Transcription factor Meis1b regulates craniofacial morphogenesis in zebrafish.

BACKGROUND: Meis family of transcription factors operates in Pbx-Meis-Hox regulatory network controlling development of various tissues including eye, limbs, heart, hindbrain or craniofacial skeletal elements originating from the neural crest. Although studies in mouse provide abundant information about Meis factors function in embryogenesis, little is known about their role in zebrafish. RESULTS: We generated zebrafish lines carrying null mutations in meis1a, meis1b, meis2a, and meis2b genes. Only meis1b mutants are lethal at larval stage around 13 dpf whereas the other mutant lines are viable and fertile. We focused on development of neural crest-derived craniofacial structures such as tendons, cranial nerves, cartilage and accompanying muscles. Meis1b mutants displayed morphogenetic abnormalities in the cartilage originating from the first and second pharyngeal arches. Meckel's cartilage was shorter and wider with fused anterior symphysis and abnormal chondrocyte organization. This resulted in impaired tendons and muscle fiber connections while tenocyte development was not largely affected. CONCLUSIONS: Loss-of-function mutation in meis1b affects cartilage morphology in the lower jaw that leads to disrupted organization of muscles and tendons.

Relationships between matrix mineralization, oxidative metabolism, and mitochondrial structure during ATDC5 murine chondroprogenitor cell line differentiation.

The mechanistic relationships between the progression of growth chondrocyte differentiation, matrix mineralization, oxidative metabolism, and mitochondria content and structure were examined in the ATDC5 murine chondroprogenitor cell line. The progression of chondrocyte differentiation was associated with a statistically significant (p ≤ 0.05) ~2-fold increase in oxidative phosphorylation. However, as matrix mineralization progressed, oxidative metabolism decreased. In the absence of mineralization, cartilage extracellular matrix mRNA expression for Col2a1, Aggrecan, and Col10a1 were statistically (p ≤ 0.05) ~2-3-fold greater than observed in mineralizing cultures. In contrast, BSP and Phex that are associated with promoting matrix mineralization showed statistically (p ≤ 0.05) higher ~2-4 expression, while FGF23 phosphate regulatory factor was significantly lower (~50%) in mineralizing cultures. Cultures induced to differentiate under both nonmineralizing and mineralizing media conditions showed statistically greater basal oxidative metabolism and ATP production. Maximal respiration and spare oxidative capacity were significantly elevated (p ≤ 0.05) in differentiated nonmineralizing cultures compared to those that mineralized. Increased oxidative metabolism was associated with both an increase in mitochondria volume per cell and mitochondria fusion, while mineralization diminished mitochondrial volume and appeared to be associated with fission. Undifferentiated and mineralized cells showed increased mitochondrial co-localization with the actin cytoskeletal. Examination of proteins associated with mitochondria fission and apoptosis and mitophagy, respectively, showed levels of immunological expression consistent with the increasing fission and apoptosis in mineralizing cultures. These results suggest that chondrocyte differentiation is associated with intracellular structural reorganization, promoting increased mitochondria content and fusion that enables increased oxidative metabolism. Mineralization, however, does not need energy derived from oxidative metabolism; rather, during mineralization, mitochondria appear to undergo fission and mitophagy. In summary, these studies show that as chondrocytes underwent hypertrophic differentiation, they increased oxidative metabolism, but as mineralization proceeds, metabolism decreased. Mitochondria structure also underwent a structural reorganization that was further supportive of their oxidative capacity as the chondrocytes progressed through their differentiation. Thus, the mitochondria first underwent fusion to support increased oxidative metabolism, then underwent fission during mineralization, facilitating their programed death.

Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

BACKGROUND: Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. METHODS AND RESULTS: Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. CONCLUSIONS: These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

Novel Function of Osteocalcin in Chondrocyte Differentiation and Endochondral Ossification Revealed on a CRISPR/Cas9 bglap-bglap2 Deficiency Mouse Model.

