Probes for INS

Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on non-vertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1 expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur, but no change in trabecular bone volume fraction (BV/TV) in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared to control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce non-vertebral fracture risk.

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.

In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized.Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts.VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses.The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.