Study of the Kinetics of Gene Expression During Differentiation of Myeloid Cells into Osteoclasts




Alex Minikes

Introduction

Osteoclasts are multinucleated cells with a very unique ability to dissolve mineralized cells. Normally, microfractures build up through normal wear and tear of bone cells. In a healthy person, these microfractures on the surface of the bone are dissolved by osteoclasts. New, healthy bone is put in its place by antagonistically-operating cells known as osteoblasts. These two cell types form a very intricate cycle that must not be disturbed to maintain good bone health. If there is an imbalance, disorders such as osteoporosis (porous bones) or osteopetrosis (thick, brittle bones) can develop.
In order to study these disorders, laboratories would require a large amount of osteoclast cells. As there are naturally issues with using primarily derived cells for large scale research on human cells (small number available, high cost, require a human donor), it is much more common in a laboratory environment to derive these cells in vitro from their natural progenitors, myeloid cells. According to Bar-Shavit et al., very early developers of a procedure to make osteoclast cells in vitro, a 72- hour period is best to allow the growing osteoclasts to fully mature. In order to see if this long wait time is required and to shed some light on the mechanisms behind osteoclastogenesis, a myeloid cell line will be exposed to TPA and Vitamin D3, and after 0, 4, 8, and 24 hours, the expression levels of RANK, TCIRG1, MMP9, and CSF1R were measured using RT-PCR and gel electrophoresis. These four genes are very important to osteoclast function and development:

RANK: the receptor for RANKL, one of the ligands that signals to begin osteoclastogenesis.
TCIRG1: encodes the protein subunits of vacuolar-ATP synthetase, an enzyme that acidifies the area of the osteoclast that will connect to the bone surface. Deleterious mutations are associated with the development of recessively-inheritable malignant osteopetrosis (Susani, et al., 2004).
MMP9: one of the many matrix metalloproteinases working in osteoclasts. This group of enzymes has the ability to metabolize components of the bone matrix, playing a major role in the osteoclast’s overall function (Bruni-Cardoso, et al. 2010).
CSF1R: a tyrosine-kinase receptor for colony stimulating factor 1, which is necessary for osteoclast differentiation (Aoki et al., 1997).

Materials & Methods

RNA Isolation: The RNA used in this experiment was donated by Dr. Sylvia Anderson. An HL60 myeloid cell line was exposed to TPA and Vitamin D3 for up to 24 hours. At 0, 4, 8 and 24 hours after exposure, cells were removed, and RNA was isolated using a QIAGEN RNeasy© Plus Mini Kit. 10 µg/mL aliquots were prepared for each sample.
Primers: Each primer extended over multiple introns to ensure that genomic DNA was not being amplified. Primers are shown in Figure 1.
Reverse Transcriptase PCR: RT-PCR was used to measure the expression levels of each gene. The QIAGEN® One Step RT-PCR Kit was used, following the manufacturer’s protocol. Each primer pair, along with GAPDH as a housekeeping gene, was used for five reactions, labeled 0, 4, 8, 24, and a control. The annealing temperature was set at 57°C, the melting temperature at 94°C, and the reaction lasted for 50 cycles.
Gel Electrophoresis: In order to visualize gene expression, a 1% agarose gel was created. This was done using 200 mL of 1x TBE buffer, 2g of agarose, and 16.5 µL of ethidium bromide, an intercalator. 5 sample was run alongside a 100bp ladder. The results were visualized using a UV camera.
PCR Purification/Spectrophotometry: 24 hour samples were purified using a QIAGEN® QIAquick PCR Purification Kit, and the manufacturer’s protocol was followed, except that dH2O was used to elute instead of Buffer EB. A 1:10 dilution was made from each purified PCR product. 100 µL of each dilution was analyzed in the spectrophotometer.
Sequencing: Using the spectrophotometer results, 10ng of product were placed in a 96-well plate and sent out for sequencing. Results were confirmed using a BLAST search.

Results

Figure 2 shows the results of the initial gel electrophoresis. TCIRG1, MMP9, and CSF1R all plateau between the 8th and 24th hour. CSF1R shows no expression when untreated, but after treatment, begins to show low levels at hour 4 and a large increase at 8, and finally steadying out at 24 hours. RANK expression shows a large peak at the 8th hour and begins to recede after that. By hour 24, expression was still elevated above the untreated levels, but much lower than it was at hour 8. To ensure this was a true result, the RANK gene was run through RT-PCR again and run on another gel. The second trial showed a very similar result, as shown in Figure 3. In order to ensure that the targeted genes were being expressed, purified PCR product was sent out for sequencing, and upon return, the data was run through an NCBI BLAST search, confirming the genes (Figure 4).