Endochondral ossification is the process by which cartilage is mineralized into bone, and is essential for the development of long bones. Osteocalcin (OCN), a protein abundant in bone matrix, also exhibits high expression in chondrocytes, especially hypertrophic chondrocytes, while its role in endochondral ossification remains unclear. Utilizing a new CRISPR/Cas9-mediated bglap-bglap2 deficiency (OCNem) mouse model generated in our laboratory, we provide the first evidence of OCN's regulatory function in chondrocyte differentiation and endochondral ossification. The OCNem mice exhibited significant delays in primary and secondary ossification centers compared to wild-type mice, along with increased cartilage length in growth plates and hypertrophic zones during neonatal and adolescent stages. These anomalies indicated that OCN deficiency disturbed endochondral ossification during embryonic and postnatal periods. Mechanism wise, OCN deficiency was found to increase chondrocyte differentiation and postpone vascularization process. Furthermore, bone marrow mesenchymal stromal cells (BMSCs) from OCNem mice demonstrated an increased capacity for chondrogenic differentiation. Transcriptional network analysis implicated that BMP and TGF-ß signaling pathways were highly affected in OCNem BMSCs, which is closely associated with cartilage development and maintenance. This elucidation of OCN's function in chondrocyte differentiation and endochondral ossification contributes to a more comprehensive understanding of its impact on skeletal development and homeostasis.

Glycosphingolipids in Osteoarthritis and Cartilage-Regeneration Therapy: Mechanisms and Therapeutic Prospects Based on a Narrative Review of the Literature.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell-surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolisms and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Collagen-Derived Dipeptide Pro-Hyp Enhanced ATDC5 Chondrocyte Differentiation under Hypoxic Conditions.

Chondrocytes are surrounded by a lower oxygen environment than other well-vascularized tissues with higher oxygenation levels. Prolyl-hydroxyproline (Pro-Hyp), one of the final collagen-derived peptides, has been previously reported to be involved in the early stages of chondrocyte differentiation. However, whether Pro-Hyp can alter chondrocyte differentiation under physiological hypoxic conditions is still unclear. This study aimed to investigate whether Pro-Hyp affects the differentiation of ATDC5 chondrogenic cells under hypoxic conditions. The addition of Pro-Hyp resulted in an approximately 18-fold increase in the glycosaminoglycan staining area compared to the control group under hypoxic conditions. Moreover, Pro-Hyp treatment significantly upregulated the expression of SOX9, Col2a1, Aggrecan, and MMP13 in chondrocytes cultured under hypoxic conditions. These results demonstrate that Pro-Hyp strongly promotes the early differentiation of chondrocytes under physiological hypoxic conditions. Therefore, Pro-Hyp, a bioactive peptide produced during collagen metabolism, may function as a remodeling factor or extracellular matrix remodeling signal that regulates chondrocyte differentiation in hypoxic cartilage.

FoxO3a cooperates with RUNX1 to promote chondrogenesis and terminal hypertrophic of the chondrogenic progenitor cells.

FoxO transcription factors (FoxOs) have recently been shown to protect against chondrocyte dysfunction and modulate cartilage homeostasis in osteoarthritis. The mechanism underlying of FoxOs regulate chondrocyte differentiation remains unknown. Runt related transcription factor 1 (RUNX1) mediated both chondrocyte and osteoblast differentiation. Our data showed that FoxO3a and RUNX1 are co-expressed in ATDC5 cells and undifferentiated mesenchyme cells and have similar high levels in chondrocytes undergoing transition from proliferation to hypertrophy. Overexpression of FoxO3a in ATDC5 cells or mouse mesenchymal cells resulted in a potent induction of the chondrocyte differentiation markers. Knockdown FoxO3a or RUNX1 potently inhibits the expressions of chondrocyte differentiation markers, including Sox9, Aggrecan, Col2, and hypertrophic chondrocyte markers including RUNX2, ColX, MMP13 and ADAMTs-5 in ATDC5 cells. Co-immunoprecipitation showed that FoxO3a binds the transcriptional regulator RUNX1. Immunohistochemistry showed that FoxO3a and RUNX1 are highly co-expressed in the proliferative chondrocytes of the growth plates in the hind limbs of newborn mice. Collectively, we revealed that FoxO3a cooperated with RUNX1 promoted chondrocyte differentiation through enhancing both early chondrogenesis and terminal hypertrophic of the chondrogenic progenitor cells, indicating FoxO3a interacting with RUNX1 may be a therapeutic target for the treatment of osteoarthritis and other bone diseases.

