Persistent expression of Twist1 in chondrocytes causes growth plate abnormalities and dwarfism in mice.

Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth.

Wnt5b regulates mesenchymal cell aggregation and chondrocyte differentiation through the planar cell polarity pathway.

Although genetic evidence has demonstrated a role for Wnt5b during cartilage and limb development, little is known about the mechanisms underlying Wnt5b-regulated chondrocyte differentiation. We observed that Wnt5b inhibited chondrocyte hypertrophy and expression of type X collagen. In addition, Wnt5b regulated the overall size of chondrogenic cultures, suggesting that Wnt5b regulates other processes involved in cartilage development. We therefore investigated the signaling pathways by which Wnt5b influences differentiation. Wnt5b activated known calcium-dependent signaling pathways and JNK, a component of the planar cell polarity pathway. Since the planar cell polarity pathway regulates process such as cell migration and cell aggregation that are involved in limb development, we assayed for effects of Wnt5b on these processes. We observed a marked increase chondroprogenitor cell migration with Wnt5b expression. This effect was blocked by inhibition of JNK, but not by inhibition of other Wnt5b-responsive factors. Expression of Wnt5b also disrupted the cellular aggregation associated with mesenchymal condensation. Decreased aggregation was associated with reduced cadherin expression as well as increased cadherin receptor turnover. This increase in cadherin receptor turnover was associated with an increase in Src-dependent beta-catenin phosphorylation downstream of Wnt5b. Our data demonstrate that not only does Wnt5b inhibit chondrocyte hypertrophy, but document a novel role for Wnt5b in modulating cellular migration through the JNK-dependent and cell adhesion through an activation of Src and subsequent cadherin receptor turnover.

WNT5A regulates chondrocyte differentiation through differential use of the CaN/NFAT and IKK/NF-kappaB pathways.

Although genetic evidence demonstrated a requirement for Wnt5a during cartilage development, little is known about the mechanisms underlying Wnt5a-regulated chondrocyte growth and differentiation. We therefore investigated the signaling pathways by which Wnt5a influences chondrogenesis and differentiation to hypertrophy. Wnt5a treatment of chondroprogenitor cells increased chondrocyte hypertrophy and was associated with an increase in nuclear factor of activated T cells (NFAT) and a decrease in nuclear factor-kappaB (NF-kappaB) activation. In contrast, Wnt5a inhibited chondrocyte hypertrophy. This inhibition of hypertrophy occurred with the reciprocal signaling activation, in that a decrease in NFAT and an increase in NF-kappaB activation was observed. Furthermore, the increase in chondroprogenitor cell differentiation with Wnt5a treatment was blocked by calmodulin kinase or NFAT loss of function. In addition, the repression of chondrocyte hypertrophy observed was abrogated by NF-kappaB loss of function. Activation of the NFAT pathway downstream of Wnt5a also negatively regulated NF-kappaB activity, providing evidence of antagonism between these two pathways. Mechanistically, Wnt5a acts to increase chondrocyte differentiation at an early stage through calmodulin kinase /NFAT-dependent induction of Sox9. Conversely, Wnt5a represses chondrocyte hypertrophy via NF-kappaB-dependent inhibition of Runx2 expression. These data indicate that Wnt5a regulates chondrogenesis and chondrocyte hypertrophy in a stage-dependent manner through differential utilization of NFAT- and NF-kappaB-dependent signal transduction.

Freeze-dried tendon allografts as tissue-engineering scaffolds for Gdf5 gene delivery.

Tendon reconstruction using grafts often results in adhesions that limit joint flexion. These adhesions are precipitated by inflammation, fibrosis, and the paucity of tendon differentiation signals during healing. In order to study this problem, we developed a mouse model in which the flexor digitorum longus (FDL) tendon is reconstructed using a live autograft or a freeze-dried allograft, and identified growth and differentiation factor 5 (Gdf5) as a therapeutic target. In this study we have investigated the potential of rAAV-Gdf5 -loaded freeze-dried tendon allografts as "therapeutically endowed" tissue-engineering scaffolds to reduce adhesions. In reporter gene studies we have demonstrated that recombinant adeno-associated virus (rAAV)-loaded tendon allografts mediate efficient transduction of adjacent soft tissues, with expression peaking at 7 days. We have also demonstrated that the rAAV-Gdf5 vector significantly accelerates wound healing in an in vitro fibroblast scratch model and, when loaded onto freeze-dried FDL tendon allografts, improves the metatarsophalangeal (MTP) joint flexion to a significantly greater extent than the rAAV-lacZ controls do. Collectively, our data demonstrate the feasibility and efficacy of therapeutic tendon allograft processing as a novel paradigm in tissue engineering in order to address difficult clinical problems such as tendon adhesions.

