ADRA2A is involved in neuro-endocrine regulation of bone resorption

Abstract

Adrenergic stimulation is important for osteoclast differentiation and bone resorption. Previous research shows that this happens through β2-adrenergic receptor (AR), but there are conflicting evidence on presence and role of α2A-AR in bone. The aim of this study was to investigate the presence of α2A-AR and its involvement in neuro-endocrine signalling of bone remodelling in humans. Real-time polymerase chain reaction (PCR) and immunohistochemistry were used to investigate α2A-AR receptor presence and localization in bone cells. Functionality of rs553668 and rs1800544 single nucleotide polymorphism SNPs located in α2A-AR gene was analysed by qPCR expression on bone samples and luciferase reporter assay in human osteosarcoma HOS cells. Using real-time PCR, genetic association study between rs553668 A>G and rs1800544 C>G SNPs and major bone markers was performed on 661 Slovenian patients with osteoporosis. α2A-AR is expressed in osteoblasts and lining cells but not in osteocytes. SNP rs553668 has a significant influence on α2A-AR mRNA level in human bone samples through the stability of mRNA. α2A-AR gene locus associates with important bone remodelling markers (BMD, CTX, Cathepsin K and pOC). The results of this study are providing comprehensive new evidence that α2A-AR is involved in neuro-endocrine signalling of bone turnover and development of osteoporosis. As shown by our results the neurological signalling is mediated through osteoblasts and result in bone resorption. Genetic study showed association of SNPs in α2A-AR gene locus with bone remodelling markers, identifying the individuals with higher risk of development of osteoporosis.

Keywords: ADRA2A; adrenergic signalling; bone; osteoblast; osteoporosis; resorption.

Figure 1

Immunolocalization of α2A-AR in human bone samples from an OP patient (A and C), OA patient (B) and an autopsy case (D). Positive α2A-AR staining was observed in cuboidal shaped osteoblasts (Ob) and lining cells (Lc). No staining was observed in any of the control slides where no α2A-AR antibody was applied (negative control, data not shown).

Figure 2

A cross-section of the human femoral head bone from the autopsy case shows no α2A-AR staining of osteocytes in the cortical and trabecular bone (A and B). No staining was observed in any of the control slides where no α2A-AR antibody was applied (negative control, data not shown).

Figure 3

Box plots depicting relative α2A-AR gene expression according to genotype in bone; expression for the reference genotype (associated with low α2A-AR gene expression) is set to 1. Numbers above the boxes indicate the number of individuals with that genotype. The bottom and top of each box indicate the 25th and 75th percentiles, respectively, while the line inside the box is the mean value. Whiskers represent the smallest and the values that are not outliers. Circles with the code of the sample indicate outliers. The P-value in red represents the anova statistical result for differences in α2A-AR gene expression according to α2A-AR genotypes subgroups, while P-values in black represent the LSD post hoc testing between each of two genotype subgroups. P-values with asterisks are statistically significant (below 0.05).

Figure 4

Left: relative expression of pOM-A and pOM-G plasmids. Right: Secondary structures formed by mRNA containing A (left structure) and G (right structure).