Mouse Growth Plate Research Articles

Meclozine and growth hormone ameliorate bone length and quality in experimental models of achondroplasia.

Achondroplasia (ACH) is a common skeletal dysplasia associated with short-limbed short stature caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Meclozine was found to inhibit FGFR3 signaling using a drug repositioning strategy. In some countries, growth hormone (GH) has been employed to ameliorate short stature in children with ACH. This study aims to investigate the effects of meclozine and GH on bone growth and quality using an experimental model of ACH. Meclozine (2mg/kg/day) and/or GH (0.35mg/kg/day) were administered to a mouse model of ACH from the age of 7 to 56days. Body length and body weight of each mouse were measured during these treatments. At the end of treatments, these mice were subjected to micro-computed tomography scans to measure the lengths of long bones and bone mineral density (BMD). The width of the growth plate was quantified by histological analysis. The body and bone length of transgenic mice significantly increased after treatment with meclozine and GH, although there was no additive effect of the combination therapy on promoting bone growth. In contrast, BMD was additively increased by the combination therapy. The width of the growth plate in transgenic mice was significantly increased by both treatments, although the hypertrophic zone was enlarged by meclozine but not by GH. Meclozine or GH may be an option for treating children with ACH to ameliorate bone length and quality, but the additive effect would be limited.

7598 A Humanized PTH Receptor Mouse Model Of Eiken Syndrome: Delayed Bone Mineralization Caused By A Receptor C-tail Truncation

Abstract Disclosure: J. Höppner: None. P. Hanna: None. M.N. Wein: None. H. Jueppner: None. T.J. Gardella: None. R. Civitelli: None. I.A. Portales-Castillo: None. Abstract The parathyroid hormone receptor-1 (PTH1R) plays a key role in bone development and acts in the growth plates in response to PTHrP ligand. Eiken syndrome is characterized by a delay in bone mineralization and is caused by homozygous PTH1R mutations. One such mutation, R485X, truncates the receptor's C-tail and removes serine phosphorylation sites involved in βarrestin binding. In HEK293 cells, R485X-hPTH1R exhibits deficient interaction with βarrestin and increased cAMP signaling both basally and in response to PTHrP (Portales-Castillo et al., Comms. Biology 2023). To understand how the R485X mutation causes delayed bone mineralization, we generated humanized R485X-hPTH1R knock-in mice. Homozygous hPTH1RR485X/R485X mice closely recapitulate the delayed bone mineralization seen in Eiken patients, as skeletal whole mount preparations from neonatal mutant mice showed reduced Alizarin red staining compared to WT controls, and metatarsal explants from the mutant mice showed a pronounced absence of mineralized bone. Tails of hPTH1RR485X/R485X mice were ∼50% shorter than those of WT littermates, and H&E-stained sections revealed only proliferative chondrocytes in the growth plates of mutant mice. This delay in growth plate chondrocyte maturation in hPTH1RR485X/R485X mice resolved with age, although older long bones and tails remained smaller in size than WT controls (Höppner et al. Presented at ASBMR 2023).Based on our in vitro findings and initial mouse characterization, we reasoned that the phenotype of Eiken mice may be explained by increased PTHrP/PTH1R signaling in growth plate chondrocyte. The class IIa histone deacetylase HDAC4 suppresses chondrocyte maturation downstream of PTH1R signaling. Therefore, we generated compound mutant mice bearing both Hdac4 deletion and the mutant R485X PTH1R allele. Indeed, Hdac4 deletion largely rescued the mineralization defect apparent in metatarsals of P1 hPTH1RR485X/R485X mice. We then assessed the contribution of endogenous PTHrP to the Eiken-like phenotype by generating hPTH1RR485X/R485X/PTHrPflox/+/Col2-Cre(tg) mice. Remarkably, these mice exhibit approximately the same tail lengths as hPTH1R-WT littermate controls, indicating that reduced PTHrP production can rescue the effects of homozygosity for R485X-PTH1R. In line, vertebral growth plates of the rescued mice exhibited normal zones of chondrocytes, including hypertrophic cells. The overall results support a disease mechanism for Eiken syndrome by which excess cAMP signaling by PTH1R-R485X, as induced in part by endogenous PTHrP, results in delayed chondrocyte differentiation and bone mineralization. Further studies employing βarr1/2-KO mice may help shed light on the specific roles of βarrestins in growth plate development by PTH1R in Eiken syndrome. Presentation: 6/3/2024

Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development.

Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. In this work, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

Apolipoprotein E is a novel marker for chondrocytes in the growth plate resting zone.

The resting zone (RZ) in mammalian growth plates is critical for maintaining and regulating chondrocyte turnover during longitudinal bone growth as a control tower and stem cell reservoir. Although recent lineage tracing studies have identified several markers for stem cells in the RZ, these markers only partially label chondrocytes in the RZ, suggesting that the resting chondrocytes (RCs) are a heterogeneous population with different types of stem cells. Since a comprehensive marker for RCs is still lacking, the RZ is generally determined based on ambiguous histological criteria, such as small and round chondrocytes without columnar formation, which may lead to inconsistencies among researchers. Therefore, in this study, we used single-cell RNA sequencing (scRNAseq) of growth plate chondrocytes followed by validation by fluorescence in situ hybridization (FISH) to precisely annotate cell clusters in scRNAseq and search for a marker of RCs. The scRNAseq analysis revealed that apolipoprotein E (Apoe) was the top-hit gene, which was ubiquitously expressed in the RC cluster. FISH confirmed that Apoe was exclusively localized to the histologically defined RZ. In newly generated Apoe-mCherry knock-in mice, we further confirmed that mCherry expression mirrored the distribution of Apoe-expressing chondrocytes in the RZ particularly after the formation of the secondary ossification center. These mCherry+ RCs were slow cycling in vivo and exhibited stem cell properties both in vitro and in vivo. Moreover, APOE was detected in human growth plate RCs. These findings suggest that Apoe is a novel pan-RC marker in both mouse and human growth plates.

Low expression of selenoprotein S induces oxidative damage in cartilages

Low levels of the indispensable trace element selenium (Se) can cause oxidative stress and disrupt environmental homeostasis in humans and animals. Selenoprotein S (Selenos), of which Se is a key component, is a member of the selenoprotein family involved in various biological processes. This study aimed to investigate whether low-level SELENOS gene expression can induce oxidative stress and decrease the antioxidative capacity of chondrocytes. Compared with control cells, SELENOS-knockdown ATDC5 cells showed substantially higher dihydroethidium, reactive oxygen species and malondialdehyde levels, and lower superoxide dismutase (SOD) expression. Knockout of the gene in C57BL/6 mice increased the 8-hydroxy-2-deoxyguanosine level considerably and decreased SOD expression in cartilages relative to the levels in wild-type mice. The results showed that the increased nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling mediated by low-level SELENOS expression was involved in oxidative damage. The proliferative zone of the cartilage growth plate of SELENOS-knockout mice was shortened, suggesting cartilage differentiation dysfunction. In conclusion, this study confirmed that low-level Selenos expression plays a role in oxidative stress in cartilages.

Protein phosphatase SCP4 regulates cartilage development and endochondral osteogenesis via FoxO3a dephosphorylation.

The regulatory mechanisms involved in embryonic development are complex and yet remain unclear. SCP4 represents a novel nucleus-resident phosphatase identified in our previous study. The primary aim of this study was to elucidate the function of SCP4 in the progress of cartilage development and endochondral osteogenesis. SCP4-/- and SCP4Col2ER mice were constructed to assess differences in bone formation using whole skeleton staining. ABH/OG staining was used to compare chondrocyte differentiation and cartilage development. Relevant biological functions were analysed using RNA-sequencing and GO enrichment, further validated by immunohistochemical staining, Co-IP and Western Blot. Global SCP4 knockout led to abnormal embryonic development in SCP4-/- mice, along with delayed endochondral osteogenesis. In parallel, chondrocyte-specific removal of SCP4 yielded more severe embryonic deformities in SCP4Col2ER mice, including limb shortening, reduced chondrocyte number in the growth plate, disorganisation and cell enlargement. Moreover, RNA-sequencing analysis showed an association between SCP4 and chondrocyte apoptosis. Notably, Tunnel-positive cells were indeed increased in the growth plates of SCP4Col2ER mice. The deficiency of SCP4 up-regulated the expression levels of pro-apoptotic proteins both in vivo and in vitro. Additionally, phosphorylation of FoxO3a (pFoxO3a), a substrate of SCP4, was heightened in chondrocytes of SCP4Col2ER mice growth plate, and the direct interaction between SCP4 and pFoxO3a was further validated in chondrocytes. Our findings underscore the critical role of SCP4 in regulating cartilage development and endochondral osteogenesis during embryonic development partially via inhibition of chondrocytes apoptosis regulated by FoxO3a dephosphorylation.