Discussion

It would seem that expression has reached a plateau somewhere between the 8th and 24th hour. Unless the expression of another gene is playing a major role in the differentiation of osteoclasts that requires more time, it would seem that following 24 hours of exposure to TPA and Vitamin D3, a myeloid cell has sufficient time to become a fully formed osteoclast. This can be investigated further by examining the living cells at each time point. Knowing the amount of time required to derive osteoclasts from myeloid cells in a laboratory environment will save a good deal of time when experimenting with osteoclasts. If, for example, it takes 24 hours instead of 48 hours for osteoclastogenesis to complete, the experiment can continue an entire day earlier.
RANK expression shows the peak at the 8th hour. This is evidence for the claim that RANK signaling promotes osteoclastogenesis. However, the decline following the peak may be because the cell no longer requires RANK to function as much once the osteoclast has differentiated.
TCIRG1 expression should be increased. However is not exclusively in osteoclasts, because it acidifies organelles. There must be a higher acid content in osteoclasts to help its enzymatic activity.
MMP9 looks to be the last gene expressed by the developing osteoclast. This makes sense, as MMP9 is one of the enzymes responsible for breaking down the bone matrix.
CSF1R is expressed in higher amounts as osteoclastogenesis continues, suggesting a possible positive feedback mechanism. There is the possibility that the RANK signaling pathway causes the expression of CSF1R, but it is more likely caused by the TPA/Vitamin D3 treatment.
Further research is required, but for all intents and purposes, osteoclast genes such as TCIRG1, MMP9, CSF1R can be expressed to their fullest capacity by 24 hours, and RANK expression has subsided for the most part, all suggesting that osteoclasts have become fully formed by this time.

References

Aoki, H., Akiyama, H., Hosoya, H., Souda, M., Morioku, T., Marunouchi, T. 1998. Transient expression of M-CSF is important for osteoclast-like cell differentiation in a monocytic leukemia cell line. Journal of Cellular Biochemistry 64: 67-76.

Bar-Shavit, Z., Teitelbaum, S.L., Reitsma, P., Hall, A., Pegg, L.E., Trial, J., Kahn, A.J. 1983. Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3. Proc. Natl. Acad. Sci. USA 80: 5907-5911.

Bruni-Cardoso, A.; Johnson, L.C., Vessella, R.L., Peterson, T.E., Lynch, C.C. 2010. Osteoclast-Derived Matrix Metalloproteinase-9 Directly Affects Angiogenesis in the Prostate Tumor–Bone Micro-environment. Mol Cancer Res 8: 459.

Mellis, D.J., Itzstein, C., Helfrich, M.H., Crockett, J.C. 2011. The Osteoclast: Role of Key Signalling (sic) Pathways In Differentiation and in Bone Resorption. Society of Endocrinology. University of Aberdeen Medical School: Aberdeen, UK.

Shinsuke, K., Daisuke, I., Kenji, H., Wilde, J., Yuji, I., Toshio, M. 2003. Expression of RANK is dependent upon differentiation into the macrophage/osteoclast lineage: induction by 1α, 25-dihydroxyvitamin D3 and TPA in a human myelomonocytic cell line, HL60. Bone: 621-629.

Susani, L., Pangrazio, A., Sobacchi, C., Taranta, A., Mortier, G., Savarirayan, R., Villa, A., Orchard, P., Vezzoni, P., Albertini, A., Frattini, A., Pagani, F. 2004. TCIRG1-dependent recessive osteopetrosis: mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA. Hum. Mutat: 225-35.

Figures


Figure 1-Primers.


Figure 2-Initial gel results.


Figure 3-Comparison of RANK between trials.


Figure 4-Sequencing results.


Abstract

Procedures have been designed to differentiate large amounts of osteoclasts from their progenitor cells, myeloid cells in vitro. However, the nature of this in vitro derivation is not very well studied. Most procedures call for a 48- to 72-hour incubation period to derive these cells, but it is unclear why this amount of time is required. The investigation of whether osteoclasts can be derived in less time was determined by monitoring the expression levels of RANK, TCIRG1, MMP9, and CSF1R; four genes vital to osteoclast function. Differentiation was achieved in phorbol-12-myristate-13-acetate (TPA) and 1,25-dihydroxyvitamin D3, or more simply, vitamin D3. RNA was extracted from cells after 0, 4, 8, and 24 hours of exposure, and expression levels were measured using RT-PCR. Analysis using gel electrophoresis showed steady levels of expression of each gene by 24 hours, except RANK, which showed a decrease in expression between 8 and 24 hours. The evidence supports the idea that following 24 hours of treatment with TPA and Vitamin D3, the myeloid cells became cells expressing osteoclast-specific genes.

Full Paper

Acknowledgments

I would like to thank Dr. Sylvia Anderson for all of her work and her generous donation of RNA. I would also like to thank Catharina Grubaugh and Katherine Reid for all of their help and guidance, and all of the hard work they put in to assure that this experiment went as smoothly as possible. Finally, I would like to thank Dr. Berish Rubin for his guidance in designing and support to make this project possible.

This work was funded in part by a grant from Familial Dysautonomia Now Foundation (FD NOW).


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