Osteoporosis is an age-related bone disease and has a severe impact on public health and economy worldwide, due to fragility fracture [
Calcium plays an important role in the development of osteoclasts with the involvement of calcium-binding proteins, such as annexins, S100 proteins, and proteins involved with calcium signaling, such as TMEM64. Three calcium-binding/associated proteins, annexin A1, S100A4, and TMEM64, are expressed in monocyte cells (Gene Expression Atlas:
The transmembrane protein, TMEM64, has been reported to interact with and modulate the activity of the protein, sarcoplasmic endoplasmic reticulum Ca2+ ATPase 2 (SERCA2), thereby enhancing RANKL-induced internal calcium oscillations that are a part of the pathway of osteoclast generation [
Thus, the aim of this study was to find out whether the levels of mRNAs for annexin A1, S100A4, and TMEM64 were significantly changed in the PBMCs of participants with osteopenia or with osteoporosis compared to nonosteoporotic controls.
The participants in this study were men and women volunteers living in Merseyside, England, in the area covered by the Royal Liverpool and Broadgreen University Hospital NHS Trust. Participants were recruited from referrals to the Nuclear Medicine Department at the Royal Liverpool University Hospital. Diagnoses of osteopenia and osteoporosis were made in accordance with the WHO guidelines. This study conformed to the principles of the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was carried out under ethical approval from the England Health Research Authority National Research Ethics Service Committee, East of England-Essex (REC reference 15/EE/0051) Ethics Committees. Informed consent was obtained from all participants prior to sample collection. Details of the participants have been described elsewhere [
Peripheral blood mononuclear cells (PBMCs) were isolated from 25 mL blood samples and stored at -80°C, as described previously [
Total RNA from human PBMCs was extracted using a combination of TRIzol reagent and PureLink RNA mini kit (Thermo Fisher, UK), according to the manufacturer’s recommendations as described previously [
Reverse transcription reactions were carried out using the RT-First Strand kit, RT-qPCR Primer Assays (Qiagen, UK), according to the manufacturer’s recommendations and as described previously [
Qiagen primers used to amplify specific mRNAs by RT-qPCR.
mRNA | Gene symbol | Accession number | Primer sequence | Qiagen catalogue |
---|---|---|---|---|
Annexin A1 | ANXA1 | NM_000700.2 | 5 |
PPH02882E |
S100 calcium-binding protein A4 | S100A4 | NM_002961 | 5 |
PPH01313E |
Transmembrane protein 64 | TMEM64 | NM_001008495.3 | 5 |
PPH10400A |
Glyceraldehyde-3-phosphate dehydrogenase | GAPDH | NM_001256799 | 5 |
PPH00150F |
Statistical analysis between two groups used Student’s
The relative levels of mRNAs of calcium-binding/associated proteins, annexin A1, S100A4, and TMEM64, in isolated PBMC preparations were each significantly different between nonosteoporosis control, osteopenia, or osteoporosis groups of participants (one-way ANOVA, annexin A1 mRNA, Figure
Relative levels of mRNAs in peripheral blood mononuclear cells associated with osteoporosis. Levels of mRNAs encoding annexin A1 (a, d, g), TMEM64 (b, e, h), and S100A4 (c, f, i) proteins were determined in the peripheral blood mononuclear cell preparations of nonosteoporotic control subjects and patients suffering from osteopenia or osteoporosis shown as box and whisker plots. The levels are shown for all participants (a, b, c), for participants who were not receiving treatment for osteoporosis (d, e, f), and for participants who were not suffering from other disorders (g, h, i). On each box and whisker plot, the black diamond shows the median value, the cross shows the mean value, and white and black circles denote outliers of 1.5 times and 3 times the interquartile range, respectively.
Summary of statistical analysis on all participants.
mRNA | Compared sample groups ( |
95% confidence interval | |
---|---|---|---|
Annexin A1 | Nonosteoporotic control ( |
0.208 to 0.476 | <0.0001 |
Nonosteoporotic control ( |
0.262 to 0.566 | <0.0001 | |
Osteoporosis ( |
-0.192 to 0.049 | 0.242 | |
TMEM64 | Nonosteoporotic control ( |
-0.004 to 0.0729 | 0.0797 |
Nonosteoporotic control ( |
0.019 to 0.106 | 0.0052 | |
Osteoporosis ( |
-0.062 to 0.006 | 0.1 | |
S100A4 | Nonosteoporotic control ( |
-0.302 to 0.47 | 0.666 |
Nonosteoporotic control ( |
0.053 to 0.925 | 0.0286 | |
Osteoporosis ( |
-0.751 to -0.057 | 0.0229 |
For annexin A1 or TMEM64, the significant associations of reduced levels of mRNA with osteopenia and osteoporosis were not affected when participants receiving treatment for osteoporosis were excluded from the analysis (annexin A1 mRNA, one-way ANOVA,
Summary of statistical analysis on participants with no osteoporosis treatment.
