Transcriptomic comparison of osteocyte cell lines - determining their utility as models to study sex hormone regulation of skeletal function.

Osteocytes are critical for bone development, and tightly regulate bone (re)modelling by directing the differentiation and activity of osteoblast and osteoclasts. Primary osteocyte isolation is challenging, therefore MLO-A5 and MLO-Y4 osteocyte-like cell lines have been instrumental in dissecting osteocytogenesis and bone mineralisation. Limited comparative analysis of these closely related in vitro models has been undertaken, therefore we used transcriptomic analysis to dissect their transcriptional similarities. Principal component analysis showed MLO-A5 and MLO-Y4 cells form distinct clusters, with 4451 differentially expressed genes. Known osteoblast markers (Alpl and Col1a1) and early osteocyte markers (Gja1, Pdpn, and Tnfsf11) predominated in MLO-A5 and MLO-Y4 cells, respectively. MLO-A5 cells expressed genes associated with extracellular matrix (re)modelling and mineralisation pathways, whilst MLO-Y4 cells were enriched with genes associated with cell signalling, consistent with MLO-A5 and MLO-Y4 cells representing different stages in osteocyte development. Androgens have anabolic effects on the bone, so their impact on the osteocyte transcriptome and osteocyte function was investigated. Surprisingly, androgen (Ar) and oestrogen receptor (Esr2) expression was significantly lower in MLO-A5 and MLO-Y4 cells compared to mouse tibiae. Treatment with androgen receptor ligands (R1881 and enzalutamide) had minimal effect on MLO-A5 or MLO-Y4 gene transcription (<20 differentially expressed genes) and treated cells showed a striking lack of differentially expressed osteoblast and osteocyte regulatory genes. This work provides a comprehensive understanding of these important in vitro osteocyte models and highlights the limitations of using MLO-A5 or MLO-Y4 cells to investigate the impact of sex hormones on osteocyte function in vitro.