Bone remodeling is a continual process necessary to maintain healthy bones and to adapt to different stresses of the body. This process is balanced by osteoclasts, cells that break down bone, and osteoblasts, cells that deposit new bone material. However, disequilibrium between osteoclast and osteoblast functions results in bone conditions, such as osteoporosis and osteopetrosis, which can affect an individual’s ability to endure daily activities.
The fundamental cause of osteoporosis is the disequilibrium between osteoclasts and osteoblasts, wherein osteoclasts break down bone faster than osteoblasts are able to rebuild the bone. The result of this disequilibrium is very porous and fragile bones that are susceptible to fractures. Vitamin D3, in conjunction with TPA, has been used for in vitro studies to induce osteoclast differentiation from HL60 progenitor cells as depicted in Figure 1. However, the individual effects of vitamin D3 and TPA themselves have yet to be defined.
This study seeks to capture the varying effects that vitamin D3 and TPA can have on individual genes necessary for osteoclast function and/or development. Comparing gene expression between reagents through gel electrophoresis may be indicative of each reagent's abiility to influence osteoclast development. Exposure of HL60 cells to each agent alone and analyzing the resultant RNA products show whether various differentiation agents, besides the standard mixture of vitamin D3 and TPA can induce maximal gene expression.
Material and Methods
HL60 progenitor cells were kindly provided by Dr. Berish Rubin of Fordham University's Department of Biological Sciences. HL60 cells were first treated with vitamin D3 alone, TPA alone and vitamin D3/TPA. RNeasy Plus Mini Kit (Qiagen) was used to obtain purified Total RNA from the treated HL60 cells.
OneStep RT-PCR Kit (Qiagen) was then used to amplify the obtained Total RNA targeted for genes using specific primers found in Figure 2. The amplification of these genes were then analyzed on a 1% agarose gel to determine differences in quantity between different exposures on a single gene.
To confirm the genes seen on the gel, QIAquick PCR Purification Kit (Qiagen) was used to purify the RT-PCR products. The purified products were sent out for sequencing to GeneWiz, Inc. and the sequences were then analyzed using the Basic Local Alignment Search Tool (BLAST) on the NCBI website.
Data from the resultant gels reveals clear amplification of all four genes upon exposure to vitamin D3, TPA and/or the standard mixture; comparisons of the strength of the bands are indicative of how much gene products were obtained. Treatments of vitamin D3 or TPA work effectively in enhancing RANK, TCIRG1 and CSF1R gene expression comparable to the combined effect of vitamin D3/TPA that is typically used in the lab setting (Figure 3). Conversely, however, MMP9 does not show any bands upon treatment of vitamin D3 or TPA, and only shows a strong band upon exposure to the mixture of both vitamin D3 and TPA indicating that gene expression is dependent on both reagents. It is worthy to note, also, that each gene is amply produced upon exposure to vitamin D3/TPA, as expected.
The standard mixture of 10nm vitamin D3 combined with 50 nm TPA has been commonly used in labs to induce differentiation of HL60 myeloid progenitor cells to functional osteoclast cells. Using specific primers, I targeted and isolated four genes needed for osteoclast differentiation or function: RANK, MMP-9, TCIRG1 and CSF1R. Based on analyses of the presence and expression of these specific genes, the ability of certain agents to produce maximal gene expression may also be indicative of their ability to induce differentiation.
Through this study, independent treatments of vitamin D3 or TPA show significant amounts of gene expression from RANK, TCIRG1 and CSF1R comparable to the mixture of vitamin D3/TPA (Figure 3). Analysis of MMP9 expression, however, reveals an aberration, wherein vitamin D3 or TPA independently do not produce any significant gene products, but together produce a robust band of gene product. The strong presence of a band for gene expression upon exposure of HL60 cells to vitamin D3/TPA indicates that sufficient gene expression depends on the availability of both reagents. The presence of the four genes analyzed in this study, though, does not necessarily qualify total osteoclast differentiation, but does demonstrate the ability, or inability, of certain compounds to enhance gene expression essential to osteoclast function. By determining the effects of vitamin D3 and TPA on gene expression, further studies can be taken on the individual effects of vitamin D3 and TPA on the overall differentiation of HL60 cells, and possibly even the mechanism through which these agents work to account for the variability between these gene products.
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