Vitamin D plays key roles for calcium homeostasis. Biallelic loss-of-function variants of CYP24A1, the enzyme that converts the active 1,25D3 into inactive metabolite, are responsible for autosomal recessive Idiopathic Infantile Hypercalcemia (IIH, ORPHA 300547, 1/80000), inducing hypercalcemia, nephrocalcinosis, nephrolithiasis and ultimately kidney failure. At the opposite of this rare disease, nephrolithiasis will affect during lifetime 20 to 30% of the general population, with a significant financial impact on health systems and an important burden of disease. The frequency of CYP24A1 heterozygosity is estimated to be 1/130 using the most frequent pathogenic variants. Thus, whether CYP24A1 heterozygosity is associated with haplo-insufficiency inducing a renal phenotype by itself or whether it is “only” a risk factor of nephrolithiasis remains debatable. Recent reports indicate that patients with heterozygous variants of CYP24A1 present increased 1,25D3 levels, and might present a higher risk of nephrolithiasis, but human data are scarce and somehow heterogeneous. In contrast, the murine model of CYP24A1 heterozygosity spontaneously displays a IIH-like phenotype, therefore positioning this pre-clinical model as a relevant tool to unravel the pathogenesis in heterozygous subjects. The objective of the translational HeteroCYP project is to deepen the understanding of heterozygous CYP24A1 variant-associated phenotypes in humans, using mouse models, clinical data and human samples. Such a demonstration will be instrumental to improve the genetic counselling and clinical care of these patients, so as to provide a personalized medicine. Last, novative therapeutic options will also be evaluated in our preclinical model, and might provide rationale for clinical trials in heterozygous patients.

Elevated serum phosphate (Pi) is a life-threatening condition which promotes the development of vascular calcifications (VC) and is associated with increased mortality in patients with chronic kidney disease (CKD). High Pi load triggers calcification of the medial layer of the arterial wall resulting in an inevitably more stiffened arterial bed and stimulates the secretion by bone of the phosphaturic hormone FGF23, whose levels increase dramatically in advanced CKD stages thereby contributing to the worsening of cardiovascular diseases. Despite elevated Pi load resulting from CKD, intestinal Pi absorption remains inappropriately unchanged, and therapeutic strategies to control transcellular intestinal Pi absorption have been unsuccessful. The PARKA project is designed to provide an integrated approach covering the entire spectrum of Pi regulation, from its intestinal absorption to its local vascular effects and systemic role, in order to decipher its mechanism of action leading to the development of VC in CKD, and to identify therapeutic targets able to counteract its deleterious effects. It is a 42-month project (624k€) based on 4 work packages: • WP0 Project’s management • WP1 Local and systemic mechanisms of action of Pi on VC during CKD • WP2 Paracellular intestinal absorption of Pi by BSP-DMP1 axis during CKD • WP3 Identification of putative therapeutic targets in mice and humans We will first focus on the role of tissue-nonspecific alkaline phosphatase (TNAP) and the sodium-Pi cotransporter PiT2. TNAP, the key enzyme for bone mineralization, was recently shown to be an early and obligatory inducer of medial calcification in a mouse model of CKD. PiT2 participates in the local effect of Pi on VCs during CKD and was characterized by Team 1 as a Pi sensor in bone controlling Pi-dependent FGF23 secretion in vivo. To study both the local and systemic effects of Pi, and the role of TNAP and PiT2, we will use conditional KOs of PiT2 in vascular cells (VSMCs) (sm22Cre;PiT2cKO) or osteoblasts (OsxCre;PiT2cKO), as well as the TNAP inhibitor SBI-425. Subtotal 5/6 nephrectomy and high-Pi diet feedings will be used to address the impact of high Pi load on medial VC during CKD. To uncover the role of intestinal absorption of Pi during CKD, we will focus on the regulation of the paracellular transport of Pi by the bone sialoprotein (BSP)-dentin matrix protein 1 (DMP1) pathway, since BSP KO mice have a dramatic reduction of paracellular intestinal absorption of Pi, together with a 10-fold increase in DMP1 intestinal expression. We expect that inactivation of TNAP and/or PiT2 in VSMCs will provide unique answers on the VC mechanisms and identify putative therapeutic targets. Deletion of PiT2 in osteoblasts will dampen the increase in FGF23 during CKD, resulting in high serum Pi levels and exacerbated VC without possible cardiovascular confounding effects of FGF23. Crossing BSPKO mice with OB-PiT2cKO mice is expected to result in normalized FGF23 and Pi serum levels in CKD conditions, alleviating the appearance of VC. The strength of PARKA is to bring together 4 teams who will share top notch expertise and specific models that will allow an integrated assessment of Pi regulation and action during CKD. This will be achieved via in vivo experiments using established and readily available in-house relevant mouse models, unbiased technical approaches (scRNA-seq and NanostringTM) and attempts to translational validation of our mouse findings in patients with advanced CKD. In vitro approaches will be used to further investigate the underlying molecular mechanisms. Fall-back solutions have been considered to include these in vitro models or organ cultures from the different animal models.

