Prechondrogenic Cells Research Articles

Methylphenidate Promotes Premature Growth Plate Closure: In Vitro Evidence

It is well known that patients with attention deficit hyperactivity disorder treated with stimulants, such as methylphenidate hydrochloride (MPH), have reduced height and weight. Even though MPH has an anorexigenic effect, an additional impact of this drug on the growth plate cannot be discarded. In this study, we aimed to determine the cellular effect of MPH on an in vitro growth plate model. We tested the effects of MPH on the viability and proliferation of a prechondrogenic cell line via an MTT assay. In vitro differentiation of this cell line was performed, and cell differentiation was evaluated through the expression of cartilage- and bone-related genes as measured via RT-PCR. MPH did not alter the viability or proliferation of prechondrogenic cells. However, it reduced the expression of cartilage extracellular matrix-related genes (type II collagen and aggrecan) and increased the expression of genes involved in growth plate calcification (Runx2, type I collagen, and osteocalcin) at different phases of their differentiation process. Our results evidence that MPH upregulates genes associated with growth plate hypertrophic differentiation. This may induce premature closure of the growth plate, which would contribute to the growth retardation that has been described to be induced by this drug.

Novel method to repair articular cartilage by direct reprograming of prechondrogenic mesenchymal stem cells

Age-related cartilage loss is worsened by the limited regenerative capacity of chondrocytes. The role of cell-based therapies using mesenchymal stem cells is gaining interest. Adipose tissue-derived mesenchymal stem cells (ADSCs) are an attractive source to generate the optimal number of chondrocytes required to repair a cartilage defect and regenerate hyaline articular cartilage. Here, we report an outstanding technique to prepare chondrocytes for cartilage repair using canine ADSCs. We hypothesized that external electrical fields promote prechondrogenic condensation without requiring genetic modifications or exogenous factors. We analyzed the effect of electrical stimulation (ES) on the differentiation of ADSC micromass into chondrocytes. Highly compact structures were formed within 3 days of ES of canine ADSC micromass. The expression of type I collagen gene was abolished in these cells compared with that in control micromass cultures and monolayer cultures. We further found that ES enhanced the production of proteoglycan, a highly produced extracellular matrix component in chondrocytes. Additionally, single-cell RNA sequencing analysis showed that canine ADSC micromass undergoing ES developed a prechondrogenic cell aggregation, suggesting their metabolic conversion, biogenesis, and calcium ion change. Collectively, our findings demonstrate the capacity of ES to drive the chondrogenesis of ADSCs in the absence of exogenous factors and confirm its commercial potential as a budget-friendly therapy for the repair of cartilage defects.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFβ to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Autophagy-related proteases accompany the transition of pre-chondrogenic cells into chondroblasts

BackgroundAutophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium. MethodsPCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture. ResultsSeveral proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression. ConclusionsThe present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

Vibration acceleration promotes endochondral formation during fracture healing through cellular chondrogenic differentiation.

Vibration acceleration through whole body vibration has been reported to promote fracture healing. However, the mechanism responsible for this effect remains unclear. Purpose of this study was to determine whether vibration acceleration directly affects cells around the fracture site and promotes endochondral ossification. Four-week-old female Wistar Hannover rats were divided into two groups (vibration [V group] and control [C group]). The eighth ribs on both sides were cut vertically using scissors. From postoperative day 3 to 11, vibration acceleration using Power Plate® (30 Hz, low amplitude [30-Low], 10 min/day) was applied in the V group. Mature calluses appeared earlier in the V group than in the C group by histological analysis. The GAG content in the fracture callus on day 6 was significantly higher in the V group than in the C group. The mRNA expressions of SOX-9, aggrecan, and Col-II in the fracture callus on day 6 and Col-X on day 9 were significantly higher in the V group than in the C group. For in vitro analysis, four different conditions of vibration acceleration (30 or 50 Hz with low or high amplitude [30-Low, 30-High, 50-Low, and 50-High], 10 min/day) were applied to a prechondrogenic cell (ATDC5) and an undifferentiated cell (C3H10T1/2). There was no significant difference in cell proliferation between the control and any of the four vibration conditions for both cell lines. For both cell lines, alcian blue staining was greater under 30-Low and 50-Low conditions than under control as well as 30-High and 50-High conditions on days 7 and 14. Vibration acceleration under 30-L condition upregulated chondrogenic gene expressions of SOX-9, aggrecan, Col-II, and Col-X. Low-amplitude vibration acceleration can promote endochondral ossification in the fracture healing in vivo and chondrogenic differentiation in vitro.

