Jillian C. Meyer
Human bones are metabolically active organs that rely on the balanced function of osteoclasts and osteoblasts to maintain their structural integrity. Normal wear and tear on the body lead to the development of microfractures that could result in serious damage if left unrepaired (Eljiro et al. 2012). The body’s response to this is the osteoclast-mediated removal of microfractures which is followed by the rebuilding of bones by osteoblasts. Osteoclasts are recruited to damaged sites and secrete protons and proteases to break down the bone, forming resorption pits. Osteoblasts are attracted to the resorption pits where they secrete the appropriate ions and collagenous fibers to rebuild the bone matrix (Jimi et al. 2012). An imbalance in this process can result in diseases like osteoporosis and osteopetrosis.
Osteoporosis is a metabolic bone disorder that results in weak brittle bones when osteoclast function greatly outweighs osteoblast function and leads to diminished bone density. It impacts 200 million people worldwide and in 2018 medical costs associated with this disorder were $13.8 billion in the US alone (Akkawi et al. 2018). However, there is a huge geographic discrepancy in incidence of osteoporotic-related fracture, even in apparently related individuals, indicating that there are environmental factors that impact the severity of the disease (Ballane et al 2013). This geographic variability may suggest that the development osteoporosis can be impacted by natural compounds found in the diet of people in different regions.
Osteopetrosis is a genetic disorder caused by mutations in osteoclast-specific genes that drastically compromises osteoclast function so that bone formation occurs at a much higher rate than bone resorption, resulting in abnormally thick bones (Calhoub et al. 2003). The OSTM1 (Osteoclastogenesis Associated Transmembrane Protein 1) gene codes for a transmembrane protein that plays a role in osteoclast differentiation, intracellular lysosomal trafficking, and exocytosis of ions and enzymes. Mutations in this gene cause severe autosomal recessive Osteopetrosis (Calhoub et al 2003). Because changes in the OSTM1 protein have been associated with impaired osteoclast function, the ability of natural compounds to modulate the alternative splicing of the OSTM1 transcript was examined.
Materials and Methods
THP1 and HL60 cells were differentiated into osteoclasts following the protocol established by Kido et al.The cells were treated with 1 of 12 different natural compounds for 48 hours. The cells were then lysed and RNA was purified using the QIAGEN RNeasy plus mini kit in the QIAcube. Reverse Transcription-PCR was performed with primers specific to the region OSTM1 transcript with known splice variants. And finally, Quantitative RT-PCR was performed with primers specific to each transcript variant.
Figure 1 represents the results of an RT-PCR performed on RNAs isolated from osteoclast models that were treated with 12 different natural compounds. The purpose of this experiment was to screen known splice altering agents for their ability to change the splicing of the OSTM1 transcript. Using primers located in exons 4 and 6, the expected product size was 292 base pairs, but there was an unexpected larger PCR product generated using RNA from cells treated with compounds 4 and 12. The products were gel purified and sequenced. Sequence analysis of the 292 base pair piece revealed the presence normal OSTM1 transcript, and sequencing of the 344 base pair piece revealed the presence of a novel 52 base pair insertion between exons 4 and 5 (Figure 2). To quantitatively assess the changes in expression of the cryptic exon-containing transcript in cells treated with compound 12, primers were designed to only amplify transcripts that had the 52 base pair insertion and Quantitative RT-PCR was performed. The results showed significant fold increases in the amount of OSTM1 transcripts that include the 52 base pair cryptic exon in treated cells as compared to untreated cells when adjusted to the Beta-2 Microglobulin housekeeping gene (Figure 3). Sequence analysis of the product of the QRT-PCR confirmed that only the transcripts containing the 52 base pair insertion had been amplified. The sequence of the +52 OSTM1 transcript was used to generate the predicted amino acid sequence. Because of the insertion of the novel exon in the RNA transcript, there would be a significant change in the reading frame, resulting in a translation that produces a truncated protein that is missing the transmembrane domain (Figure 4).
The RT-PCR analysis (Fig. 1) revealed the ability of compounds 4 and 12 to facilitate the inclusion of a 52 base pair cryptic exon in the OSTM1 transcript (Fig. 2). In cells treated with compound 12, Quantitative RT-PCRs confirmed the increased production of the transcript containing this novel exon, showing a 7.21 and 3.86 fold increase in the amount of this transcript in HL60 and THP1 cells, respectively (Fig. 3). The inclusion of these 52 base pairs results in a significant shift in the reading frame of the RNA. Therefore the insertion of the cryptic exon will result in the generation of a truncated protein that lacks a transmembrane domain (Fig. 4). The possible therapeutic benefit of compound 12 is supported by the observation that individuals who produce a transcript that contains this same 52 bp insertion (as a result of a mutation at the +5 position of intron 5 of the OSTM1 transcript) develop severe osteopetrosis because of osteoclast dysfunction (Calhoub et al 2003).
While the complete loss of functional OSTM1 causes disease in people with osteopetrosis, interfering with at least some OSTM1 in people who are at risk of osteoporosis can serve as a therapy. Compounds 4 and 12 have the potential to be used as therapeutic agents because of their ability to induce the inclusion of the cryptic exon. By interfering with OSTM1 in this way, the treatments disturb osteoclast function. This would be favorable to osteoporosis patients because inhibiting osteoclasts can slow down the rate of bone resorption to a point that is more comparable to the rate of bone formation.
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