Autoimmunity and bone.

Focal erosions of cartilage and bone, which occur in the joints of patients with autoimmune inflammatory arthritis (i.e., rheumatoid arthritis (RA) and psoriatic arthritis [PsA]), represent the most debilitating and irreversible components of the disease. Over the last decade, seminal breakthroughs in our understanding of the cells and signal transduction pathways central to this process have been elucidated. From this information an established paradigm has been developed to explain focal erosions in which osteoclasts responsible for erosions are derived from bone marrow-derived myeloid precursors. Using the tumor necrosis factor (TNF) transgenic mouse model of erosive arthritis and anti-TNF clinical trials with PsA patients, we have demonstrated that systemic TNF induces the migration of CD11b+ osteoclast precursors (OCP) from the bone marrow into peripheral blood. These OCP can then enter the joints in blood vessels, translocate across the receptor activator of NF-kappaB ligand (RANKL) rich inflamed synovium, and differentiate into active osteoclasts. In direct contrast to this, systemic lupus erythematosus (SLE) patients appear to have an innate resistance to bone resorption. Our hypothesis to explain this phenomenon is that systemic interferon-alpha (IFN-alpha) diverts the bone marrow-derived myeloid precursors away from the osteoclast lineage and stimulates their differentiation into dendritic cells (DC). In support of this model, several labs have used microarray analyses to define the IFN-induced transcriptome in peripheral blood mononuclear cells (PBMC) from SLE patients. Here we propose the hypothesis that systemic TNF induces osteoclastic differentiation of PBMC in PsA patients that correlates with their erosive disease, and that the innate immune TNF/IFN axis in patients with autoimmune disease dictates their erosive phenotype. To demonstrate this, we injected wild-type C57B/6 and TNF-Tg mice with poly I:C, which is known to induce systemic IFN responses, and show its dominant effects on increasing the number of circulating CD11b+/CD11c+ precursor dendritic cells (pDC), concomitant with a dramatic reduction in CD11b+/CD11c- OCP. Thus, systemic factors produced by autoimmunity have a dramatic impact on active myelopoiesis and bone homeostasis.

Safety and efficacy of ultraviolet-a light-activated gene transduction for gene therapy of articular cartilage defects.

BACKGROUND: Gene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo. METHODS: The effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or beta-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for beta-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with +/-6000 J/m2 of ultraviolet A one week after direct injection of 10(7) transducing units of rAAV-eGFP. RESULTS: Ultraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m2. Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transduction effects in vitro, which were dose-dependent (p < 0.05). In vivo, ultraviolet A mediated a tenfold increase in transduction in which 40.8% of the superficial chondrocytes adjacent to the defect stained positive for green fluorescent protein compared with 5.2% in the knees treated with no ultraviolet A (p < 0.006). CONCLUSIONS: These results provide what we believe is the first formal demonstration of an agent that can induce rAAV transduction in the complete absence of cytotoxicity and DNA modification. They also suggest that the mechanism by which long-wavelength ultraviolet light mediates site-specific gene expression is by means of the induction of intracellular reactive oxygen species. Finally, laser-derived ultraviolet A can be readily transferred through a fiberoptic cable to mediate light-activated gene transduction in vivo.

Fibroblasts express RANKL and support osteoclastogenesis in a COX-2-dependent manner after stimulation with titanium particles.

