ABSTRACT Early‐onset osteoporosis (EOOP) has been associated with several genes, including LRP5, coding for a coreceptor in the Wnt pathway. Variants in LRP5 were also described in osteoporosis pseudoglioma syndrome, combining severe osteoporosis and eye abnormalities. Genomewide‐association studies (GWAS) showed that LRP5 p.Val667Met (V667M) variant is associated with low bone mineral density (BMD) and increased fractures. However, despite association with a bone phenotype in humans and knockout mice, the impact of the variant in bone and eye remains to be investigated. Here, we aimed to evaluate the bone and ocular impact of the V667M variant. We recruited 11 patients carrying the V667M variant or other loss‐of‐function variants of LRP5 and generated an Lrp5V667M mutated mice. Patients had low lumbar and hip BMD Z‐score and altered bone microarchitecture evaluated by HR‐pQCT compared with an age‐matched reference population. Murine primary osteoblasts from Lrp5V667M mice showed lower differentiation capacity, alkaline phosphatase activity, and mineralization capacity in vitro. Ex vivo, mRNA expression of Osx, Col1, and osteocalcin was lower in Lrp5V667M bones than controls (all p < 0.01). Lrp5V667M 3‐month‐old mice, compared with control (CTL) mice, had decreased BMD at the femur (p < 0.01) and lumbar spine (p < 0.01) with normal microarchitecture and bone biomarkers. However, Lrp5V667M mice revealed a trend toward a lower femoral and vertebral stiffness (p = 0.14) and had a lower hydroxyproline/proline ratio compared with CTL, (p = 0.01), showing altered composition and quality of the bone matrix. Finally, higher tortuosity of retinal vessels was found in the Lrp5V667M mice and unspecific vascular tortuosity in two patients only. In conclusion, Lrp5V667M variant is associated with low BMD and impaired bone matrix quality. Retinal vascularization abnormalities were observed in mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Osteocytes are mechanosensitive cells that control bone remodeling in response to mechanical loading. To date, specific signaling pathways modulated by mechanical loading in osteocytes are not well understood. Yes associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the main effectors of the Hippo pathway, are reported to play a role in mechanotransduction and during osteoblastogenesis. Here, we hypothesized that YAP/TAZ signaling mediates osteocyte mechanosensing to target genes of the bone remodeling process. We aimed to investigate the contribution of YAP/TAZ in modulating the gene expression in an osteocyte-like cell line MLO-Y4. We developed a 3D osteocyte compression culture model from an MLO-Y4 osteocyte cell line embedded in concentrated collagen hydrogel. 3D-mechanical loading led to the increased expression of mechanosensitive genes and a subset of chemokines, including M-csf, Cxcl1, Cxcl2, Cxcl3, Cxcl9, and Cxcl10. The transcription regulators YAP and TAZ translocated to the nucleus and upregulated their target genes and proteins. RNAseq analysis revealed that YAP/TAZ knockdown mediated the regulation of several genes including osteocyte dendrite formation. Use of YAP/TAZ knockdown partially blunted the increase in M-csf and Cxcl3 levels in response to MLO-Y4 compression. These findings demonstrate that YAP/TAZ signaling is required for osteocyte-like cell mechano-transduction, regulates the gene expression profiles and controls chemokine expression.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in the SLC26A2 gene encoding for a sulfate/chloride transporter. When SLC26A2 is impaired intracellular level of sulfate is reduced leading to the synthesis of undersulfated proteoglycans. In normal chondrocytes, the main source of intracellular sulfate is the extracellular uptake through SLC26A2, but a small amount comes from the catabolism of sulfur-containing amino acids and other thiols. Here N-acetylcysteine (NAC), an extensively used drug, is proposed as alternative source of intracellular sulfate in an animal model of DTD (dtd mouse). Mutant and wild type mice were treated twice a day with hypodermic injections of 250 mg NAC/kg body weight for one week after birth. At the end of the treatment, an improvement trend in cartilage proteoglycan sulfation and in the skeletal phenotype of treated dtd mice were observed. Thus, a longer treatment lasted three weeks starting from birth was performed. Treated mutant mice showed a significant increase of cartilage proteoglycan sulfation and a relevant improvement of the skeletal phenotype based on measurements of several bony elements and bone quality by DEXA and micro CT. Moreover, the amelioration of the overall growth plate morphology in treated dtd mice suggested a partial rescue of the endochondral ossification process. Overall, the results prove that NAC is an effective source of intracellular sulfate for dtd mice in the postnatal period. This finding paves the way for a potential pharmacological treatment of DTD patients taking advantage from a drug repositioning strategy.