TMCO3, a Putative K+ :Proton Antiporter at the Golgi Apparatus, Is Important for Longitudinal Growth in Mice and Humans.

Isolated short stature, defined as short stature without any other abnormalities, is a common heterogeneous condition in children. Exome sequencing identified the homozygous nonsense variant c.1832G>A/p.(Trp611*) in TMCO3 in two sisters with isolated short stature. Radiological studies, biochemical measurements, assessment of the skeletal status, and three-dimensional bone microarchitecture revealed no relevant skeletal and bone abnormalities in both sisters. The homozygous TMCO3 variant segregated with short stature in the family. TMCO3 transcript levels were reduced by ~50% in leukocyte-derived RNA of both sisters compared with controls, likely due to nonsense-mediated mRNA decay. In primary urinary cells of heterozygous family members, we detected significantly reduced TMCO3 protein levels. TMCO3 is functionally uncharacterized. We ectopically expressed wild-type TMCO3 in HeLa and ATDC5 chondrogenic cells and detected TMCO3 predominantly at the Golgi apparatus, whereas the TMCO3W611* mutant did not reach the Golgi. Coordinated co-expression of TMCO3W611* -HA and EGFP in HeLa cells confirmed intrinsic instability and/or degradation of the mutant. Tmco3 is expressed in all relevant mouse skeletal cell types. Highest abundance of Tmco3 was found in chondrocytes of the prehypertrophic zone in mouse and minipig growth plates where it co-localizes with a Golgi marker. Knockdown of Tmco3 in differentiated ATDC5 cells caused reduced and increased expression of Pthlh and Ihh, respectively. Measurement of long bones in Tmco3tm1b(KOMP)Wtsi knockout mice revealed significant shortening of forelimbs and hindlimbs. TMCO3 is a potential member of the monovalent cation:proton antiporter 2 (CPA2) family. By in silico tools and homology modeling, TMCO3 is predicted to have an N-terminal secretory signal peptide, forms a dimer localized to the membrane, and is organized in a dimerization and a core domain. The core domain contains the CPA2 motif essential for K+ binding and selectivity. Collectively, our data demonstrate that loss of TMCO3 causes growth defects in both humans and mice. © 2023 American Society for Bone and Mineral Research (ASBMR).

Blocking FGF23 signaling improves the growth plate of mice with X-linked hypophosphatemia.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone. X-linked hypophosphatemia (XLH) is the most prevalent inherited phosphate wasting disorder due to mutations in the PHEX gene, which cause elevated circulating FGF23 levels. Clinically, it is characterized by growth impairment and defective mineralization of bones and teeth. Treatment of XLH is challenging. Since 2018, neutralizing antibodies against FGF23 have dramatically improved the therapy of XLH patients, although not all patients fully respond to the treatment, and it is very costly. C-terminal fragments of FGF23 have recently emerged as blockers of intact FGF23 signaling. Here, we analyzed the effect on growth and bone of a short 26 residues long C-terminal FGF23 (cFGF23) fragment and two N-acetylated and C-amidated cFGF23 peptides using young XLH mice (Phex C733RMhda mice). Although no major changes in blood parameters were observed after 7 days of treatment with these peptides, bone length and growth plate structure improved. The modified peptides accelerated the growth rate probably by improving growth plate structure and dynamics. The processes of chondrocyte proliferation, death, hypertrophy, and the cartilaginous composition in the growth plate were partially improved in young treated XLH mice. In conclusion, these findings contribute to understand the role of FGF23 signaling in growth plate metabolism and show that this may occur despite continuous hypophosphatemia.