Multiple factors, systemic and local, concur to the biomechanical competence of the bone mineralized matrix, which is a major determinant of bone strength. Matrix components play a key role, and in particular the proteins of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoproteins) family. Among these, bone sialoprotein (BSP) and osteopontin (OPN) are highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. Single knock-out (KO) of their respective genes have shown them to be key regulators of the process of bone formation, mineralization and turnover, with distinct, although partly overlapping sets of functions. However, given their structure similarities and coexpression in various cell types, partial functional substitution/compensation in a single KO model by the cognate protein is likely, and will impair proper understanding of the redundancy and complementarity of their roles in bone metabolism and mechanical resistance. Indeed, the study of SIBLING functions through the independent analysis of each gene has reached its limits, and the understanding of interplay between these factors now requires multiple targeting approaches. Our goal in this project is to analyse mice with a double extinction (DE) of OPN (Spp1 gene) and BSP (Ibsp gene), to describe their skeletal biology with multiple experimental approaches, comparing it with single extinctions of either gene, generated in the same genetic background. To generate DE mice, we shall use a BSP KO mouse line on a mixed, outbred 129sv/CD1 background, as well as mice produced by the EUCOMM consortium on the C57-Bl/6 genetic background, which display an insertion in the first intron of the Ibsp (BSP) gene that extinguishes its expression (BSPtme1). Using targeted mutagenesis with TALENs, we shall realise a KO of the Spp1 gene in BSP KO and BSPtme1 one-cell embryos, then generate through a genetic screening and breeding program, mice expressing neither BSP nor OPN (DE mice), as well as single OPN-/- mice on the same genetic background as the DE. The mouse lines will then be used to study in the same genetic background the impact of single and double mutation of OPN and BSP on major aspects of skeletal physiology and disease in which these two proteins have been shown to be involved, namely : (a) the bone turnover and (b) the bone microenvironment (vascularisation, haematopoiesis) and their response to challenges, (c) bone marrow ablation and (d) bone mechanical stimulation. (e) The bone mineralised matrix structure and composition as well as (f) its biomechanical resistance, will also be studied, and their response to intense mechanical strain (hypergravity) will be analysed. This work will be performed by two laboratories which have already collaborated in recent years to the elucidation of the BSP KO phenotype and collectively master the diversity of expertise necessary to this endeavour. Overall, this project will be a breakthrough in the elucidation of the respective roles of SIBLING proteins, which appear more and more as major regulators of bone and mineral metabolism, and are involved in an increasing number of other physiological mechanisms.

Wider research content: Osteoporosis is a common metabolic disease, causing high morbidity and major economic costs. Obesity is often linked to bone deterioration leading to a greater risk of peripheral fragility fractures and clinical complications in these patients. The association between obesity and osteoporosis is complex and usually the fracture risk is underestimated in obese patients. Thus, understanding this relationship could improve the diagnosis and treatment of susceptible individuals and provide insights into the mechanisms involved in bone loss. Our preliminary data suggest that diet-induced changes in bile acid metabolism could be related to poor bone health, possibly via changes in the farnesoid X receptor (FXR)-driven metabolic pathways. Objectives: We aim to investigate the mechanisms underlying bile acid-induced metabolic rewiring in bone, especially focusing on changes in carnitine and amino acid pathways. We also aim to uncover novel metabolic biomarkers for bone loss that could be used to better predict fracture risk and provide stratified clinical care as well as to identify therapeutic targets with potential interest for personalised treatments in osteoporosis, including obesity-related bone loss. Methods: Bile acid-related metabolic alterations in carnitines and amino acids will be assessed in bone cells and biofluids of a diet-induced rat model for obesity and further investigated in an Fxr-/- murine model treated with rescue diets. Associated molecular, metabolic, and microbiome-related biomarkers will be identified in both models and osteoporotic patients via comprehensive quantitative panels and untargeted -omics methods. Validation of common targets will be carried out in primary human osteoblasts and osteoclasts. A separate analysis of markers for obesity-related bone loss will be performed. Level of originality: Osteoporosis and obesity are closely linked phenomena, but the underlying mechanisms are not fully understood, hampering the clinical assessment and appropriate management of these patients. We have identified for the first time that obesogenic diet-induced bone loss correlates with changes in bile acid, carnitine and amino acid metabolism in rats. Based on these results, BAMBinO aims to investigate this connection via the bile acid receptor FXR, using innovative methodological approaches and appropriate preclinical models. Moreover, the collaboration with the French groups will allow us to validate the findings in osteoporotic patients and human bone cells, which will ensure the biomarkers found in the project will have clinical applicability to develop targeted medical care. The comprehensive analysis proposed here will uncover metabolic targets to develop novel pharmacological, nutritional and microbiome-based therapies for osteoporosis.