Background/Introduction: Site-2 protease (S2P), encoded by MBTPS2, is a Golgi transmembrane proprotein convertase of membrane-bound transcription factors, involved in cholesterol metabolism.We previously identified an X-R form of osteogenesis imperfecta (type XVIII OI) with mutations in S2P causing impaired regulated intramembrane proteolysis (RIP) of SREBP, ATF6 and OASIS, and decreased type I collagen secretion.Purpose: We identified probands 3 and 4 with type XVIII OI: 2y4m boy with S2Pp.N459S (c.1376ANG), and 1y5m boy with novel S2P p.L455Q (c.1364TNA) mutation.Methods: Bone and primary osteoblasts (OB) with p.N459S were investigated with qBEI, histomorphometry, qPCR, RNAseq.Results: Male with S2P p.N459S had LE bowing on 20 wk US.He has short stature, blue sclerae, fractures of ribs, clavicles, limbs, vertebral compressions, rhizomelia of UE and LE.His L2-L4 BMD zscore b -2 (0.167 g/cm 2 ).The S2P p.L455Q mutation is associated with short stature, blue sclerae, limb fractures and deformity, undertubulated long bones with LE rhizomelia, vertebral compressions, and L1-L4 DXA z-score= -7.36 (0.128 g/cm2).Total (1574 IU/L) and bone-specific (420 mcg/L) ALP and osteocalcin (68.9 ng/ml) were elevated.Oasis processing in proband FB revealed decreased 50kD S1P/S2P cleavage product.Cortical bone from S2Pp.N459S proband had notable marrow fibrosis and was not hypermineralized, distinct from classical OI.Histomorphometry revealed increased osteoblast (14.1%; control 8.5±4.1) and osteoid surface (46.8%; control 34±6.7).In vitro, the S2P p.N459S mutation hampered osteoblastogenesis.Early osteoblast markers were downregulated in primary OB, whereas, late osteoblast/early osteocyte markers were upregulated.In vitro mineralization was severely delayed in proband OB.Transcript profiling revealed that p.N459S alters expression of genes encoding ECM constituents and involved in ECM organization.Conclusion(s): These MBTPS2 missense mutations support a critical role of RIP in normal bone development.The distinctive features of type XVIII OI bone tissue and OB will reveal insights into the tissue-specific mechanism of RIP.

Osteoarthritis is characterized by cartilage loss resulting from the activation of chondrocytes associated with a synovial inflammation. Activated chondrocytes promote an increased secretion of matrix proteases and proinflammatory cytokines leading to cartilage breakdown. Since natural products possess anti-inflammatory properties, we investigated the direct effect of Rubus idaeus extracts (RIE) in chondrocyte metabolism and cartilage loss. The effect of RIE in chondrocyte metabolism was analyzed in murine primary chondrocytes and cartilage explants. We also assessed the contribution of RIE in an inflammation environment by culturing mice primary chondrocytes with the supernatant of Raw 264.7 macrophage-like cells primed with RIE. In primary chondrocytes, RIE diminished chondrocyte hypertrophy (Col10), while increasing the expression of catabolic genes (Mmp-3, Mmp-13) and reducing anabolic genes (Col2a1, Acan). In cartilage explants, Rubus idaeus prevented the loss of proteoglycan (14.84 ± 3.07% loss of proteoglycans with IL1 alone vs. 3.03 ± 1.86% with IL1 and 100 µg/mL of RIE), as well as the NITEGE neoepitope expression. RIE alone reduced the expression of Il1 and Il6 in macrophages, without changes in Tnf and Cox2 expression. The secretome of macrophages pre-treated with RIE and transferred to chondrocytes decreases the gene and protein expression of Mmp-3 and Cox2. In conclusion, these data suggest that RIE may protect from chondrocyte catabolism and cartilage loss in inflammatory conditions. Further evaluations are need before considering RIE as a candidate for the treatment for osteoarthritis.

