Zuqiang Wang

FGFR3 Deficiency Causes Multiple Chondroma-like Lesions by Upregulating Hedgehog Signaling.

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.

Conditional Deletion of Fgfr3 in Chondrocytes leads to Osteoarthritis-like Defects in Temporomandibular Joint of Adult Mice

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.

A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis.

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.

Fibroblast Growth Factor Receptor 3 Inhibits Osteoarthritis Progression in the Knee Joints of Adult Mice.

Fibroblast growth factor (FGF) signaling is involved in articular cartilage homeostasis. This study was undertaken to investigate the role and mechanisms of FGF receptor 3 (FGFR‐3) in the pathogenesis of osteoarthritis (OA) caused by surgery and aging in mice.

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL-1β/NF-κB pathway

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.

A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice

OBJECTIVE We previously reported that genetic ablation of (Fibroblast Growth Factors Receptors) FGFR1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice, which suggests that FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM). DESIGN Binding ability between R1-P1 and FGFR1 protein was evaluated by enzyme-linked immuno sorbent assay (ELISA) and molecular docking. Alterations in cartilage were evaluated histologically. The expression levels of molecules associated with articular cartilage homeostasis and FGFR1 signaling were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC). The chondrocyte apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently inhibited extracellular signal-regulated kinase (ERK)1/2 pathway in mouse primary chondrocytes. In addition, R1-P1 attenuated the IL-1β induced significant loss of proteoglycan in full-thickness cartilage tissue from human femur head. Moreover, this peptide can significantly restore the IL-1β mediated loss of proteoglycan and type II collagen (Col II) and attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the loss of proteoglycan and the destruction of articular cartilage and decreased the expressions of extracellular matrix (ECM) degrading enzymes and apoptosis in articular chondrocytes of mice underwent DMM surgery. CONCLUSIONS R1-P1, a novel inhibitory peptide for FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a potential leading molecule for the treatment of OA.

Adeno-Associated Virus-Mediated RNAi against Mutant Alleles Attenuates Abnormal Calvarial Phenotypes in an Apert Syndrome Mouse Model

Apert syndrome (AS), the most severe form of craniosynostosis, is caused by missense mutations including Pro253Arg(P253R) of fibroblast growth factor receptor 2 (FGFR2), which leads to enhanced FGF/FGFR2-signaling activity. Surgical correction of the deformed skull is the typical treatment for AS. Because of constant maldevelopment of sutures, the corrective surgery is often executed several times, resulting in increased patient challenge and complications. Biological therapies targeting the signaling of mutant FGFR2 allele, in combination with surgery, may bring better outcome. Here we screened and found a small interfering RNA (siRNA) specifically targeting the Fgfr2-P253R allele, and we revealed that it inhibited osteoblastic differentiation and matrix mineralization by reducing the signaling of ERK1/2 and P38 in cultured primary calvarial cells and calvarial explants from Apert mice (Fgfr2+/P253R). Furthermore, AAV9 carrying short hairpin RNA (shRNA) (AAV9-Fgfr2-shRNA) against mutant Fgfr2 was delivered to the skulls of AS mice. Results demonstrate that AAV9-Fgfr2-shRNA attenuated the premature closure of coronal suture and the decreased calvarial bone volume of AS mice. Our study provides a novel practical biological approach, which will, in combination with other therapies, including surgeries, help treat patients with AS while providing experimental clues for the biological therapies of other genetic skeletal diseases.

Postnatal deletion of Alk5 gene in meniscal cartilage accelerates age-dependent meniscal degeneration in mice: WANG et al.

