Abstract
PKC-δ is an important molecule for B-cell proliferation and tolerance. B cells have long been recognized to play a part in osteoimmunology and pathological bone loss. However, the role of B cells with PKC-δ deficiency in bone homeostasis and the underlying mechanisms are unknown. We generated mice with PKC-δ deletion selectively in B cells by crossing PKC-δ-loxP mice with CD19-Cre mice. We studied their bone phenotype using micro-CT and histology. Next, immune organs were obtained and analyzed. Western blotting was used to determine the RANKL/OPG ratio in vitro in B-cell cultures, ELISA assay and immunohistochemistry were used to analyze in vivo RANKL/OPG balance in serum and bone sections respectively. Finally, we utilized osteoclastogenesis to study osteoclast function via hydroxyapatite resorption assay, and isolated primary calvaria osteoblasts to investigate osteoblast proliferation and differentiation. We also investigated osteoclast and osteoblast biology in co-culture with B-cell supernatants. We found that mice with PKC-δ deficiency in B cells displayed an osteopenia phenotype in the trabecular and cortical compartment of long bones. In addition, PKC-δ deletion resulted in changes of trabecular bone structure in association with activation of osteoclast bone resorption and decrease in osteoblast parameters. As expected, inactivation of PKC-δ in B cells resulted in changes in spleen B-cell number, function, and distribution. Consistently, the RANKL/OPG ratio was elevated remarkably in B-cell culture, in the serum and in bone specimens after loss of PKC-δ in B cells. Finally, in vitro analysis revealed that PKC-δ ablation suppressed osteoclast differentiation and function but co-culture with B-cell supernatant reversed the suppression effect, as well as impaired osteoblast proliferation and function, indicative of osteoclast–osteoblast uncoupling. In conclusion, PKC-δ plays an important role in the interplay between B cells in the immune system and bone cells in the pathogenesis of bone lytic diseases.
Highlights
Osteoimmunology was an interdisciplinary subject coined by Arron in 2000 to highlight the reciprocal interactions between the skeletal and immune systems[1].Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, accumulating evidence indicated an important role for B cells in osteoimmunological regulation[2]
Our study aimed to investigate the important role of Protein kinase C δ (PKC-δ) in B cells and its subsequent effects on OC and OB biology by using a Cre-loxP-based conditional knockout technology to selectively inactivate PKC-δ in B cells, which could help to shed more light on our understanding of osteoimmunology-related disease, such as rheumatoid arthritis and osteoporosis
To determine the contribution of PKC-δ conditional knockout (cKO) in B cells in skeletal development and bone homeostasis, we firstly analyzed the gross appearance of 3-month-old PKC-δ cKO mice
Summary
Osteoimmunology was an interdisciplinary subject coined by Arron in 2000 to highlight the reciprocal interactions between the skeletal and immune systems[1].Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, accumulating evidence indicated an important role for B cells in osteoimmunological regulation[2]. In 2002, PKC-δ null mice, which develop systemic autoimmunity revealed an essential role for this kinase in B-cell homeostasis and tolerance[9,10]. Biallelic mutations in PRKCD (the gene that encodes PKC-δ) are associated with lupus and lymphoproliferative diseases because PKC-δ displays proapoptotic activity and is crucial to eliminate self-reactive transitional B cells[11,12,13,14]. These findings further confirmed PKC-δ as a critical proapopotic molecule essential in B-cell survival and apoptosis
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