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Osteoarthritis (OA) is the most common joint disease. The surface of joint cartilage is a defensive and first affected structure of articular cartilage (AC) during the pathogenesis of OA. Alk5 signaling is critical for maintaining AC homeostasis, however, the role and underlying mechanism for the involvement of Alk5 signaling in the phenotypes of articular cartilage stem cells (ACSCs) at the surface of AC is still unclear. The role of Alk5 in OA development was explored using an ACSCs-specific Alk5-deficient (cKO) mouse model. Alterations in cartilage structure were evaluated histologically. Senescence was detected by SA-β-gal, while reactive oxygen species (ROS), MitoTracker, and LysoTracker staining were used to detect changes related to senescence. In addition, mice were injected intra-articularly with ganciclovir to limit the detrimental roles of senescent cells (SnCs). Alk5 cKO mice showed a decreased number of the slow-cell cycle cells and less lubricant secretion at the surface accompanied with drastically accelerated cartilage degeneration under ageing and surgically induced OA conditions. Further studies showed that Alk5 deficient ACSCs exhibited senescence-like manifestations including decreased proliferation and differentiation, more SA-β-gal-positive cells and ROS production, as well as significantly swollen mitochondria and lysosome breakdown. We further found that local limitation of the detrimental roles of SnCs can attenuate the development of posttraumatic OA. Taken together, our findings suggest that Alk5 signaling acts as an important regulator of the SnCs in the superficial layer during AC maintenance and OA initiation.

The clinical application of small interfering RNA (siRNA) drugs provides promising opportunities to develop treatment strategies for autoimmune inflammatory diseases. In this study, siRNAs targeting the endoplasmic reticulum to nucleus signaling 1 (ERN1) gene (siERN1) were screened. Two cationic polymers, polyethylenimine (PEI) and poly(β-amino amine) (PBAA), which can improve the efficiency of the siRNA transfection, were used as siERN1 delivery carriers. They were implemented to construct a nanodrug delivery system with macrophage-targeting ability and dual responsiveness for the treatment of autoimmune inflammatory diseases. In terms of the mechanism, siERN1 can regulate the intracellular calcium ion concentration by interfering with the function of inositol 1,4,5-trisphosphate receptor 1/3 (IP3R1/3) and thus inducing M2 polarization of macrophages. Furthermore, siERN1-nanoprodrug [FA (folic acid)-PEG-R(RKKRRQRRR)-NPs(ss-PBAA-PEI)@siERN1] acts as a conductor of macrophage polarization by controlling the calcium ion concentration and is an inhibitor of MyD88-dependent Toll-like receptor signaling. The results revealed that the FA-PEG-R-NPs@siERN1 has universal biocompatibility, long-term drug release responsiveness, superior targeting properties, and therapeutic effects in mouse collagen-induced arthritis and inflammatory bowel disease models. In conclusion, this study reveals a potential strategy to treat autoimmune inflammatory disorders.

Background Intervertebral disc degeneration (IVDD) can cause low back pain, a major public health concern. IVDD is characterized with loss of cells especially those in nucleus pulposus (NP), due to the limited proliferative potential and regenerative ability. Few studies, however, have been carried out to investigate the in vivo proliferation events of NP cells and the cellular contribution of a specific subpopulation of NP during postnatal growth or regeneration. Methods We generated FGFR3-3*Flag-IRES-GFP mice and crossed FGFR3-CreERT2 mice with Rosa26-mTmG, Rosa26-DTA and Rosa26-Confetti mice, respectively, to perform inducible genetic tracing studies. Results Expression of FGFR3 was found in the outer region of NP with co-localized expressions of proliferating markers. By fate mapping studies, FGFR3-positive (FGFR3+) NP cells were found proliferate from outer region to inner region of NP during postnatal growth. Clonal lineage tracing by Confetti mice and ablation of FGFR3·+ NP cells by DTA mice further revealed that the expansion of the FGFR3+ cells was required for the morphogenesis and homeostasis of postnatal NP. Moreover, in degeneration and regeneration model of mouse intervertebral disc, FGFR3+ NP cells underwent extensive expansion during the recovery stage. Conclusion Our present work demonstrates that FGFR3+ NP cells are novel subpopulation of postnatal NP with long-existing proliferative capacity shaping the adult NP structure and participating in the homeostasis maintenance and intrinsic repair of NP. These findings may facilitate the development of new therapeutic approaches for IVD regeneration.