Whole Aspect of Runx2 Functions in Skeletal Development.

Runt-related transcription factor 2 (Runx2) is a fundamental transcription factor for bone development. In endochondral ossification, Runx2 induces chondrocyte maturation, enhances chondrocyte proliferation through Indian hedgehog (Ihh) induction, and induces the expression of vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) in the terminal hypertrophic chondrocytes. Runx2 inhibits the apoptosis of the terminal hypertrophic chondrocytes and induces their transdifferentiation into osteoblasts and osteoblast progenitors. The transdifferentiation is required for trabecular bone formation during embryonic and newborn stages but is dispensable for acquiring normal bone mass in young and adult mice. Runx2 enhances the proliferation of osteoblast progenitors and induces their commitment to osteoblast lineage cells through the direct regulation of the expressions of a hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathway genes and distal-less homeobox 5 (Dlx5), which all regulate Runx2 expression and/or protein activity. Runx2, Sp7, and Wnt signaling further induce osteoblast differentiation. In immature osteoblasts, Runx2 regulates the expression of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and induces osteoblast maturation. Osteocalcin (Bglap/Bglap2) is required for the alignment of apatite crystals parallel to the collagen fibers; however, it does not physiologically work as a hormone that regulates glucose metabolism, testosterone synthesis, or muscle mass. Thus, Runx2 exerts multiple functions essential for skeletal development.

Regulation of FGF-2, FGF-18 and Transcription Factor Activity by Perlecan in the Maturational Development of Transitional Rudiment and Growth Plate Cartilages and in the Maintenance of Permanent Cartilage Homeostasis.

The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environments. Nuclear perlecan may modulate chromatin structure and transcription factor access to DNA and gene regulation. Snail-1, a mesenchymal marker and transcription factor, signals through FGFR-3 to promote chondrogenesis and maintain Acan and type II collagen levels in articular cartilage, but prevents further tissue expansion. Pre-hypertrophic growth plate chondrocytes also express high Snail-1 levels, leading to cessation of Acan and CoI2A1 synthesis and appearance of type X collagen. Perlecan differentially regulates FGF-2 and FGF-18 to maintain articular cartilage homeostasis, rudiment and growth plate cartilage growth, and maturational changes including mineralization, contributing to skeletal growth.

Wnt5a is a transcriptional target of Gli3 and Trps1 at the onset of chondrocyte hypertrophy.

During endochondral ossification, the differentiation of proliferating into hypertrophic chondrocytes is a key step determining the pace of bone formation and the future length of the skeletal elements. A variety of transcription factors are expressed at the onset of hypertrophy coordinating the expression of different signaling molecules like Bmps, Ihh and Wnt proteins. In this study, we characterized the murine Wnt5a promoter and provide evidence that two alternative Wnt5a transcripts, Ts1 and Ts2, are differentially expressed in the developing skeletal elements. Ts2 expression decreases while Ts1 expression increases during chondrocyte differentiation. The transcription factor Trps1 and the activator form of Gli3 (Gli3A), which is a mediator of Hedgehog signaling, activate Wnt5a expression. In Chromatin Immunoprecipitation and reporter gene assays, we identified two upstream regulatory sequences (URS) in the Wnt5a promoter mediating either activating or repressive functions. The activating URS1 is bound by Trps1 and Gli3A in vitro and in vivo to upregulate Wnt5a expression. Loss of both transcription factors decreases endogenous Wnt5a mRNA and protein levels during chondrocyte differentiation, thereby identifying Wnt5a as a target gene of Trps1 and Gli3A in chondrocytes.

