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Introduction: Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts., Methods: We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D 3 analog WY 1048 or with 1,25(OH) 2 D 3 ., Results and Discussion: We show that Nrp2 expression is induced by 1,25(OH) 2 D 3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D 3 analog WY 1048 (0.4 μg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH) 2 D 3 (0.5 μg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Verlinden, Doms, Janssens, Meyer, Pike, Carmeliet and Verstuyf.)

Active vitamin D 3 , 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH) 2 D 3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D 3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER + ) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER + (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER + BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D 3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER + BC both in vitro and in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vanhevel, Verlinden, Loopmans, Doms, Janssens, Bevers, Stegen, Wildiers and Verstuyf.)