UNLABELLED: Synovial fibroblasts are possible mediators of osteolysis. Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Fibroblasts pretreated with titanium or PGE2 stimulated osteoclast formation, showing the functional importance of RANKL induction. Synovial fibroblasts and their activation pathways are potential targets to prevent osteolysis. INTRODUCTION: Bone loss adjacent to the implant is a major cause of joint arthroplasty failure. Although the cellular and molecular response to microscopic wear debris particles is recognized as causative, little is known concerning role of synovial fibroblasts in these events. MATERIALS AND METHODS: Murine embryonic fibroblasts and knee synovial fibroblasts in culture stimulated with titanium particles were examined by FACS, real time RT-PCR, Northern blot, and Western blot for expressions of vascular cell adhesion molecule (VCAM)1, RANKL, cyclooxygenase (COX)-1, and COX-2, and the four prostaglandin E2 (PGE2) receptor isoforms. Experiments were performed in the presence and absence of COX inhibitors, protein kinase A (PKA) and protein kinase C (PKC) inhibitors, and various EP receptor agonists. Osteoclast formation was examined in co-cultures of pretreated glutaraldehyde-fixed fibroblasts and primary murine spleen cells treated with macrophage-colony stimulating factor (M-CSF) for 7-days. RESULTS: TNF-alpha stimulated VCAM1 expression, consistent with a synovial fibroblast phenotype. Titanium particles stimulated RANKL gene and protein expressions in fibroblasts in a dose-dependent manner. Gene expression was increased 5-fold by 4 h, and protein levels reached a maximum after 48 h. Within 1 h, titanium particles also induced COX-2 mRNA and protein levels, whereas both indomethacin and celecoxib blocked the stimulation of RANKL, suggesting a COX-2-mediated event. Furthermore, PGE2 induced RANKL gene and protein expression and rescued RANKL expression in titanium-treated cultures containing COX-2 inhibitors. Fibroblast cultures pretreated with either PGE2 or titanium particles enhanced osteoclast formation, indicating the functional importance of RANKL induction. EP4 was the most abundant PGE2 receptor isoform, EP1 and EP2 were expressed at low levels, and EP3 was absent. The EP1 selective agonist iloprost and the EP2 selective agonist butaprost minimally stimulated RANKL. In contrast, the EP2 and EP4 agonist misoprostol induced RANKL to a magnitude similar to PGE2. Finally, PKA antagonism strongly repressed RANKL stimulation by PGE2. CONCLUSION: Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Thus, the synovial fibroblast is important mediator of osteolysis and target for therapeutic strategies.

Overlapping expression of Runx1(Cbfa2) and Runx2(Cbfa1) transcription factors supports cooperative induction of skeletal development.

Identifying the genetic pathways that regulate skeletal development is necessary to correct a variety of cartilage and bone abnormalities. The Runx family of transcription factors play a fundamental role in organ development and cell differentiation. Initial studies have shown that both Runx1 and Runx2 are expressed in pre-chondrogenic mesenchyme of the developing embryo at E12.5. Abrogation of the Runx2 gene completely inhibits bone formation yet the cartilage anlagen in these mice is fully formed. In the present study, we hypothesized that Runx1 may compensate for the lack of Runx2 in vivo to induce the early stages of skeletal formation and development. Histologic beta-gal stained sections using the Runx1(+/-)-Lac-Z mice demonstrate Runx1 promoter activity in pre-chondrocytic cell populations. In situ hybridization using Runx1 and Runx2 specific probes indicate that both factors are expressed in mesenchymal stem cell progenitors during early embryonic development. During later stages of mouse skeletal formation, Runx1 is excluded from the hypertrophic cartilage while Runx2 is present in these matured chondrocyte populations. Quantification of Runx expression by real time RT-PCR and Western blot analyses reveals that Runx1 and Runx2 are differentially modulated during embryogenesis suggesting a temporal role for each of these transcriptional regulators during skeletal formation. We provide evidence that haploinsufficiency results in normal appearing embryo skeletons of heterozygote Runx2 and Runx1 mutant mouse models; however, a delay in bone formation was identified in the calvarium. In summary, our results support a function for Runx1 and Runx2 during skeletal development with a possible role for Runx1 in mediating early events of endochondral and intramembranous bone formation, while Runx2 is a potent inducer of late stages of chondrocyte and osteoblast differentiation.

Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Several factors regulate maturation via the activity of downstream signaling pathways that target specific transcription factors and regulate chondrocyte-specific genes. In this study, we investigated the mechanisms involved in the regulation of chick lower sternal chondrocyte maturation upon stimulation by retinoic acid (RA) and the bone morphogenetic protein BMP2. RA-induced Runx2 in lower sternal chondrocyte cultures and over-expression of wild-type (WT) Runx2 enhanced colX and alkaline phosphatase activity, while over-expression of dominant negative Runx2 was inhibitory. Furthermore, WT Runx2 enhanced the effects of both BMP2 and RA on colX expression, while the effects of both growth factors were completely blocked in cultures over-expressing dominant negative Runx2. Similarly, WT Runx2 enhanced the induction of colX by Smad1. Smad1 and Runx2 were found to act cooperatively at the chicken type X collagen promoter and elimination of either the putative Smad binding site or Runx2 binding site eliminated responsiveness to BMP2, RA, or either of the transcription factors. Altogether the results show cross talk between the BMP-associated Smads and Runx2 during chondrocyte differentiation and dependence upon both signals for induction of the type X collagen promoter. Factors or signals that alter either of these transcription factors regulate the rate of chondrocyte differentiation.