The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted geneslike testis- or placenta-specific genesis frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells.We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.

Background: Osteoporosis which affects 200 million women worldwide is the consequence of an imbalance of low anabolism to high catabolism, causing a risk of fracture. Impaired anabolism involved reduced osteoblast differentiation. The osteoblast differentiation is mediated by transcription factors, including Dlx5 and Dlx6. Dlx5 is known to have a role in osteoblast/osteoclast couple and as a promotor of osteoblast lineage commitment [1, 2]. Thus, Dlx5 is a transcriptional actor of Runx2 [3], a key element of osteoblastic differentiation. Objectives: The goal of this project is to expand our knowledge about bone formation and cellular precursors of osteoblasts, focusing on Dlx5 and Dlx6. Methods: We analyze the kinetic expression of Dlx5, Dlx6 and osteoblastic markers during the osteoblastic differentiation from murine osteoblastic progenitor derived from calvaria and bone marrow. Same analysis was carried out in osteoblastic precursors from control cells, Dlx5/Dlx6 cells with ex vivo recombination or from KO mice in parallel to human bone marrow cells. We analyzed the bone phenotype of mutated mice in the absence of expression of Dlx5 and Dlx6 under Osx promoter. Results: Dlx5 and Dlx6 increases at D7 during osteoblastic differentiation in the murine bone marrow and then was stable to D21. The absence of Dlx5/6 in cells derived from calvaria and bone marrow resulted in decreased levels of osteocalcin and alkaline phosphatase. Dlx5/6fl/fl Osx-Cre mice were lethal. Dlx5/6 fl/+ Osx Cre mice does not affect cortical and trabecular parameters at 6 weeks but had a significant lower cortical thickness and also lower Tb. BV/TV and Tb. Th along with a lower BMD at 3 months in both sexes. Moreover, periosteal volume was also lower in mutated mice. The skulls revealed a lack of sutures closures and dental abnormalities at 6 weeks and 3 months in both sexes. Conclusion: The deletion of these transcription factors under the action of the Osterix promoter generates lethality, in favor of an essential role in bone development. Heterozygous mutation show impaired bone acquisition during growth. To obtain a total deletion of Dlx5 and Dlx6 in osteoblastic precursor cells, a new murine model of conditional induced deletion is generated. Dlx5 and Dlx6 promote osteoblastic differentiation with an effect on late bone markers, in favor of a role in terminal differentiation. Analysis in vitro of Dlx5 and Dlx6 will be confirmed by ongoing in vivo experimentation. References [1] - Samee N, Geoffroy V, Marty C, Schiltz C, Vieux-Rochas M, Levi G, et al. Dlx5, a positive regulator of osteoblastogenesis, is essential for osteoblast-osteoclast coupling. Am J Pathol2008;173(3):773-80. [2] - Samee N, Geoffroy V, Marty C, Schiltz C, Vieux-Rochas M, ClementLacroix P, et al. Increased bone resorption and osteopenia in Dlx5 heterozygous mice. J Cell Biochem2009;107(5):865-72. [3] - Hassan MQ, Tare RS, Lee SH, Mandeville M, Morasso MI, Javed A, et al. BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. J Biol Chem2006;281(52):40515-26. Acknowledgement: With the support of the french rheumatology society Disclosure of Interests: Camille Blandin: None declared, morgane Bourmaud: None declared, eris Hay: None declared, giovanni Levi: None declared, martine Cohen Solal Speakers bureau: Amgen and Lilly