Acquired heterotopic ossification (HO) is the extraskeletal bone formation after trauma. Various mesenchymal progenitors are reported to participate in ectopic bone formation. Here we induce acquired HO in mice by Achilles tenotomy and observe that conditional knockout (cKO) of fibroblast growth factor receptor 3 (FGFR3) in Col2+ cells promote acquired HO development. Lineage tracing studies reveal that Col2+ cells adopt fate of lymphatic endothelial cells (LECs) instead of chondrocytes or osteoblasts during HO development. FGFR3 cKO in Prox1+ LECs causes even more aggravated HO formation. We further demonstrate that FGFR3 deficiency in LECs leads to decreased local lymphatic formation in a BMPR1a-pSmad1/5-dependent manner, which exacerbates inflammatory levels in the repaired tendon. Local administration of FGF9 in Matrigel inhibits heterotopic bone formation, which is dependent on FGFR3 expression in LECs. Here we uncover Col2+ lineage cells as an origin of lymphatic endothelium, which regulates local inflammatory microenvironment after trauma and thus influences HO development via FGFR3-BMPR1a pathway. Activation of FGFR3 in LECs may be a therapeutic strategy to inhibit acquired HO formation via increasing local lymphangiogenesis., Different types of mesenchymal progenitors participate in ectopic bone formation. Here, the authors show Col2+ lineage cells adopt a lymphatic endothelium cell fate, which regulates local inflammatory microenvironment after trauma, thus influencing heterotopic ossification (HO) development via a FGFR3-BMPR1a pathway.

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.

ObjectivesThis study aims to investigate the role and mechanism of FGFR3 in macrophages and their biological effects on the pathology of arthritis.MethodsMice with conditional knockout of FGFR3 in myeloid cells (R3cKO) were generated. Gait behaviours of the mice were monitored at different ages. Spontaneous synovial joint destruction was evaluated by digital radiographic imaging and μCT analysis; changes of articular cartilage and synovitis were determined by histological analysis. The recruitment of macrophages in the synovium was examined by immunostaining and monocyte trafficking assay. RNA-seq analysis, Western blotting and chemotaxis experiment were performed on control and FGFR3-deficient macrophages. The peripheral blood from non-osteoarthritis (OA) donors and patients with OA were analysed. Mice were treated with neutralising antibody against CXCR7 to investigate the role of CXCR7 in arthritis.ResultsR3cKO mice but not control mice developed spontaneous cartilage destruction in multiple synovial joints at the age of 13 months. Moreover, the synovitis and macrophage accumulation were observed in the joints of 9-month-old R3cKO mice when the articular cartilage was not grossly destructed. FGFR3 deficiency in myeloid cells also aggravated joint destruction in DMM mouse model. Mechanically, FGFR3 deficiency promoted macrophage chemotaxis partly through activation of NF-κB/CXCR7 pathway. Inhibition of CXCR7 could significantly reverse FGFR3-deficiency-enhanced macrophage chemotaxis and the arthritic phenotype in R3cKO mice.ConclusionsOur study identifies the role of FGFR3 in synovial macrophage recruitment and synovitis, which provides a new insight into the pathological mechanisms of inflammation-related arthritis.

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.

