Characterization and Classifcation of HDACs in Aging Osteoclasts

Abstract

Background: As humans age, bone mass is lost due to increased osteoclast and decreased osteoblast function. Bone cell differentiation is regulated by epigenetic changes within histones through acetylation/ de-acetylation. Histone deacetylase removes an acetyl group from a histone, repressing transcription. Several studies have demonstrated that loss of HDAC expression enhances osteoclast activity. There are 18 currently identified HDACs which are divided in 3 classes: I, II, and IV. This study aims to examine expression levels of HDAC class I and II in osteoclasts at 1 and 24-months of age. We hypothesize that osteoclasts from older mice will exhibit lower HDAC expression. This will increase our understanding of how HDAC expression changes in osteoclasts from aging mice. These changes may suggest a possible mechanism by which osteoclast activity is increased in aging osteoclasts. Methods: Bone marrow cells were flushed from femurs and tibiae of either male or female 1- or 24-month mice. BMMs were harvested and differentiated into osteoclasts at days 0, 2, and 4. They were then lysed to isolate RNA and reverse transcriptase was added to yield cDNA. Samples were subjected to qRT-PCR. Data analysis yielded expression coefficients with standard deviations. True expression was calculated for data sets and examined in graph form showing average with +/- standard deviation. Multiple group comparison ANOVA tests were run with significance set at p< 0.05. Results: For HDAC 4, expression at day 4 of differentiation of 24-month females was significantly higher than the 1-month females (p= 0.0273). For HDAC 11, expression at day 4 of differentiation of 1-month males was significantly higher than that of the 24-month males. No other group comparison yielded significance. Overall, expression was similar between age groups and sexes. Expression levels were shown to differ between days of differentiation. Conclusions: This study functions as a pilot study regarding HDAC classification and expression. To the knowledge of the author, there are no studies to date examining HDAC expression between young and advanced age subjects. The acquired data has numerous outliers which may be disguising areas of significance. The data does not support our hypothesis that expression is lower in advanced age subjects. Limitations of the study include number of test subjects, quality/quantity of cDNA, and accuracy of sample preparation. On a broader scope, the data does not depict distinct expression patterns for the varying classes of HDACs. It is suggested that each HDAC has varying expression levels at different days of differentiation and might have roles at various stages during expression. This study provides a groundwork for moving forward with more targeted studies based on conditions such as osteoporosis and periodontitis.