run in TBE £0.5 at room temperature for 2 h at 150 V. The following two (Q/q) 27-bp unmethylated oligonucleotides were used: 5 0 -GATCCTTCGCCTAGGCTC(A/G)CAGCG CGGGAGCGA-3 0 . A methylated q probe (q*) was generated by incorporating a methylated cytosine at the mutated CpG site during oligonucleotide synthesis. Transient transfection assayThe constructs contained 578 bp from IGF2 intron 3 (nucleotides 2868-3446), followed by the IGF2 P3 promoter (nucleotides 2222 to þ45 relative to the start of transcription) 12 and a luciferase reporter. C2C12 myoblast cells were grown to approximately 80% confluence. Cells were transiently co-transfected with the firefly luciferase reporter construct (4 mg) and a Renilla luciferase control vector (phRG-TK, Promega; 80 ng) using 10 mg Lipofectamine 2000 (Invitrogen). Cells were incubated for 25 h before lysis in 100 ml Triton lysis solution. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The results are based on four triplicate experiments using two independent plasmid preparations for each construct. Statistical analysis was done with an analysis of variance.

Understanding the molecular mechanisms by which cartilage formation is regulated is essential toward understanding the physiology of both embryonic bone development and postnatal bone growth. Although much is known about growth factor signaling in cartilage formation, the regulatory role of noncollagenous matrix proteins in this process are still largely unknown. In the present studies, we present evidence for a critical role of DMP1 (dentin matrix protein 1) in postnatal chondrogenesis. The Dmp1 gene was originally identified from a rat incisor cDNA library and has been shown to play an important role in late stage dentinogenesis. Whereas no apparent abnormalities were observed in prenatal bone development, Dmp1-deficient (Dmp1 ؊/؊ ) mice unexpectedly develop a severe defect in cartilage formation during postnatal chondrogenesis. Vertebrae and long bones in Dmp1-deficient (Dmp1 ؊/؊ ) mice are shorter and wider with delayed and malformed secondary ossification centers and an irregular and highly expanded growth plate, results of both a highly expanded proliferation and a highly expanded hypertrophic zone creating a phenotype resembling dwarfism with chondrodysplasia. This phenotype appears to be due to increased cell proliferation in the proliferating zone and reduced apoptosis in the hypertrophic zone. In addition, blood vessel invasion is impaired in the epiphyses of Dmp1 ؊/؊ mice. These findings show that DMP1 is essential for normal postnatal chondrogenesis and subsequent osteogenesis.

Dentin Matrix Protein 1 (Dmp1) was originally identified from dentin. However, its expression and function in vivo are not clear. To clarify these two issues, we have generated mice carrying a truncated Dmp1 gene by using gene targeting to replace exon 6 with a lacZ gene. Northern blot analysis shows the expected 5.8-kb Dmp1-lacZ fusion transcript and loss of the wild-type 2.8-kb Dmp1 transcript, confirmed by a lack of immunostaining for the protein. Using heterozygous animals, we demonstrate that Dmp1 is specific for mineralized tissues. Not previously shown, Dmp1 is also expressed in pulp cells. Dmp1-deficient embryos and newborns display no apparent gross abnormal phenotype, although there are a modest expansion of the hypertrophic chondrocyte zone and a modest increase in the long bone diameter. This suggests that DMP1 is not essential for early mouse skeletal or dental development.

Synovitis, a common clinical symptom for osteoarthritis (OA) patients, is highly related to OA pathological progression and pain manifestation. The activated synovial macrophages have been demonstrated to play an important role in synovitis, but the mechanisms about macrophage activation are still not clear. In this study, we found that the exosome-like vesicles from osteoarthritic chondrocytes could be a new biological factor to stimulate inflammasome activation and increase mature IL-1β production in macrophages. The degraded cartilage explants produced more exosome-like vesicles than the nondegraded ones, while the exosome-like vesicles from chondrocytes could enter into joint synovium tissue and macrophages. Moreover, the exosome-like vesicles from osteoarthritic chondrocytes enhanced the production of mature IL-1β in macrophages. These vesicles could inhibit ATG4B expression via miR-449a-5p, leading to inhibition of autophagy in LPS-primed macrophages. The decreased autophagy promoted the production of mitoROS, which further enhanced the inflammasome activation and subsequent IL-1β processing. Ultimately, the increase of mature IL-1β may aggravate synovial inflammation and promote the progression of OA disease. Our study provides a new perspective to understand the activation of synovial macrophages and synovitis in OA patients, which may be beneficial for therapeutic intervention in synovitis-related OA patients.

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.

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.

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.

The purpose of this study was to evaluate the negative influence of excessive exercise on immunity, substance and energy metabolism as well as gut microbiota in mice. Firstly, an overtraining model of Male Kunming mice was established by high-intensity swimming exercise for 4 weeks. Then, a series of evaluation indicators, including the routine blood analysis, immune organ coefficient, digestive enzymes, and aquaporins expression levels of small intestine and colon tissue, histological examinations of liver, spleen, small intestine, and colon, were determined based on this model. Furthermore, 16S rRNA gene sequencing was also employed to measure the microbial composition in gut. The results found that immune parameters, substance and energy metabolism of all mice was altered and disturbed after high-intensity swimming for 4 weeks, led to an atrophy of thymus and spleen as well as abnormal structural changes in liver when compared to non-swimming mice. Besides, excessive swimming mice had lower microbial diversity compared to non-swimming mice. However, there was no significant difference in gut microbial taxa between the two groups. The data indicated that excessive exercise exhibits negative impacts on immunity, substance and energy metabolism as well as gut microbial diversity.