Apert syndrome (AS) is a common genetic syndrome in humans characterized with craniosynostosis. Apert patients and mouse models showed abnormalities in sutures, cranial base and brain, that may all be involved in the pathogenesis of skull malformation of Apert syndrome. To distinguish the differential roles of these components of head in the pathogenesis of the abnormal skull morphology of AS, we generated mouse strains specifically expressing mutant FGFR2 in chondrocytes, osteoblasts, and progenitor cells of central nervous system (CNS) by crossing Fgfr2+/P253R-Neo mice with Col2a1-Cre, Osteocalcin-Cre (OC-Cre), and Nestin-Cre mice, respectively. We then quantitatively analyzed the skull and brain morphology of these mutant mice by micro-CT and micro-MRI using Euclidean distance matrix analysis (EDMA). Skulls of Col2a1-Fgfr2+/P253R mice showed Apert syndrome-like dysmorphology, such as shortened skull dimensions along the rostrocaudal axis, shortened nasal bone, and evidently advanced ossification of cranial base synchondroses. The OC-Fgfr2+/P253R mice showed malformation in face at 8-week stage. Nestin-Fgfr2+/P253R mice exhibited increased dorsoventral height and rostrocaudal length on the caudal skull and brain at 8 weeks. Our study indicates that the abnormal skull morphology of AS is caused by the combined effects of the maldevelopment in calvarias, cranial base, and brain tissue. These findings further deepen our knowledge about the pathogenesis of the abnormal skull morphology of AS, and provide new clues for the further analyses of skull phenotypes and clinical management of AS.

Summary: PTH stimulates bone formation in Fgfr3 knockout mice through promotion of proliferation and differentiation in osteoblasts Introduction: Previous studies showed that endogenous fibroblast growth factor 2 (FGF-2) is required for parathyroid hormone (PTH)-stimulated bone anabolic effects, however, the exact mechanisms by which PTH stimulate bone formation and the function of FGF receptors in mediating these actions are not fully defined. FGF receptor 3 (FGFR3) has been characterized as an important regulator of bone metabolism and is confirmed to cross-talk with PTH/PTHrP signal in cartilage and bone development. Methods: Fgfr3 knockout and wild-type mice at 2-month-old and 4-month-old were intraperitoneally injected with PTH intermittently for 4 weeks and then the skeletal responses to PTH were assessed by dual energy X-ray absorptiometry (DEXA), micro-computed tomography (μCT) and bone histomorphometry. Results: Intermittent PTH treatment improved bone mineral density (BMD) and femoral mechanical properties in both Fgfr3-/- and wild-type mice. Histomorphometric analysis showed that bone formation and bone resorption were increased in both genotypes following PTH treatment. PTH treatment increased trabecular bone volume (BV/TV) in WT and Fgfr3-deficient mice. The anabolic response in Fgfr3-deficient and wild-type bone is characterized by an increase of both bone formation and resorption-related genes following PTH treatment. In addition, we found that Fgfr3 null osteoblasts (compared to wild-type controls) maintained normal abilities to response to PTH-stimulated increase of proliferation, differentiation, expression of osteoblastic marker genes (Cbfa1, Osteopontin and Osteocalcin), and phosphorylation of Erk1/2. Conclusions: Bone anabolic effects of PTH were not impaired by the absence of FGFR3, suggesting that the FGFR3 signaling may not be required for osteoanabolic effects of PTH activities. Refereed/Peer-reviewed

Bone fracture healing is processed through multiple biological stages that partly recapitulates the skeletal development process. FGFR3 is a negative regulator of chondrogenesis during embryonic stage and plays an important role in both chondrogenesis and osteogenesis. We have investigated the role of FGFR3 in fracture healing using unstabilized fracture model and found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis during cartilage callus formation. Here, we created closed, stabilized proximal tibia fractures with an intramedullary pin in Fgfr3-/-mice and their littermate wild-type mice. Fracture healing was evaluated by radiography, micro-CT, histology, and real-time polymerase chain reaction (RT-PCR) analysis. The fractured Fgfr3-/- mice had increased formation of cartilaginous callus, more fracture callus, and more rapid endochondral ossification in fracture sites with up-regulated expressions of chondrogenesis related gene. The fractures of Fgfr3-/- mice healed faster with accelerated fracture callus mineralization and up-regulated expression of osteoblastogenic genes. The healing of fractures in Fgfr3-/- mice was accelerated in the stage of formation of cartilage and endochondral ossification. Downregulation of FGFR3 activity can be considered as a potential bio-therapeutic strategy for fracture treatment.

The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1 (FGFR1) in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis (OA). The goal of the current study was to investigate the effect of a novel non-ATP-competitive FGFR1 inhibitor, G141, on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA. G141 was screened and identified via cell-free kinase-inhibition assay. In the in vitro study, G141 decreased the mRNA levels of catabolic markers ADAMTS-5 and MMP-13, the phosphorylation of Erk1/2, JNK and p38 MAPK and the protein level of MMP-13 in human articular chondrocytes. In the ex vivo study, proteoglycan loss was markedly reduced in G141 treated human cartilage explants. For the in vivo study, intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus (DMM) induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice. Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression.