Prechondrogenic ATDC5 cell response to graphene/multi-walled carbon nanotube-containing porous polycaprolactone biocomposite scaffolds

In the study graphene and multi- walled carbon nanotube (MWCNT) - containing polycaprolactone (PCL) scaffolds were prepared by solvent casting-particulate leaching method for cartilage tissue engineering applications. Graphene nanopowders or MWCNTs were added to the system at different concentrations namely 1, 3, 5 and 10 wt%. Mechanical, thermal and electrical properties of the scaffolds were measured as a function of additive concentration. Response of prechondrogenic ATDC5 cells seeded on the composite constructs were tested using XTT method and live-dead cell viability assay. Alkaline phosphatase activity of the cells was also tested to investigate differentiation behaviour. Results showed that electrically conductive composite scaffolds prepared in the study have potential to be used in tissue engineering.

Chicken knee cartilage extract promotes proliferation of the mouse pre-chondrogenic cell line, ATDC5

Chicken knee cartilage extract promotes proliferation of the mouse pre-chondrogenic cell line, ATDC5

MiR-27 regulates chondrogenesis by suppressing focal adhesion kinase during pharyngeal arch development

Cranial neural crest cells are a multipotent cell population that generate all the elements of the pharyngeal cartilage with differentiation into chondrocytes tightly regulated by temporal intracellular and extracellular cues. Here, we demonstrate a novel role for miR-27, a highly enriched microRNA in the pharyngeal arches, as a positive regulator of chondrogenesis. Knock down of miR-27 led to nearly complete loss of pharyngeal cartilage by attenuating proliferation and blocking differentiation of pre-chondrogenic cells. Focal adhesion kinase (FAK) is a key regulator in integrin-mediated extracellular matrix (ECM) adhesion and has been proposed to function as a negative regulator of chondrogenesis. We show that FAK is downregulated in the pharyngeal arches during chondrogenesis and is a direct target of miR-27. Suppressing the accumulation of FAK in miR-27 morphants partially rescued the severe pharyngeal cartilage defects observed upon knock down of miR-27. These data support a crucial role for miR-27 in promoting chondrogenic differentiation in the pharyngeal arches through regulation of FAK.

Deferoxamine synergizes with transforming growth factor-β signaling in chondrogenesis.

Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, is an epidemic disease that affects millions of people worldwide. Despite extensive recent work on the cellular biology of osteoarthritis, the precise mechanisms involved are still poorly understood and there is no effective treatment for this disease. The role of transforming growth factor-beta (TGF-β) in promoting chondrogenesis and inducing the expression of cartilage-specific extracellular matrix molecules to form cartilage is well-established. Historically, TGF-β has been considered to prevent osteoarthritis, but recent work suggests that TGF-β overexpression accelerates the progression of osteoarthritis in vivo. Clinically, it is therefore important to limit TGF-β expression while still providing effective treatment of osteoarthritis. One possible approach to achieve this effect would be to use a combination of TGF-β with other small molecular chemical compounds. Hypoxia promotes chondrogenesis and the usefulness of deferoxamine, a chelating agent that mimics hypoxia, in stimulating chondrogenesis has been investigated in clinical trials. In this study, we investigated the role of deferoxamine in TGF-β-induced chondrogenesis in pre-chondrogenic cells and examined whether deferoxamine synergizes with the TGF-β signaling pathway to promote chondrocyte differentiation.

Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System.

Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

FLRT2 Interacts With Fibronectin in the ATDC5 Chondroprogenitor Cells

Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development.

Bone‐Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro

Autografts are used for bone reconstruction in regenerative medicine including oral and maxillofacial surgery. Bone grafts release paracrine signals that can reach mesenchymal cells at defect sites. The impact of the paracrine signals on osteogenic, adipogenic, and chondrogenic differentiation of mesenchymal cells has remained unclear. Osteogenesis, adipogenesis, and chondrogenesis were studied with murine ST2 osteoblast progenitors, 3T3-L1 preadipocytes, and ATDC5 prechondrogenic cells, respectively. Primary periodontal fibroblasts from the gingiva, from the periodontal ligament, and from bone were also included in the analysis. Cells were exposed to bone-conditioned medium (BCM) that was prepared from porcine cortical bone chips. BCM inhibited osteogenic and adipogenic differentiation of ST2 and 3T3-L1 cells, respectively, as shown by histological staining and gene expression. No substantial changes in the expression of chondrogenic genes were observed in ATDC5 cells. Primary periodontal fibroblasts also showed a robust decrease in alkaline phosphatase and peroxisome proliferator-activated receptor gamma (PPARγ) expression when exposed to BCM. BCM also increased collagen type 10 expression. Pharmacologic blocking of transforming growth factor (TGF)-β receptor type I kinase with SB431542 and the smad-3 inhibitor SIS3 at least partially reversed the effect of BCM on PPARγ and collagen type 10 expression. In support of BCM having TGF-β activity, the respective target genes were increasingly expressed in periodontal fibroblasts. The present work is a pioneer study on the paracrine activity of bone grafts. The findings suggest that cortical bone chips release soluble signals that can modulate differentiation of mesenchymal cells in vitro at least partially involving TGF-β signaling.