THE IMPACT OF SEX ON BONE STRUCTURE AND ANGIOGENESIS IN SYNDECAN-1 DEFICIENT MICE

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised.Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel.Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT.Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner.

Presence of type IIB procollagen in mouse articular cartilage and growth plate is revealed by immuno-histochemical analysis with a novel specific antibody

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen.

Compressive stress induces spinal vertebral growth plate chondrocytes apoptosis via Piezo1.

Many clinical studies have indicated an association between biomechanical factors and the incidence and pathological progression of adolescent idiopathic scoliosis (AIS). However, at present, the research on AIS is mainly focused on the etiology, and there are few studies reporting the causes of progressive aggravation of AIS. In the present study, we aim to investigate the role of Piezo1 in compressive stress-induced mouse spinal vertebral growth plate chondrocytes apoptosis. First, a scoliosis mouse model was established, and the expression of Piezo1 as well as the degree of apoptosis were investigated. We found that the expression of Piezo1 and the degree of apoptosis were significantly higher on the concave sides than that on the convex sides of the vertebral growth plate in mice with scoliosis. Spinal vertebral growth plate chondrocytes were further isolated and treated with Yoda1 to mimic Piezo1 overload. Excess Piezo1 significantly promoted apoptosis of spinal vertebral growth plate chondrocytes. Moreover, static gas compressive stress was used to simulate the increased concave compressive stress in the process of scoliosis with or without GsMTx4, a Piezo inhibitor. It was observed that with the increase of static compressive stress, the expression of Piezo1 increased, and the chondrocytes of vertebral growth plate treated with Piezo1 inhibitor GsMTx4 weakened the above phenomena. In conclusion, our results indicated that compressive stress is strongly associated with the different degrees of apoptosis on both sides on the convex and concave sides of the vertebral growth plate in scoliosis via inducing different expressions of Piezo1. Reducing the expression of Piezo1 in the concave side of the vertebral growth plate and inhibiting the apoptosis of chondrocytes in the bilateral vertebral growth plate caused by asymmetric stress on both sides of the concave vertebral body may be a promising treatment strategy for AIS.

Do not overwork: cellular communication network factor 3 for life in cartilage.

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

TNF overexpression and dexamethasone treatment impair chondrogenesis and bone growth in an additive manner

Children with chronic inflammation are often treated with glucocorticoids (GCs) and many of them experience growth retardation. It is poorly understood how GCs interact with inflammatory cytokines causing growth failure as earlier experimental studies have been performed in healthy animals. To address this gap of knowledge, we used a transgenic mouse model where human TNF is overexpressed (huTNFTg) leading to chronic polyarthritis starting from the first week of age. Our results showed that femur bone length and growth plate height were significantly decreased in huTNFTg mice compared to wild type animals. In the growth plates of huTNFTg mice, increased apoptosis, suppressed Indian hedgehog, decreased hypertrophy, and disorganized chondrocyte columns were observed. Interestingly, the GC dexamethasone further impaired bone growth, accelerated chondrocyte apoptosis and reduced the number of chondrocyte columns in huTNFTg mice. We conclude that TNF and dexamethasone separately suppress chondrogenesis and bone growth when studied in an animal model of chronic inflammation. Our data give a possible mechanistic explanation to the commonly observed growth retardation in children with chronic inflammatory diseases treated with GCs.

Novel Peptides Derived from Sea Cucumber Intestine Promotes Osteogenesis by Upregulating Integrin-Mediated Transdifferentiation of Growth Plate Chondrocytes to Osteoblasts.

