NBCe1 (electrogenic Na⁺/HCO₃ – cotransporter 1) is a product of gene SLC4A4 and has five splice variants, NBCe1-A through NBCe1-E. In agreement with an essential role of NBCe1 in cellular pH regulation, human families carrying missense mutations of gene SLC4A4 show proximal renal tubular acidosis. Some of them exhibit brain function-related symptoms, such as migraine and mental retardation, but physiological roles of NBCe1 in brain function remain unclear. To gain insights into NBCe1-specific functions in the brain, we herein identified proteins that specifically bind to a unique C-terminal region of NBCe1-C, a brain-specific NBCe1 isoform. We found that a catalytic subunit of calcineurin binds to the C terminus of NBCe1-C in the mouse cerebellum. Heterologous-coexpression experiments revealed that calcineurin binds to NBCe1-C via a “PQIRIE” motif at its C terminus. The interaction enhanced cell surface expression of NBCe1-C, resulting in an increase of its transporter activity, for which the phosphatase activity of calcineurin was essential. When NBCe1-C was stably expressed in HeLa cells, its cell surface expression was enhanced by an intracellular Ca²⁺ concentration increase and was suppressed by FK506, a specific inhibitor of calcineurin. These mechanisms of surface expression and transport activity of NBCe1-C regulated by the Ca²⁺–calcineurin axis indicate specialized functions of NBCe1-C in the brain.

Anthocyanins are plant-derived pigments, and their aglycons are called anthocyanidin. Anthocyanidins have shown to exhibit various biological functions, such as anti-oxidant effects. However, their structure-activity relationship in bone tissue is not known. In this study, we examined the effects of three anthocyanidins, delphinidin, cyanidin and pelargonidin, on osteoclast differentiation and bone resorption to elucidate the structure-activity relationship. Anthocyanidins suppressed both IL-1 and LPS induced osteoclast differentiation in cocultures of bone marrow cells and primary osteoblasts, and bone resorbing activity in calvarial organ cultures. In osteoblasts, anthocyanidins inhibited prostaglandin (PG) E₂ production via the downregulation of membrane-bound PGE synthase (mPGES)-1, leading to the suppression of PGE₂-mediated receptor-activator of nuclear factorkappa B (NF-κB) ligand (RANKL) expression. In osteoclasts, anthocyanidins inhibited RANKL-induced osteoclast differentiation through the downregulation of osteoclast differentiation marker genes, nuclear factor of activated T-cells 1 (NFATc1), cathepsin K and tartrate-resistant acid phosphatase (TRAP). We further found that anthocyanidins suppressed the inhibitor of NF-κB kinase (IKK) activity in vitro assay, a signal component of NF-κB pathway, suggesting IKK was a novel target molecule of anthocyanidins. We found that delphinidin exerted the most potent inhibitory activity in these experiments, compared with cyanidin and pelargonidin. Anthocyanidins exhibits inhibitory activity in bone resorption, which may depend on the number of hydroxide residues.