Comparative pelvic development of the axolotl (Ambystoma mexicanum) and the Australian lungfish (Neoceratodus forsteri): conservation and innovation across the fish-tetrapod transition

BackgroundThe fish-tetrapod transition was one of the major events in vertebrate evolution and was enabled by many morphological changes. Although the transformation of paired fish fins into tetrapod limbs has been a major topic of study in recent years, both from paleontological and comparative developmental perspectives, the interest has focused almost exclusively on the distal part of the appendage and in particular the origin of digits. Relatively little attention has been paid to the transformation of the pelvic girdle from a small unipartite structure to a large tripartite weight-bearing structure, allowing tetrapods to rely mostly on their hindlimbs for locomotion. In order to understand how the ischium and the ilium evolved and how the acetabulum was reoriented during this transition, growth series of the Australian lungfish Neoceratodus forsteri and the Mexican axolotl Ambystoma mexicanum were cleared and stained for cartilage and bone and immunostained for skeletal muscles. In order to understand the myological developmental data, hypotheses about the homologies of pelvic muscles in adults of Latimeria, Neoceratodus and Necturus were formulated based on descriptions from the literature of the coelacanth (Latimeria), the Australian Lungfish (Neoceratodus) and a salamander (Necturus).ResultsIn the axolotl and the lungfish, the chondrification of the pelvic girdle starts at the acetabula and progresses anteriorly in the lungfish and anteriorly and posteriorly in the salamander. The ilium develops by extending dorsally to meet and connect to the sacral rib in the axolotl. Homologous muscles develop in the same order with the hypaxial musculature developing first, followed by the deep, then the superficial pelvic musculature.ConclusionsDevelopment of the pelvic endoskeleton and musculature is very similar in Neoceratodus and Ambystoma. If the acetabulum is seen as being a fixed landmark, the evolution of the ischium only required pubic pre-chondrogenic cells to migrate posteriorly. It is hypothesized that the iliac process or ridge present in most tetrapodomorph fish is the precursor to the tetrapod ilium and that its evolution mimicked its development in modern salamanders.

Growth and Differentiation of Prechondrogenic Cells on Bioactive Self-Assembled Peptide Nanofibers

Restoration of cartilage defect remains a challenge, as the current treatments are ineffective to return tissue to its health. Thus, developing therapies for treatment of cartilage tissue damage caused by common joint diseases including osteoarthritis, rheumatoid arthritis, and accidents is crucial. Sulfated glycosaminoglycan molecules are vital constituents of both developing and mature cartilage extracellular matrix. The interplay between regulator proteins and glycosaminoglycan molecules has an essential role in coordinating differentiation, expansion, and patterning during cartilage development. In this study, we exploited the functional role of an extracellular matrix on chondrogenic differentiation by imitating extracellular matrix both chemically by imparting functional groups of native glycosaminoglycans and structurally through peptide nanofiber network. For this purpose, sulfonate, carboxylate, and hydroxyl groups were incorporated on self-assembled peptide nanofibers. We observed that when ATDC5 cells were cultured on functional peptide nanofibers, they rapidly aggregated in insulin-free medium and formed cartilage-like nodules and deposited sulfated glycosaminoglycans shown by Safranin-O staining. Moreover, collagen II and aggrecan gene expressions revealed by qRT-PCR were significantly enhanced, which indicated the remarkable bioactive role of this nanofiber system on chondrogenic differentiation. Overall, these results show that glycosaminoglycan mimetic peptide nanofiber system provides a promising platform for cartilage regeneration.