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

Histological and molecular characterization of the growing nasal septum in mice.

The nasal septum is a cartilaginous structure that serves as a pacemaker for the development of the midface. The septum is a hyaline cartilage which is surrounded by a perichondrium and epithelium. It remains cartilaginous anteriorly, but posteriorly it undergoes endochondral ossification to form the perpendicular plate of the ethmoid. Understanding of hyaline cartilage differentiation stems predominantly from investigations of growth plate cartilage. It is currently unclear if the morphological and molecular properties of the differentiating nasal septum align with what is known from the growth plate. In this study, we describe growth, molecular, and cellular characteristics of the nasal septum with reference to hyaline cartilage differentiation. The nasal septum grows asynchronous across its length with phases of rapid growth interrupted by more stagnant growth. Growth appears to be driven predominantly by acquisition of chondrocyte hypertrophy. Similarly, cellular differentiation is asynchronous, and differentiation observed in the anterior part precedes posterior differentiation. Overall, the nasal septum is structurally and molecularly heterogeneous. Early and extensive chondrocyte hypertrophy but no ossification is observed in the anterior septum. Onset of hypertrophic chondrocyte differentiation coincided with collagen fiber deposition along the perichondrium. Sox9, Col2, Col10, Mmp13, Sp7, and Runx2 expression was heterogeneous and did not always follow the expected pattern established from chondrocyte differentiation in the growth plate. The presence of hypertrophic chondrocytes expressing bone-related proteins early on in regions where the nasal septum does not ossify displays incongruities with current understanding of hyaline cartilage differentiation. Runx2, Collagen II, Collagen X, and Sp7 commonly used to mark distinct stages of chondrocyte maturation and early bone formation show wider expression than expected and do not align with expected cellular characteristics. Thus, the hyaline cartilage of the nasal septum is quite distinct from growth plate hyaline cartilage, and caution should be taken before assigning cartilage properties to less well-defined cartilage structures using these commonly used markers. Beyond the structural description of the nasal cartilage, this study also provides important information for cartilage tissue engineering when using nasal septal cartilage for tissue regeneration.

Effects of Normal Synovial Fluid and Interferon Gamma on Chondrogenic Capability and Immunomodulatory Potential Respectively on Equine Mesenchymal Stem Cells.

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-ß1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-ß1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.

PRMT5 is necessary to form distinct cartilage identities in the knee and long bone.

During skeletal development, limb progenitors become specified as chondrocytes and subsequently differentiate into specialized cartilage compartments. We previously showed that the arginine dimethyl transferase, PRMT5, is essential for regulating the specification of progenitor cells into chondrocytes within early limb buds. Here, we report that PRMT5 regulates the survival of a separate progenitor domain that gives rise to the patella. Independent of its role in knee development, PRMT5 regulates several distinct types of chondrocyte differentiation within the long bones. Chondrocytes lacking PRMT5 have a striking blockage in hypertrophic chondrocyte differentiation and are marked by abnormal gene expression. PRMT5 remains important for articular cartilage and hypertrophic cell identity during adult stages, indicating an ongoing role in homeostasis of these tissues. We conclude that PRMT5 is required for distinct steps of early and late chondrogenic specialization and is thus a critical component of multiple aspects of long bone development and maintenance.

YAP1 regulates chondrogenic differentiation of ATDC5 promoted by temporary TNF-α stimulation through AMPK signaling pathway.