The sea cucumber intestine is a major by-product of sea cucumber processing and contains high levels of protein. In this study, we isolated and identified 28 novel osteogenic peptides from sea cucumber intestinal hydrolysis by the activity-tracking method for the first time. In vitro experimental results showed that compared with high molecular weight, the peptides from sea cucumber intestine (SCIP) with molecular weight <3 kDa more significantly promoted the proliferation and mineralized nodules of MC3T3-E1 cell and exhibited potential integrin binding capacity. In vivo experimental results showed that the SCIP supplement significantly increased the longitudinal bone length and elevated the height of the growth plate (especially the hypertrophic zone, 37.2%, p < 0.01) in adolescent mice. Further, immunofluorescence labeling results indicated that the SCIP supplement increased chondrocyte transdifferentiate to osteoblast in the growth plate close to the diaphysis. Mechanistically, transcriptome analysis revealed that the SCIP supplement induced the dedifferentiation of chondrocyte to osteoprogenitor cell via integrin-mediated histone acetylation and then redifferentiated to osteoblast via integrin-mediated Wnt/β-catenin signaling. These results reported for the first time that sea cucumber intestine had the potential to develop into a dietary supplement for promoting osteogenic, and provide new evidence for the mechanism of dietary promotes chondrocyte to osteoblast transdifferentiation.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, μCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.

Suppressor of fused-restrained Hedgehog signaling in chondrocytes is critical for epiphyseal growth plate maintenance and limb elongation in juvenile mice.

Hedgehog (Hh) signaling plays multiple critical roles in regulating chondrocyte proliferation and differentiation during epiphyseal cartilage development. However, it is still unclear whether Hh signaling in chondrocytes is required for growth plate maintenance during juvenile growth, and whether sustained activation of Hh signaling in chondrocytes promotes limb elongation. In this study, we first utilized Hh reporter mice to reveal that Hh signaling was activated in resting and columnar chondrocytes in growth plates of juvenile and adult mice. Next, we genetically modulated Hh signaling by conditionally deleting Smo or Sufu in all or a subpopulation of growth plate chondrocytes, and found that ablation of either Smo or Sufu in chondrocytes of juvenile mice caused premature closure of growth plates and shorter limbs, whereas Osx-Cre-mediated deletion of either of these two genes in prehypertrophic chondrocytes did not lead to obvious growth plate defects, indicating that Hh signaling mainly functions in resting and/or columnar chondrocytes to maintain growth plates at the juvenile stage. At the cellular level, we found that chondrocyte-specific ablation of Smo or Sufu accelerated or suppressed chondrocyte hypertrophy, respectively, whereas both decreased chondrocyte proliferation and survival. Thus, our study provided the first genetic evidence to establish the essential cell-autonomous roles for tightly-regulated Hh signaling in epiphyseal growth plate maintenance and limb elongation during juvenile growth.

Targeted Deletion of the Claudin12 Gene in Mice Increases Articular Cartilage and Inhibits Chondrocyte Differentiation.

To study the role of Claudin (CLDN)12 in bone, we developed mice with a targeted deletion of exon2 in the Cldn12 gene for skeletal phenotype analysis. Micro-CT analysis of the secondary spongiosa of distal femurs of mice with targeted disruption of the Cldn12 gene and control littermates showed no significant genotype-specific differences in either cortical or trabecular bone parameters for either gender in 13-week-old mice. Immunohistochemistry revealed that while CLDN12 was expressed in both differentiating chondrocytes and osteoblasts of the secondary spongiosa of 3-week-old wild-type mice, its expression was restricted to differentiating chondrocytes in the articular cartilage and growth plate in adult mice. Articular cartilage area at the knee were increased by 47% in Cldn12 knockout (KO) mice compared to control littermates. Micro-CT analyses found that while the trabecular number was increased by 9% and the trabecular spacing was reduced by 9% in the femoral epiphysis of Cldn12 KO mice, neither bone volume nor bone volume adjusted for tissue volume was different between the two genotypes. The expression levels of Clusterin, Lubricin and Mmp13 were increased by 56%, 46%, and 129%, respectively, in primary articular chondrocytes derived from KO compared to control mice. Our data indicate that targeted deletion of the Cldn12 gene in mice increases articular cartilage, in part, by promoting articular chondrocyte phenotype.

