5α-dihydrotestosterone upregulates interferon signaling and limits osteoclast differentiation during inflammatory state.

PURPOSE: Rheumatoid arthritis is characterized by inflammatory erosions and increased prevalence in females. Androgen, the dominant sex hormone in males, is protective against bone loss. Here, we investigated the cellular targets of androgen and hypothesized that androgen negatively regulates TNFα-driven osteoclastogenesis.

MATERIALS AND

METHODS

Bone marrow-derived cells from a C57BL/6J mouse femur and tibia were plated with 10-9M of 5α-dihydrotestosterone (DHT) or vehicle then collected for flow cytometry and flow sorting. Osteoclast precursors (OCPs), myeloid-like CD11b+Gr-1- cells, were grown to osteoclasts then fixed and TRAP-stained. Osteoclasts were quantified by cell counting. Single-cell RNA-sequencing was performed on DHT or vehicle-treated TNFα-stimulated cells, and the differences in cell populations, gene expression, gene ontology, and cell trajectory were analyzed via bioinformatic approaches. RT-qPCR validated sequencing results.

RESULTS

DHT-treated OCPs were decreased versus vehicle-treated OCPs (Vehicle = 12.43 ± 0.53%, DHT = 9.93 ± 0.85%, p < 0.05), and there was decreased differentiation from OCPs to osteoclasts (Vehicle = 403.3 ± 141.5, DHT = 10.67 ± 14.36, p < 0.01). Analysis identified osteoclastogenesis-related clusters that varied between samples and interferon-related processes upregulated with DHT treatment. Analysis also determined that DHT treatment may be associated with expression of the transcription factor Irf7. Isg15, the most differentially expressed gene in the DHT-treated clusters, may also be involved in osteoclastogenesis.

CONCLUSIONS

In summary, androgen targets OCPs and limits their differentiation into osteoclasts. In myeloid populations, DHT treatment results in differential expression of interferon-related processes and genes and alters the trajectory of cells, potentially regulating osteoclastogenesis.