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a common degenerative disease in adult, which is characterized by progressive destruction of the articular cartilage. To investigate the role of FGFR3 in the homeostasis of TMJ cartilage during adult stage, we generated Fgfr3f/f; Col2a1-CreERT2 (Fgfr3 cKO) mice, in which Fgfr3 was deleted in chondrocytes at 2 months of age. OA-like defects were observed in Fgfr3 cKO TMJ cartilage. Immunohistochemical staining and quantitative real-time PCR analyses revealed a significant increase in expressions of COL10, MMP13 and AMAMTS5. In addition, there was a sharp increase in chondrocyte apoptosis at the Fgfr3 cKO articular surface, which was accompanied by a down-regulation of lubricin expression. Importantly, the expressions of RUNX2 and Indian hedgehog (IHH) were up-regulated in Fgfr3 cKO TMJ. Primary Fgfr3 cKO chondrocytes were treated with IHH signaling inhibitor, which significantly reduced expressions of Runx2, Col10, Mmp13 and Adamts5. Furthermore, the IHH signaling inhibitor partially alleviated OA-like defects in the TMJ of Fgfr3 cKO mice, including restoration of lubricin expression and improvement of the integrity of the articular surface. In conclusion, our study proposes that FGFR3/IHH signaling pathway plays a critical role in maintaining the homeostasis of TMJ articular cartilage during adult stage.

In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for combination, deletion or insertion of DNA fragments.

Knock-out (KO) mouse models have been increasingly used to dissect the roles of genes in development, diseases, and injuries. The conventional KO approach allows study of the role of the targeted genes in all cells, but it sometimes results in embryonic lethality. Using the classical conditional KO approach, reseachers can avoid embryonic lethality, but they cannot modulate genes in a temporally controllable way. The inducible KO technique, which has been used to study the role of a gene in life processes at the adult stage, avoids the potential interfering role of changed structures and functions of the tissues/organs resulting from the early KO of the gene in the non-inducible conditional knock-out approach. However, it is difficult to develop clinically applicable therapies for some diseases or injuries based on the results obtained from inducible KO studies since the total summed role of the genes of interest in those diseases or injuries cannot be determined and, therefore, the potential therapeutic effects of the applied modulators of the activity of the targeted genes cannot be predicted. To solve this problem of the classical conditional and inducible KO approaches, researchers need to simultaneously knock out a gene in all cells locally-a process called the localized all-cell KO (LACKO) strategy. We describe the concept of this new strategy in detail in this article.

Achondroplasia (ACH) and thanatophoric dysplasia (TD) are caused by gain-of-function mutations of fibroblast growth factor receptor 3 (FGFR3) and they are the most common forms of dwarfism and lethal dwarfism, respectively. Currently, there are few effective treatments for ACH. For the neonatal lethality of TD patients, no practical effective therapies are available. We here showed that systemic intermittent PTH (1-34) injection can rescue the lethal phenotype of TD type II (TDII) mice and significantly alleviate the retarded skeleton development of ACH mice. PTH-treated ACH mice had longer naso-anal length than ACH control mice, and the bone lengths of humeri and tibiae were rescued to be comparable with those of wild-type control mice. Our study also found that the premature fusion of cranial synchondroses in ACH mice was partially corrected after the PTH (1-34) treatment, suggesting that the PTH treatment may rescue the progressive narrowing of neurocentral synchondroses that cannot be readily corrected by surgery. In addition, we found that the PTH treatment can improve the osteopenia and bone structure of ACH mice. The increased expression of PTHrP and down-regulated FGFR3 level may be responsible for the positive effects of PTH on bone phenotype of ACH and TDII mice. Refereed/Peer-reviewed

Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair, and may exert a beneficial effect on the clinical outcome of sepsis. Osteoblasts act as a component of "niche" in bone marrow, which provides a nest for stem/progenitor cells and are involved in the formation and maintenance of stem/progenitor cells. Fibroblast growth factor receptor 1 (FGFR1) can regulate osteoblast activity and influence bone mass. So we explored the role of FGFR1 in EPC mobilization. Male mice with osteoblast-specific knockout of Fgfr1 (Fgfr1(fl/fl);OC-Cre) and its wild-type littermates (Fgfr1(fl/fl) ) were used in this study. Mice intraperitoneally injected with lipopolysaccharide (LPS) were used to measure the number of circulating EPCs in peripheral blood and serum stromal cell-derived factor 1α (SDF-1α). The circulating EPC number and the serum level of SDF-1α were significantly higher in Fgfr1(fl/fl);OC-Cre mice than those in Fgfr1(fl/fl) mice after LPS injection. In cell culture system, SDF-1α level was also significantly higher in Fgfr1(fl/fl);OC-Cre osteoblasts compared with that in Fgfr1(fl/fl) osteoblasts after LPS treatment. TRAP staining showed that there was no significant difference between the osteoclast activity of septic Fgfr1(fl/fl) and Fgfr1(fl/fl);OC-Cre mice. This study suggests that targeted deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood through up-regulating SDF-1α secretion from osteoblasts.

Osteoarthritis (OA) is one of the most prevalent chronic joint diseases and is characterized by progressive cartilage destruction and insufficient extracellular matrix synthesis. Thus far, none of the strategies employed to prevent and treat OA have been effective. The final option for advanced OA is the surgical approach of total joint replacement (1). Studies have shown that changes in growth factor signalings and their downstream target genes may be involved in the development of OA (2–5). However, the pathogenic mechanisms of OA are still largely unknown. Better understanding of the molecular events in OA development would provide important information that could facilitate the identification of novel therapeutic targets for the prevention and treatment of OA. Chondrocytes are the only cell type in adult human articular cartilage in which the main responsibility is the synthesis and degradation of extracellular matrix (6). Numerous molecules and pathways, such as hypoxia-inducible factor 2α, discoidin domain receptor 2, and hedgehog signaling, in the articular chondrocytes have been shown to be involved in cartilage metabolism (7–11). Recently, the role of fibroblast growth factor (FGF) family members in the regulation of cartilage homeostasis has received specific attention (12). FGF-18 is a well-established anabolic growth factor that contributes to the metabolism of articular cartilage (13,14). Although several lines of evidence support the tight association between FGF-2 and articular cartilage metabolism, the role of FGF-2 in cartilage homeostasis is controversial (12,15). Some studies of human articular chondrocytes have demonstrated that FGF-2 stimulates the production of matrix metalloproteinase 13 (MMP-13), which is the main collagenase responsible for collagen degradation (16). Results of other studies have suggested that FGF-2 functions as a chondroprotective factor in cartilage homeostasis (17–19). It has been reported that FGF-2 can suppress interleukin-1 (IL-1)–induced catabolic effects on human cartilage (18). In mice with Fgf2 deficiency, spontaneous OA, as well as instability-induced OA, is accelerated (19). In response to tissue injury or mechanical compression, FGF-2 can be released from the extracellular matrix to activate intracellular ERK signaling and regulate expression of chondrocyte-specific genes, suggesting that FGF-2 plays a homeostatic role in articular chondrocytes (20). The involvement of FGF ligands in the maintenance of articular cartilage suggests that FGF receptors (FGFRs) may also play an important role in articular cartilage homeostasis. Notably, FGFR-1 and FGFR-3 are highly expressed in human articular chondrocytes and have been implicated in cartilage metabolism (21). Valverde-Franco et al showed that Fgfr3-knockout (KO) mice exhibited abnormal cartilage metabolism and early signs of OA, suggesting that FGFR-3 signaling may have a chondroprotective role during the development of OA (22). It is hypothesized that activation of FGFR-1 may exert antianabolic and procatabolic effects on adult human articular cartilage (12). However, since conditional deletion of Fgfr1 is known to have a lethal effect on mouse embryos (23), it is impossible to investigate the in vivo function of FGFR-1 in articular cartilage homeostasis using conventional Fgfr1-KO mice. In this study, we investigated the role of FGFR-1 in articular cartilage function postnatally, using mice with tamoxifen-inducible and cartilage-specific conditional knockout (cKO) of the Fgfr1 gene (hereinafter referred to as Fgfr1 cKO mice). We evaluated the effects of FGFR-1 on the function of articular chondrocytes and the degeneration of articular cartilage in mice using 2 models of OA, as well as an antigen-induced arthritis (AIA) model. Our study demonstrated that Fgfr1 deficiency in mice attenuates articular cartilage degeneration in all 3 arthritis models. In addition, we found that blockade of FGFR-1 signaling could antagonize the IL-1β–induced up-regulation of MMP-13 and enhance the expression of FGFR-3 in human articular chondrocytes.