Activation of transforming growth factor‐β (TGF‐β) signaling has been used to enhance healing of meniscal degeneration in several models. However, the exact role and molecular mechanism of TGF‐β signaling in meniscus maintenance and degeneration are still not understood due to the absence of in vivo evidence. In this study, we found that the expression of activin receptor‐like kinases 5 (ALK5) in the meniscus was decreased with the progression of age and/or osteoarthritis induced meniscal degeneration. Col2α1 positive cells were found to be specifically distributed in the superficial and inner zones of the anterior horn, as well as the inner zone of the posterior horn in mice, indicating that Col2α1‐CreERT2 mice can be a used for studying gene function in menisci. Furthermore, we deleted Alk5 in Col2α1 positive cells in meniscus by administering tamoxifen. Alterations in the menisci structure were evaluated histologically. The expression levels of genes and proteins associated with meniscus homeostasis and TGF‐β signaling were analyzed by quantitative real‐time PCR analysis (qRT‐PCR) and immunohistochemistry (IHC). Our results revealed severe and progressive meniscal degeneration phenotype in 3‐ and 6‐month‐old Alk5 cKO mice compared with Cre‐negative control, including aberrantly increased hypertrophic meniscal cells, severe fibrillation, and structure disruption of meniscus. qRT‐PCR and IHC results showed that disruption of anabolic and catabolic homeostasis of chondrocytes may contribute to the meniscal degeneration phenotype observed in Alk5 cKO mice. Thus, TGF‐β/ALK5 signaling plays a chondro‐protective role in menisci homeostasis, in part, by inhibiting matrix degradation and maintaining extracellular matrix proteins levels in meniscal tissues.

A novel FGFR1-binding peptide exhibits anti-tumor effect on lung cancer by inhibiting proliferation and angiogenesis

It has been reported that overactivation of fibroblast growth factor receptor 1 (FGFR1) is an important characteristic found in most non-small cell lung cancer (NSCLC) samples. Here, we identified a FGFR1 inhibitory peptide R1-P2 and investigated its effects on the lung cancer cells growth and angiogenesis in vitro and in vivo. Our results demonstrate that R1-P2 bound to human FGFR1 protein, and efficiently blocked the binding of FGF2 to FGFR1 in A549 and NCI-H460 cells. Moreover, this peptide significantly decreased the proliferation, migration and invasion, but promoted the apoptosis in both cell lines. In addition, R1-P2 treatment effectively inhibited the tumor growth and neovascularization in nude mice with xenografted A549 cells, and R1-P2 also significantly inhibited the FGF2-induced angiogenesis in tube formation experiment and CAM model. We further demonstrated that R1-P2 suppressed lung tumor growth through anti-angiogenic and anti-proliferative activity. Our data may provide a novle leading molecule with potential application in the treatment of FGFR1 activation related lung cancers.

Inducible activation of FGFR2 in adult mice promotes bone formation after bone marrow ablation

Apert syndrome is one of the most severe craniosynostoses, resulting from gain‐of‐function mutations in fibroblast growth factor receptor 2 (FGFR2). Previous studies have shown that gain‐of‐function mutations of FGFR2 (S252W or P253R) cause skull malformation of human Apert syndrome by affecting both chondrogenesis and osteogenesis, underscoring the key role of FGFR2 in bone development. However, the effects of FGFR2 on bone formation at the adult stage have not been fully investigated. To investigate the role of FGFR2 in bone formation, we generated mice with tamoxifen‐inducible expression of mutant FGFR2 (P253R) at the adult stage. Mechanical bone marrow ablation (BMX) was performed in both wild‐type and Fgfr2 mutant (MT) mice. Changes in newly formed trabecular bone were assessed by micro‐computed tomography and bone histomorphometry. We found that MT mice exhibited increased trabecular bone formation and decreased bone resorption after BMX accompanied with a remarkable increase in bone marrow stromal cell recruitment and proliferation, osteoblast proliferation and differentiation, and enhanced Wnt/β‐catenin activity. Furthermore, pharmacologically inhibiting Wnt/β‐catenin signaling can partially reverse the increased trabecular bone formation and decreased bone resorption in MT mice after BMX. Our data demonstrate that gain‐of‐function mutation in FGFR2 exerts a Wnt/β‐catenin‐dependent anabolic effect on trabecular bone by promoting bone formation and inhibiting bone resorption at the adult stage.