Bone as the major structural support for the human body, must undergo a continuous cycle of remodeling to ensure the bone stays strong. Osteoclasts are cells responsible for the breakdown of bone so that it can later be rebuilt by osteoblast cells(Crockett,2011). Osteoporosis is a metabolic bone disease, which occurs when osteoclast activity is higher than the activity of osteoblasts. It affects 50% of women and 25% of men over 50 years of age (Teitlebaum, 2007). Osteoporosis is characterized by weak and porous bone, which leads to more frequent breaks, and long term disability. Osteoclasts are differentiated from myeloid precursor cells. It has been shown that differentiation can be induced in vitro by treating myeloid cells with a combination of the compounds vitamin D3 and tetradecanoyl phorbol acetate(TPA) (kido,2003). The gene that initiates this differentiation is RANK.
RANK is a gene that encodes for a receptor, that when bound to RANK ligand, initiates the signal for myeloid precursors to differentiate into mature osteoclasts (Xing, 2005). Two genes important in mature osteoclasts are MMP9 and tartrate resistant acid phosphatase(TRAP). MMP9 is a gene that encodes for a protein that mature osteoclasts secrete. The MMP9 protein is a metaloprotease that aids in the breakdown of bone matrix (Brunni-Cardoso, 2010). The biological function of the protein encoded by the TRAP gene is not well characterized, but TRAP has been widely used as a marker for the presence of mature osteoclasts. (Minkin, 1982).
The goal of the Rubin lab is to find compounds that can compromise osteoclast gene expression. In order to better develop these treatments for osteoporosis, the induction of gene expression of several osteoclast genes during differentiation needs to be better understood. In this study, the induction of three osteoclast specific genes: RANK, MMP9, and TRAP was observed over a 24 hour period via quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). In future studies this knowledge will allow us to determine the effectiveness of treatments designed to alter osteoclast gene expression.
Materials and Methods
HL-60 cells were generously donated by Dr. Sylvia Anderson. At time zero cells were treated with 10nM vitamin D3 and 50nM TPA. Untreated cells were used as a control.
At the time points of 0,0.5,1,2,4,8,12,and 24 hours after treatment, cells were transferred to a 1.5 ml Eppendorf tube, and centrifuged at 2 rpm for 6 minutes. The media was then taken off top of the cells, and cells were resuspended in 350 ul of RLT lysis buffer plus B-mercaptoethanol.
All samples were placed through a Qiagen qia-shredder column and spun at 16 x g for 2 minutes to remove genomic DNA. The RNA was then purified using Qiagen's RNeasy plus mini kit by the Qiagen Qiacube automated system, following the manufacturer's protocol.
qRT-PCR was performed for the following genes(figure 1):
RANK: RANK mRNA expression started to increase at two hours after treatment with vitamin D3 and TPA. RANK expression continued to increase up to 12 hours post treatment.
MMP9: MMP9 expression began to increase at 4 hours after treatment. MMP9 continued to increase up to 12 hours after treatment, where it levels off.(Figure 2)
TRAP" TRAP expression increase started around 8 hours after treatment, and continued to increase through the 24 hour time point (Figure 2).
It was seen that RANK, was induced first, followed by MMP9 and TRAP. There is an increase in all three genes at 12 hours after treatment.(Figure 4)
RANK expression started to increase at around 2 hours after treatment with vitamin D3 and TPA, indicating it does not take very long for D3 and TPA to begin to induce the differentiation of the myeloid cells into osteoclasts. RANK expression continued to increase up to 12 hours, after which it began to decrease. This could indicate that the cells are reaching full differentiation into osteoclasts around that time, therefore no longer needing the RANK signal.
MMP9 expression started to increase at 4 hours and continued to increase up to 12 hours, where the expression no longer changed. Further studies need to be done to determine whether or not this pattern is mimicked in the production of the MMP9 protein.
TRAP expression started to increase at 8 hours and continued to increase through 24 hours.
The expression of RANK before MMP9 and TRAP expression was expected because it is known that RANK initiates the signal for differentiation.
The increase of all three genes through the 12 hour time point suggests that, for future studies, the effectiveness of compounds designed to impact osteoclasts gene expression can be assessed following following a 12 hour incubation with vitamin D3 and TPA.
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