Juvenile mice exhibit inverse catch‐up growth but retain disorganized growth plates following a high‐fat to low‐fat diet intervention

Obesity can negatively impact the skeleton. We previously reported that a high‐fat diet causes rapid and dysregulated bone elongation. Our goal was to determine whether a diet intervention could mitigate or even reverse some of these effects on skeletal growth plates. We tested theHYPOTHESISthat rapid and dysregulated growth caused by a high‐fat diet will be mitigated by a low‐fat diet intervention. We expected to see a reduction in growth rate and changes in growth plate morphology that matched controls after switching to a low‐fat diet.METHODS3‐week old C57BL/6 mice (N=96 mixed sex) were fed control (10% kcal fat) or high‐fat (60% kcal fat) diets for one week. Some high‐fat diet mice were switched to control diet and examined at 5‐, 6‐, and 8‐weeks age (at least N=5/diet/age). Sex by diet interactions were tested at 5‐weeks. Body mass was recorded. Tibial elongation rate was measured from oxytetracycline labeled (7.5 mg/kg IP) slab sections. Growth plate height and columnar orientation (angle of proliferative column relative to long axis) were quantified in ImageJ. Statistical significance (p&lt;0.05) was determined in SPSS by ANOVA with post‐hoc tests.RESULTSThere were no sex by diet interactions, so females were analyzed separately to discriminate diet effects. High‐fat and intervention groups were both heavier than controls at 4‐weeks before the intervention (F=7.7, p&lt;.001). Body mass declined sharply after the diet switch and intervention mice were similar to controls at 5‐, 6‐, and 8‐weeks, while high‐fat diet mice remained heavier (p&lt;0.001). Tibial elongation rate was faster in high‐fat diet mice (F=12.2, p&lt;0.001), but slower in the intervention group compared to controls at both 5‐ (p&lt;.001) and 6‐weeks age (p&lt;0.01). Relative to controls, the most significant changes in growth plate height (F=10.7, p&lt;0.001) were enlarged growth plates in high‐fat diet mice at 5‐weeks (p&lt;0.001) and smaller growth plates in intervention mice at 6‐weeks (p=0.003). Chondrocyte columns in controls aligned within 2 degrees of the long axis at 5 weeks, while columns in both high‐fat and intervention mice deviated over 10 degrees (F=17.3, p&lt;0.001). Columns remained disorganized in high‐fat and intervention groups at 6‐ and 8‐weeks (p&lt;0.01).DISCUSSIONAfter switching from high‐ to low‐fat diet, intervention mice exhibited a rapid decrease in body mass, tibial elongation rate, and growth plate height. While we hypothesized that diet intervention would attenuate these to control levels, we surprisingly found that tibial elongation rate in the intervention group was actually slower than that of controls, resembling “inverse catch‐up growth” to achieve a potential target growth rate. However, chondrocyte columns remained disorganized, suggesting a possible lasting consequence of high‐fat diet.SIGNIFICANCEThese results demonstrate that the skeleton is exquisitely sensitive to diet in the earliest stages of postnatal bone elongation and are relevant for establishing interventions to prevent childhood obesity and its negative effects on bone structure.

Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum Phosphate and Improve Skeletal Abnormalities in Hyp Mice.

Excess fibroblast growth factor (FGF) 23 causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the fibroblast growth factor receptor/α-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design, we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogs with enhanced drug-like properties. In this study, we tested in a preclinical Hyp mouse homolog of XLH a direct connect analog [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (8n), which exhibited the greatest stability in microsomal assays, and [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] (13a), which exhibited increased in vitro potency. Using cryo-electron microscopy structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541, and its analogs (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation-fluorescence resonance energy transfer assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders. SIGNIFICANCE STATEMENT: This study used structure-based drug design and medicinal chemistry approaches to identify and optimize small molecules with different stability and potency, which antagonize excessive actions of fibroblast growth factor 23 (FGF23) in hereditary hypophosphatemic rickets. The findings confirmed that these antagonists bind to the N-terminus of FGF23 to inhibit its binding to and activation of the fibroblast growth factor receptors/α-Klotho signaling complex. Administration of these lead compounds improved phosphate homeostasis and abnormal skeletal phenotypes in a preclinical Hyp mouse model.