Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations.

Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4(V620I) channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of the TRPV4(V620I) mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4(T89I) mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.

Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.

Temporomandibular joint disorder (TMJD) is known for its mastication-associated pain. TMJD is medically relevant because of its prevalence, severity, chronicity, the therapy-refractoriness of its pain, and its largely elusive pathogenesis. Against this background, we sought to investigate the pathogenetic contributions of the calcium-permeable TRPV4 ion channel, robustly expressed in the trigeminal ganglion sensory neurons, to TMJ inflammation and pain behavior. We demonstrate here that TRPV4 is critical for TMJ-inflammation-evoked pain behavior in mice and that trigeminal ganglion pronociceptive changes are TRPV4-dependent. As a quantitative metric, bite force was recorded as evidence of masticatory sensitization, in keeping with human translational studies. In Trpv4(-/-) mice with TMJ inflammation, attenuation of bite force was significantly less than in wildtype (WT) mice. Similar effects were seen with systemic application of a specific TRPV4 inhibitor. TMJ inflammation and mandibular bony changes were apparent after injections of complete Freund adjuvant but were remarkably independent of the Trpv4 genotype. It was intriguing that, as a result of TMJ inflammation, WT mice exhibited significant upregulation of TRPV4 and phosphorylated extracellular-signal-regulated kinase (ERK) in TMJ-innervating trigeminal sensory neurons, which were absent in Trpv4(-/-) mice. Mice with genetically-impaired MEK/ERK phosphorylation in neurons showed resistance to reduction of bite force similar to that of Trpv4(-/-) mice. Thus, TRPV4 is necessary for masticatory sensitization in TMJ inflammation and probably functions upstream of MEK/ERK phosphorylation in trigeminal ganglion sensory neurons in vivo. TRPV4 therefore represents a novel pronociceptive target in TMJ inflammation and should be considered a target of interest in human TMJD.

Synovial fluid concentrations and relative potency of interleukin-1 alpha and beta in cartilage and meniscus degradation.

Cartilage degeneration with osteoarthritis (OA) is believed to involve the activities of interleukin-1 (IL-1), which exists as alpha and beta isoforms. The goal of this study was to measure the concentrations of both isoforms of IL-1 in the synovial fluid of normal and spontaneously osteoarthritic porcine knees, and to test the hypothesis that physiologic concentrations of IL-1α and IL-1ß exhibit different potencies in activating calcium signaling, the production of matrix metalloproteinases and nitric oxide, and the loss of proteoglycans and tissue mechanical properties in cartilage and meniscus. Median concentrations of IL-1α were 0.043 ng/ml with mild OA and 0.288 ng/ml with moderate OA, whereas IL-1ß concentrations were 0.109 ng/ml with mild OA and 0.122 ng/ml with moderate OA. Both isoforms induced calcium signaling in chondrocytes and meniscal cells at all concentrations. Overall, cartilage and meniscus catabolism was significantly more sensitive to IL-1α than IL-1ß at concentrations of 1 ng/ml or less, while few differences were observed between the two forms at 10 ng/ml. These data provide a range of physiologic IL-1 concentrations that can serve as a framework for the comparison of various in vitro studies, as well as providing further insight for the development of anti-cytokine therapies for OA.

Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration.

INTRODUCTION: Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-ß1 (TGF-ß1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-ß1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. METHODS: A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-ß1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-ß1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. RESULTS: IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-ß1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system, while TGF-ß1 had no effect on either measure. CONCLUSIONS: Meniscal cell proliferation in vivo may be diminished following joint injury due to the up-regulation of inflammatory cytokines, thereby limiting native cellular repair of meniscal lesions. Therefore, therapies that can promote meniscal cell proliferation have promise to enhance meniscal repair and improve tissue engineering strategies.