Local injection of tumor necrosis factor-alpha (TNF-α) at bone fracture sites during the early stage of the inflammatory response is reported to improve fracture repair in a murine model. However, the underlying mechanism is unclear. Endochondral bone formation, a process that is highly related to fracture repair, requires a certain amount of chondrocyte hypertrophy. This study aimed to investigate the effect of TNF-α on the differentiation of murine chondrogenic ATDC5 cells and the underlying mechanism. In this study, improved chondrogenic differentiation of ATDC5 cells was achieved by brief TNF-α stimulation. Moreover, the expression of Yes-associated protein 1 (YAP1) was suppressed after brief TNF-α stimulation. The expressions of inflammatory mediators and chondrogenic and hypertrophic-associated genes in ATDC5 cells triggered by TNF-α were suppressed in the YAP1 overexpression group but enhanced in the YAP1 knockdown group. Mechanistically, TNF-α-induced activation of the 5' AMP-activated protein kinase (AMPK) signaling pathway was regulated by YAP1, as revealed by the phosphorylated-AMPK/AMPK change ratios in the YAP1 overexpression and knockdown groups, respectively. Moreover, the potential for TNF-α to enhance chondrogenic differentiation could be partially reversed with an AMPK inhibitor. Taken together, we demonstrate, for the first time, that YAP1 modulates the ability of TNF-α to enhance chondrocyte differentiation partly through AMPK signaling.

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.

Genetic deletion of GIT2 prolongs functional recovery and suppresses chondrocyte differentiation in rats with rheumatoid arthritis.

The current study was conducted for investigating the mechanism by which GIT2 gene deletion affects the functional recovery and chondrocyte differentiation in rats with rheumatoid arthritis (RA). Thirty-two rats were randomly divided into normal, model, GIT2 gene knockout (GIT2-KO), and model + GIT2-KO groups. Hematoxylin-eosin (HE) staining was performed for the observation of synovial tissues. Immunohistochemistry examinations were conducted to determine type II collagen expression as well as identify chondrocyte differentiation. qRT-PCR and Western blotting techniques were adopted in order to expressions of interleukin-1ß (1L-1ß), tumor necrosis factor-α (TNF-α), Aggrecan, and Sry-related HMG box 9 (Sox9). A tape measure and Vernier caliper were used to measure the degree of swelling. Compared with synovial tissues in the model group, those in the model + GIT2-KO group, were thicker and comprised of a mass of inflammatory cells (P < 0.05). Compared with the model group, the type II collagen expressions of the cartilage tissues of the rats decreased in the model + GIT2-KO group (P < 0.05). In terms of the degree of swelling in cartilage tissues, the model group displayed a lesser degree of swelling than in that of the model + GIT2-KO group (P < 0.05). When compared with the model + GIT2-KO group, the mRNA expressions of 1L-1ß, TNF-α, Aggrecan, Sox9 and the relevant protein expressions were lower in the model group (all P < 0.05). GIT2 gene deletion might weaken chondrocyte differentiation in rats with RA, as a result acting to ultimately prolong the functional recovery of RA.

Dlx2 overexpression enhanced accumulation of type II collagen and aggrecan by inhibiting MMP13 expression in mice chondrocytes.

Genetic studies revealed a crucial role of Distal-homebox (Dlx) genes in skeletal development, and our previous study demonstrated overexpressing Dlx2 in neural crest cells led to abnormal cartilage structure, including ectopic cartilage in the maxillary region and nasal bone in mice. The aim of this study was to investigate how Dlx2 overexpression affects chondrogenesis in mouse chondroblast cell line TMC23 and the underlying mechanism. We first demonstrated that Dlx2 expression was upregulated during chondrogenesis in TMC23 cells. Moreover, forced overexpression of Dlx2 in TMC23 cells led to increased accumulation of aggrecan and type II collagen, markers of early chondrocyte differentiation, but had little effect on mRNA and protein levels of Aggrecan and Col2α1, type II collagen gene. Importantly, Dlx2 overexpression decreased mRNA and protein levels of MMP13, a major collagenase degrading aggrecan and type II collagen during late stages of chondrogenesis. Luciferase-reporter and Chromatin-immunoprecipitation analysis demonstrated that MMP13 promoter contained two Dlx2-response elements, and Dlx2 inhibited MMP13 expression by directly binding to these two elements. Based on these observations, we propose that forced overexpression of Dlx2 enhances early chondrocyte differentiation by increasing accumulation of type II collagen and aggrecan, but interferes later stages of chondrocyte differentiation through inhibiting MMP13 expression.