Gain-of-function mutations in fibroblast growth factor receptor-3 (FGFR3) lead to several types of human skeletal dysplasia syndromes including achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Currently, there are no effective treatments for these skeletal dysplasia diseases. In this study, we screened, using FGFR3 as a bait, a random 12-peptide phage library and obtained 23 positive clones that share identical amino acid sequences (VSPPLTLGQLLS), named as peptide P3. This peptide had high binding specificity to the extracellular domain of FGFR3. P3 inhibited tyrosine kinase activity of FGFR3 and its typical downstream molecules, extracellular signal-regulated kinase/mitogen-activated protein kinase. P3 also promoted proliferation and chondrogenic differentiation of cultured ATDC5 chondrogenic cells. In addition, P3 alleviated the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII). Finally, P3 reversed the neonatal lethality of TDII mice. Thus, this study identifies a novel inhibitory peptide for FGFR3 signaling, which may serve as a potential therapeutic agent for the treatment of FGFR3-related skeletal dysplasia. Refereed/Peer-reviewed

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.

Most cartilaginous tumors are formed during skeletal development in locations adjacent to growth plates, suggesting that they arise from disordered endochondral bone growth. Fibroblast growth factor receptor (FGFR)3 signaling plays essential roles in this process; however, the role of FGFR3 in cartilaginous tumorigenesis is not known. In this study, we found that postnatal chondrocyte-specific Fgfr3 deletion induced multiple chondroma-like lesions, including enchondromas and osteochondromas, adjacent to disordered growth plates. The lesions showed decreased extracellular signal-regulated kinase (ERK) activity and increased Indian hedgehog (IHH) expression. The same was observed in Fgfr3-deficient primary chondrocytes, in which treatment with a mitogen-activated protein kinase (MEK) inhibitor increased Ihh expression. Importantly, treatment with an inhibitor of IHH signaling reduced the occurrence of chondroma-like lesions in Fgfr3-deficient mice. This is the first study reporting that the loss of Fgfr3 function leads to the formation of chondroma-like lesions via downregulation of MEK/ERK signaling and upregulation of IHH, suggesting that FGFR3 has a tumor suppressor-like function in chondrogenesis., Author Summary Benign cartilaginous tumors, especially osteochondromas and enchondromas, are the most common primary bone tumors in humans. Several hereditary diseases are characterized by the development of cartilaginous tumors, including hereditary multiple exostoses, metachondromatosis, and enchondromatosis, which are caused by mutations in genes such as exostosin 1 and 2, tyrosine protein phosphatase non-receptor type 11, parathyroid hormone receptor 1, and isocitrate dehydrogenase 1 and 2. The proteins encoded by these genes are directly or indirectly linked to fibroblast growth factor (FGF) signaling. In addition, osteochondroma was found in several members of a family with camptodactyly, tall stature, and hearing loss syndrome, a rare inherited disorder caused by a heterozygous missense mutation in Fgfr3. In this study, we found that Fgfr3 deficiency leads to the formation of cartilaginous tumors, including osteochondromas and enchondromas, likely due to dysregulated endochondral ossification in growth plates. We also show that cartilaginous tumorigenesis in Fgfr3-deficient mice results from excessive Indian hedgehog production mediated by the activation of mitogen-associated protein kinase signaling. Based on these results, we propose a model for cartilaginous tumor development in which FGFR3 functions as a tumor suppressor. Our findings also suggest that modulation of FGFR3 and related signaling pathways is a potential therapeutic strategy for treating benign cartilaginous tumors.