mRNA | Compared sample groups ( |
95% confidence interval | |
---|---|---|---|
Annexin A1 | Nonosteoporotic control ( |
0.18 to 0.478 | <0.0001 |
Nonosteoporotic control ( |
0.232 to 0.603 | <0.0001 | |
Osteoporosis ( |
-0.248 to 0.071 | 0.272 | |
TMEM64 | Nonosteoporotic control ( |
-0.00096 to 0.0784 | 0.056 |
Nonosteoporotic control ( |
0.011 to 0.109 | 0.0168 | |
Osteoporosis ( |
-0.063 to 0.02 | 0.305 | |
S100A4 | Nonosteoporotic control ( |
-0.273 to 0.556 | 0.498 |
Nonosteoporotic control ( |
-0.124 to 0.906 | 0.134 | |
Osteoporosis ( |
-0.694 to 0.195 | 0.267 |
Summary of statistical analysis on participants without nonosteoporotic medical disorders.
mRNA | Compared sample groups ( |
95% confidence interval | |
---|---|---|---|
Annexin A1 | Nonosteoporotic control ( |
0.176 to 0.477 | <0.0001 |
Nonosteoporotic control ( |
0.202 to 0.557 | <0.0001 | |
Osteoporosis ( |
-0.206 to 0.1 | 0.491 | |
TMEM64 | Nonosteoporotic control ( |
-0.013 to 0.075 | 0.166 |
Nonosteoporotic control ( |
0.013 to 0.117 | 0.016 | |
Osteoporosis ( |
-0.078 to 0.01 | 0.132 | |
S100A4 | Nonosteoporotic control ( |
-0.224 to 0.54 | 0.413 |
Nonosteoporotic control ( |
-0.077 to 0.828 | 0.102 | |
Osteoporosis ( |
-0.608 to 0.172 | 0.269 |
However, for S100A4, the significant association between the reduction in S100A4 mRNA and osteoporosis was lost when participants either receiving treatment for osteoporosis or participants with nonosteoporotic medical disorders were excluded from the analysis (osteoporosis treatment removed, one-way ANOVA,
To find out whether the same results were obtained in female osteoporosis patients aged over 50 years specifically, the analyses were confined to the 63 female subjects over 50 years of age. All three mRNAs showed the same trend of a decrease in the mRNA level in the order: nonosteoporotic control, osteopenia, and osteoporosis observed when all patients were included. Annexin A1 and TMEM64 mRNAs showed a significant reduction between nonosteoporotic, osteopenia, and osteoporosis participants (annexin A1 mRNA, one-way ANOVA,
Relative levels of mRNAs in peripheral blood mononuclear cells associated with osteoporosis in females over the age of 50. Levels of mRNAs encoding annexin A1 (a), TMEM64 (b), and S100A4 (c) proteins were determined in peripheral blood mononuclear cell preparations of nonosteoporotic control subjects and patients suffering from osteopenia or osteoporosis. On each box and whisker plot, the black diamond shows the median value, the cross shows the mean value, and white and black circles denote outliers of 1.5 times and 3 times the interquartile range, respectively.
Summary of statistical analysis on female participants over the age of 50.