O-GlcNAc protein modification stimulates chondrogenesis in vitro and chondrocyte hypertrophy in mouse

Backgroundand objectives Chondrocyte differentiation that allows endocondral ossification sequentially includes cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralisation, vascular invasion and eventually apoptosis, allowing cartilage remodeling into bone. Most of these processes have been also associated to the development of osteoarthritis. Insulin ameliorate impaired bone growth and impaired bone healing both in vitro and in vivo, and is able to induce chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-GlcNAc glycosylated proteins and in the expression of the key enzymes of this pathway: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). Furthermore, the authors studied if O-GlcNAc-accumulation per se was able to induce prehypertrophic chondrocyte differentiation both in vitro and in vivo. Methods Prechondrogenic ATDC5 cells were cultured in 5% FBS DMEM/F12. Insulin-induced differentiation was studied for 24 days. The accumulation of O-GlcNAcylated proteins was induced employing the OGA inhibitor Thiamet-G (TG). The gene expression of the differentiation markers collagen X (ColX), PTH receptor (PTHR), Indian hedgehog protein (IHH), Runx-2 and alkaline phosphatase (AP) was studied by real time-PCR. O-GlcNAcylation, OGT, OGA, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were studied by western-blot. The effect of the in vivo accumulation of O-GlcNAc proteins in the tibial growth plate was studied in 23-day old C57/bl mice that received 20mg/kg TG during 15 days. Results Insulin-induced differentiation paralleled with a gradual increase in the accumulation of O-GlcNacylated proteins that was appreciable since day 3, and with an increase in the expression of OGT and OGA. These increases occurred previously to any modification in the differentiation markers. O-GlcNAc accumulation per se induced by TG, in the absence of insulin, induced ATDC5 differentiation measured by the increase in the expression of differentiation markers. TG cells also induced an increase in the activity of MMP-1,-2 and -9, similar to that evoked by insulin. TG activated pERK, p-JNK and p-p38 in a similar extent to insulin. The administration of TG to C57/bl mice induced an accumulation of O-GlcNAcylated proteins in brain, liver, muscle and knee joint. TG induced a significant expansion in the growth plate height and in the hypertropic zone height, in comparison to untreated mice, without a significant modification of body weight. Conclusions TG induced the expression of chondrogenic markers in ATDC5 cells, and increased chondrogenic differentiation in vivo. Our results show that o-GlcNAc glycosylation has chondromodulating activity.

FGF signaling specifies pre‐chondrogenic identity in neural crest derived mesenchyme, but initiation of chondrogenesis requires BMP4 signaling

The chick middle ear bone, columella, derives from neural crest mesenchyme located proximally in the second pharyngeal arch. Subjacent pharyngeal endoderm is hypothesized as the source of the local signals that specify the pre‐chondrogenic identity of this tissue. Sox9 is induced and maintained in pre‐chondrogenic cells during condensation formation and endochondral ossification. We show that pharyngeal endoderm was sufficient, but not necessary for specifying pre‐chondrogenic identity. Many Fgf genes are expressed specifically in the pharyngeal endoderm subjacent to neural crest derived mesenchyme. Our results show that FGF signaling is both sufficient and required for specification of Sox9 expression and specification of pre‐chondrogenic identity. Further, FGF signaling, by itself, is insufficient for the induction of the chondrogenic marker, Col2a1. BMP4 signaling can induce Sox9 expression, but only in combination with FGF signaling induced Col2a1 expression and, thus, chondrogenesis. Given the spatio‐temporal expression patterns of FGFs and BMPs in the pharyngeal arches, we suggest that this may represent a general mechanism of specifying pre‐chondrogenic identity and chondrogenesis.Grant Funding Source: NIH/NIDCD

Fibroblast growth factor and bone morphogenetic protein signaling are required for specifying prechondrogenic identity in neural crest‐derived mesenchyme and initiating the chondrogenic program

The pharyngeal endoderm is hypothesized as the source of local signals that specify the identity of neural crest-derived mesenchyme in the arches. Sox9 is induced and maintained in prechondrogenic cells during condensation formation and endochondral ossification. Using explant culture, we determined that pharyngeal endoderm was sufficient, but not necessary for specifying prechondrogenic identity, as surrounding tissues including the otic vesicle can compensate for signals from the pharyngeal endoderm. Multiple Fgf genes are expressed specifically in the pharyngeal endoderm subjacent to the neural crest-derived mesenchyme. Fibroblast growth factor (FGF) signaling is both sufficient and required for specification of Sox9 expression and specification of prechondrogenic identity, as demonstrated by the addition of recombinant FGF protein or the FGF receptor inhibitor (SU5402) to explanted tissue, respectively. However, FGF signaling cannot maintain Sox9 expression or initiate the chondrogenic program as indicated by the absence of Col2a1 transcripts. Bone morphogenetic protein (BMP) 4 signaling can induce and maintain Sox9 expression in isolated mesenchyme, but only in combination with FGF signaling induce Col2a1 expression, and thus, chondrogenesis. Given the spatiotemporal expression patterns of FGFs and BMPs in the pharyngeal arches, we suggest that this may represent a general mechanism of local signals specifying prechondrogenic identity and initiation of the chondrogenic program.

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm 2 for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2α mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2α is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover

Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse prechondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type II and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.