoblastic niche and attached to N-cadherin+ osteoblast cells in irradiated mice (). Lo Celso et al (2009) used a combination of high resolution confocal microscopy and two-photon video imaging technology that allowed observation of homing of individual HSC in calvarial bone marrow of living mice in real time. HSCs have been observed closer to bone and osteoblasts in irradiated and recipient mice (). It is only by using modern microscope that has
N-cadherin Structure of N-cadherin cell-cell adhesion molecule N-cadherin is a member of a Ca2+-dependent cell-cell adhesion receptor family known as cadherin 2, containing extracellular domain, transmembrane domain and cytoplasmic domain (Figure 1.8). The extracellular domain of N-cadherin contains five extracellular repeats of ~110 amino acids each. The cytoplasmic domain directly binds to p120 catenin, β-catenin and α-catenin cleaved by γ-secretase protein and translocate the carboxy-terminal fragment of N-cadherin with β-catenin to the nucleus (Figure 1.9). Cleaved N-cadherin mediates signalling pathway following cell-cell adhesion (, ). N-cadherin expression in bone cells Within the skeleton there are four basic types of bone cells; osteoclasts, osteoblast, bone lining cells and osteocytes. Mbalaviele et al (1995) showed that E-cadherin was expressed by osteoclasts, but no expression of N-cadherin was detected in these cells (). In osteoblasts, Ferrari et al (2000) demonstrated that N-cadherin was expressed and this was increased during osteoblastogenesis. This study suggested a role for in the late stage of osteoblast differentiation (). Kawaguchi et al (2001) also showed that N-cadherin was expressed by osteoblasts and this expression was increased during osteoblastogenesis (). Marie (2002) demonstrated that the marked increases in
Table 2.2: Primers designed and used to amplify target genes Gene Accession number Forward 5`- 3` Reverse 5`- 3` Expected size N-cad NM_007664.4 CAGGTAGCTGTAAACCTGAG CCATTCAGGGCATTTGGATC 560 bp GAPDH NM_008084.2 TTGTCAGCAATGCATCCTGC GCTTCACCACCTTCTTGATG 354 bp
Zeiss 510 multiphoton laser scanning microscope. To block N-cadherin, myeloma cells, 5T33MM and 5TGM1, were incubated with anti-N-cadherin 40 µg/ml antibody (Sigma, UK) for 30 minutes at 37 °C (). Mouse IgG1 antibody (Dako, Denmark) was used as a negative control. Cells were mixed every 5 minutes. After 30 minutes, myeloma cells were centrifuged at 1000 rpm for 4 minutes, washed twice with PBS to remove the antibody and re-suspended in 1 ml PBS. Cells were counting (see section 2.2.1) to determine the percentage of dead and live cells after blocking. Inoculation of MM cells into mice 4-6 week old male C57BL/KaLwRij mice were used. Mice were handled carefully. Mice were put in rodent restrainer, a plastic that hold mice body and keep their tails free for induction, and their tails were anesthetized using EMLATM cream 5% (AstraZeneca, UK). After 5 minutes, 200 µl of MM cell lines: 5T33MM and 5TGM1 (2x106 cells/200 µl) were inoculated into each mouse via their tail veins. Mice were harvested after various times according to the aims of each study.
osteoblasts. Cells were incubated in different formulations and regimens of osteogenic medium as described in the following table (Table 3.1). Table 3.1: Different formulation and regimens of osteogenic medium Series 1st formulation and regimens (Days 0-14) 2nd formulation and regimens (Days 15-28) 1 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid. As 1st formulation 2 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid. α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid + 10-8 M of dexamethasone. 3 α-MEM medium + 10% FCS + 10 mM of β-glycerophosphate + 50 µg/ml of ascorbic acid + 10-8 M of dexamethasone. As 1st formulation 4 α-MEM medium + 10% FCS, 10 mM of β-glycerophosphate, 50 µg/ml of ascorbic acid + H2O. As 1st formulation Molecular biology RNA isolation and quantitation For osteoblasts, nearly confluent T25 flasks were washed twice with PBS and total RNA was extracted from primary osteoblasts at different stages of development using TriZol. For myeloma cell lines 5T33MM and 5TGM1 cells around 1x106 cells were washed twice with PBS and total RNA was extracted using TriZol. Total RNA was precipitated using isopropanol and re-suspended in RNase and DNase free H2O as described in section 2.2.5.1. NanoDrop ND 1000 Spectrophotometer (NanoDrop
Immuno-localization of N-cadherin protein in myeloma cell lines, 5T33MM and 5TGM1 A rabbit monoclonal anti-mouse-N-cadherin antibody (Millipore, UK) and an isotype control, normal rabbit IgG antibody (R&D systems, UK) were used to determine possible localization of N-cadherin in 5T33MM and 5TGM1 myeloma cells. Northern light anti-rabbit IgG (NL637) antibody, (secondary antibody for immunofluorescence) or biotinylated anti-rabbit IgG antibody, (secondary antibody for immunocytochemistry) was used for detection of primary antibody. Figure 3.27 panel A shows positive staining (red fluorescence) of N-cadherin in 5T33MM cells and no staining with isotype control (Figure 3.27 panel B). Data determined that the
Discussion In this chapter I developed and optimized a co-culture system to study the interactions of MM cells with the osteoblasts via adhesion molecule, N-cadherin, in vitro. 5T33MM cells and 5TGM1 cells were cultured separately in vitro as described in methods. Mouse primary osteoblasts were obtained from calvarial bones of 2-4 day old C57Bl/6J mice, (Harlan, UK) and cultured in vitro as described in methods. Osteoblasts were differentiated into mature functional osteoblasts using an in vitro model of