mRNA | Compared sample groups ( |
95% confidence interval | |
---|---|---|---|
Annexin A1 | Nonosteoporotic control ( |
0.114 to 0.493 | 0.0021 |
Nonosteoporotic control ( |
0.125 to 0.539 | 0.0022 | |
Osteoporosis ( |
-0.175 to 0.118 | 0.699 | |
TMEM64 | Nonosteoporotic control ( |
0.008 to 0.108 | 0.0245 |
Nonosteoporotic control ( |
0.041 to 0.15 | 0.0009 | |
Osteoporosis ( |
-0.077 to 0.0009 | 0.056 | |
S100A4 | Nonosteoporotic control ( |
-0.406 to 0.601 | 0.7 |
Nonosteoporotic control ( |
-0.095 to 1.01 | 0.103 | |
Osteoporosis ( |
-0.7486 to 0.033 | 0.072 |
Osteoporosis has been associated with chronic inflammatory disorders. In order to find out whether the reduction in the levels of mRNAs for annexin A1, TMEM64, or S100A4 in peripheral blood mononuclear cells was associated with the presence of chronic diseases, the participants with osteopenia or osteoporosis were divided into those without and those with reported chronic diseases or undergoing treatment with steroids. The levels of annexin A1, TMEM64, or S100A4 mRNAs in PBMCs were not significantly different between those with and those without known chronic inflammatory disorders (Supplementary Figure
The levels of mRNAs for annexin A1, S100A4, and TMEM64 were reduced in PBMCs from osteoporosis patients compared to nonosteoporotic controls; thus, the presence of these mRNAs marked the nonosteoporotic condition. ROC curve analysis for nonosteoporotic controls and osteoporosis showed that, when all participants were included, the areas under the ROC curves for annexin A1, TMEM64, and S100A4 were 0.893, (Figure
Diagnostic values of annexin A1, TMEM64, and S100A4 mRNAs for nonosteoporotic controls vs osteoporosis. ROC curves between nonosteoporotic controls and osteoporosis sufferers are shown for the mRNAs of annexin A1 (a, d), TMEM64 (b, e), and S100A4 (c, f) for all participants (a, b, c) and female participants over the age of 50 (d, e, f). The circled point indicates the optimum cut-off.
Diagnostic values of annexin A1 for nonosteoporotic controls vs osteopenia and osteoporosis. ROC curves are shown for the mRNA for annexin A1 for all participants (a, b) and female participants over the age of 50 (c, d), comparing nonosteoporotic controls with osteopenia (a, c) and nonosteoporotic controls with osteopenia and osteoporosis (b, d). The circled point indicates the optimum cut-off.
When the analysis was confined to females over the age of 50 years, similar results were obtained, with areas under the ROC curves for annexin A1, TMEM64, and S100A4 mRNAs being 0.809 (Figure
Three mRNAs, encoding calcium-binding/associated proteins, ANXA1, S100A4, and TMEM64, were found to be at a lower level in the PBMC preparations from osteoporosis patients than nonosteoporotic controls. The reductions of S100A4 mRNA levels were found to be due to participants with other disorders, those receiving existing treatments for osteoporosis, males, and under 50-year olds. In contrast, the observed reductions in annexin A1 or TMEM64 mRNAs were not affected by any of these factors.
Annexin A1 protein and, to a lesser extent, TMEM64 protein are found in the CD14+/CD16-negative monocytes [
Annexin A1 has been reported to be present in the human U937 cultured monocyte cell line, where it has been reported to reduce adhesion of U937 cells to bone marrow endothelial cells by directly interacting with
However, the PBMC fractions consist of only 20-40% monocytes and a low percentage of polymorphonuclear cells (1-4%), but a majority (60-80%) of cells in PBMC preparations are lymphocytes [
ROC curve analysis for the changes in these mRNAs showed moderate accuracy for the areas under the curves [
In conclusion, this study demonstrates for the first time that the levels of mRNAs of annexin A1 and TMEM64 in peripheral blood mononuclear cells are reduced in association with osteoporosis, at least in this group of patients with loss of annexin A1 mRNA in peripheral blood mononuclear cells being a good marker for osteopenia/osteoporosis.
The data used to support the findings of this study are available from the corresponding author upon request.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the England Health Research Authority National Research Ethics Service Committee, East of England-Essex (REC reference 15/EE/0051) Ethics Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
The current address of Abdullah Y. Mandourah is Al Hada Armed Forces Hospital, Taif, Saudi Arabia. The current address of Ayed Dera is Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia.
The authors declare that they have no conflicts of interest.
D.L.B. conceived the study and designed the experiments. A.A.D. and A.Y.M. performed the experiments; A.A.D. analysed the data. L.R., R.B., S.V., and S.H. provided intellectual guidance in the study and interpretation of data. D.L.B. and R.B. wrote and prepared the manuscript with contribution from all coauthors. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
We thank the healthy volunteers and patients who participated in this study. We thank the members of the Unit of Clinical Chemistry and Department of Nuclear Medicine at the Royal Liverpool and Broadgreen University Hospital NHS Trust and Institute of Ageing and Chronic Disease at the University of Liverpool for all their support. A.A.D. and A.Y.M. were supported by Ph.D. scholarships from the Saudi Arabian Embassy.
Supplementary Figure S1: relative levels of mRNAs for annexin A1, TMEM64, and S100A4 were determined in the peripheral blood mononuclear cells of osteopenia and osteoporosis participants. Box and whisker plots show the levels of mRNAs in participants with or without chronic inflammatory disorders/treatment with steroids. Supplementary Table S1: summary of characteristics of clinical samples.