Osteoporosis is a common age-related disorder characterized by low bone mass and deterioration in bone microarchitecture, leading to increased skeletal fragility and fracture risk. Age-related osteoporosis fractures are increasing in link with the proportion of elderly population is increasing, thus resulting in human burden for the health system. Beside the efficacy of antiresorbing drugs at stopping bone loss, the major question arising now is how is it possible to restore lost bone? For this aim, there is a crucial need for identifying new targets especially on bone forming cells (osteoblasts). Our project will focus on the lysophosphatidic acid (LPA)-producing enzyme Autotaxin (ATX) because LPA has an anabolic action on bone by activating osteoblasts. However, LPA also promotes osteoclastic bone resorption. Thus, specific therapeutic blocking of LPA’s catabolic activity could promote its anabolic action on bone tissue. ATX is the main producer of LPA in the organism. Remarkably, global deletion of ATX gene (Enpp2) is lethal at the embryonic stage making impossible the use of mice for bone study. Nevertheless, the choice of ATX was supported because of our recent observations in MC3T3 cell line and calvaria primary osteoblasts that express high levels of ATX making osteoblasts a potent source of LPA at the bone site. The first objective of the project is to elucidate the role of ATX/LPA axis during bone mass acquisition in youth and bone loss in aging and to determine its impact on osteoblast function and bone quality. For this aim we will analyze ATXdeltaOb mice already generated by crossing Enpp2flox/flox mice with Osx:GFP-Cre/+ animals allowing specific invalidation of ATX expression in osteoblast progenitors and hypertrophic chondrocytes. These animals present a remarkable low bone mass phenotype. Animal bone phenotype will be characterized based on technics used in routine in partners’ laboratories such as microCT, histology, immunohistochemistry, fluorescence imaging. The second objective of the project is to characterize novel signaling pathways that control the anabolic activity of osteoblasts and that connect osteoblast to osteoclast functions. Our project will decipher in primary mouse and human osteoblasts the molecular connections between Wnt/beta-catenin and ATX/LPA signaling pathways that have recently been revealed in malignant cells allowing the identification of potential new therapeutic targets. The third objective of the project is to develop new therapeutic tools promoting bone gain. To this aim we will characterize at molecular levels domains of ATX that bind to cell surface of osteoblasts and osteoclasts because of ATX new role emerging as a docking molecule required for the proper presentation of LPA to the cell surface, leading to the activation of specific LPA receptors. We and other have shown that ATX binds to beta-1, beta-3 integrins and heparan sulfate proteoglycans. We have demonstrated that ATX controls osteoclastic bone erosion in inflammatory conditions. Because of the paramount role of beta-3 integrin in osteoclast function, we will develop a unique strategy in the field by performing in silico proteochemometrics studies based on artificial intelligence followed by co-crystallography and biochemical analyses of ATX with identified beta-3 integrins interactants that will be validated both in osteoclast activity and osteoblast/osteoclast coculture assays. Functionally active peptides will be PEGylated to increase stability in vivo and used in our preclinical animal models of osteoporosis as a proof of concept in the development of new therapies against bone loss. Altogether, based on animal whole body bone characterization, histology, osteoblast and osteoclast cell biology, in silico proteochemometrics and crystallography analyses, the project will fully characterize the molecular of actions of ATX on bone cells and will develop new tools dedicated to bone regeneration therapies.