Depleting transforming growth factor beta receptor 2 signalling in the cartilage of itga1-null mice attenuates spontaneous knee osteoarthritis.

Objectives: Integrin α1ß1 protects against osteoarthritis (OA) when it is upregulated in the superficial zone of cartilage in the early stages of disease. However, the mechanism behind this protection is unknown. Integrin α1ß1 moderates transforming growth factor ß receptor II (TGFBR2) signalling, a critical regulator of chondrocyte anabolic activity. To this end, mice lacking integrin α1ß1 have increased baseline activation of TGFBR2 signalling and overall fibrosis. The purpose of this study was to evaluate the interplay between integrin α1ß1 and TGFBR2 in the development of spontaneous OA. We hypothesized that dampening TGFBR2 signalling in the cartilage of itga1-null mice would attenuate OA. Methods: Behavioural and histological manifestations of spontaneous knee OA were measured at 4, 8, 12 and 16 months in mice with and without a ubiquitous itga1 deletion and with and without a tamoxifen-induced cartilage specific TGFBR2 depletion. Results: Knee cartilage degeneration, collateral ligament ossification and pain responses increased with age. Itga1-null mice with intact TGFBR2 signalling developed earlier and more severe OA compared to controls. In agreement with our hypothesis, depleting TGFBR2 signalling in the cartilage of itga1-null mice attenuated OA progression. Conclusion: Intact TGFBR2 signalling drives early and worse knee OA in itga1-null mice. This result supports the hypothesis that the increased expression of integrin α1ß1 by superficial zone chondrocytes early in OA development dampens TGFBR2 signalling and thus protects against degeneration.

Chondrocyte primary cilia lengthening and shortening in response to mediators of osteoarthritis; a role for integrin α1ß1 and focal adhesions.

Objective: Integrin α1ß1 protects against osteoarthritis when it is upregulated in the early stages of disease, however, the mechanism behind this is currently unknown. Hypo-osmotic stress, interleukin-1 (IL-1) and transforming growth factor ß (TGFß) influence chondrocyte signaling and are important mediators of osteoarthritis. Evidence for primary cilia as a signaling hub for these factors and the involvement of the F-actin cytoskeleton in this response is growing. The purpose of this study was to investigate the role of integrin α1ß1 in the response of primary cilia and the F-actin cytoskeleton to these osteoarthritic mediators. Design: Primary cilia length and the number of F-actin peaks were measured in ex vivo wild type and itga1-null chondrocytes in response to hypo-osmotic stress, IL-1, and TGFß alone or in combination, and with or without focal adhesion kinase inhibitor. Results: We show that integrin α1ß1 and focal adhesions are necessary for cilial lengthening and increases in F-actin peaks with hypo-osmotic stress and IL-1, but are not required for cilial shortening with TGFß. Furthermore, we established that the chondrocyte primary cilium has a resting length of 2.4 â€‹µm, a minimum length of 2.1 â€‹µm corresponding to the thickness of the pericellular matrix, and a maximum length of 3.0 â€‹µm. Conclusions: While integrin α1ß1 is not necessary for the formation of chondrocyte primary cilia and cilial shortening in response to TGFß, it is necessary for the mediation of cilial lengthening and the formation of F-actin peaks in response to hypo-osmotic stress and IL-1.

Sexual dimorphism in reactive oxygen species production and a role for integrin α1ß1 in estrogen receptor α and ß expression in articular cartilage.

BACKGROUND: Osteoarthritis (OA) is a debilitating disease involving cartilage degradation. A need remains for the discovery of new molecular targets in cartilage for pharmaceutical intervention of OA. One potential target is integrin α1ß1 that protects against OA when it is upregulated by chondrocytes early in the disease process. Integrin α1ß1 offers this protection by dampening epidermal growth factor receptor (EGFR) signaling, and its effects are more robust in females compared to males. The aim of this study, therefore, was to measure the impact of itga1 on chondrocyte EGFR activity and downstream reactive oxygen species (ROS) production in male and female mice. Furthermore, chondrocyte expression of estrogen receptor (ER) α and ERß was measured to investigate the mechanism for sexual dimorphism in the EGFR/integrin α1ß1 signaling axis. We hypothesized that integrin α1ß1 would decrease ROS production and pEGFR and 3-nitrotyrosine expression, with this effect being greater in females. We further hypothesized that chondrocyte expression of ERα and ERß would be greater in females compared to males, with a greater effect seen in itga1-null compared to wild-type mice. MATERIALS AND METHODS: Femoral and tibial cartilage of male and female, wild-type and itga1-null mice were processed for ex vivo confocal imaging of ROS, immunohistochemical analysis of 3-nitrotyrosine, or immunofluorescence of pEGFR and ERα and ERß. RESULTS: We show that ROS-producing chondrocytes are more abundant in female itga1-null compared to wild-type mice ex vivo; however, itga1 had limited influence on the percent of chondrocytes stained positively for 3-nitrotyrosine or pEGFR in situ. In addition, we found that itga1 influenced ERα and ERß expression in femoral cartilage from female mice, and that ERα and ERß were coexpressed as well as colocalized in chondrocytes. Finally, we show sexual dimorphism in ROS and 3-nitrotyrosine production, but surprisingly not in pEGFR expression. CONCLUSIONS: Together these data highlight sexual dimorphism in the EGFR/integrin α1ß1 signaling axis and underline the need for further investigation into the role of ERs in this biological paradigm. Understanding the molecular mechanisms underlying the development of OA is essential for the development of individualized, sex-specific treatments in this age of personalized medicine.

Sexual dimorphism in knee osteoarthritis: Biomechanical variances and biological influences.

Objective: Osteoarthritis (OA) is a degenerative joint disease that is more prevalent in women than men, especially later in life. This suggests that sexual dimorphism may be present in the pathogenesis of the disease. The purpose of this review is to discuss evidence of sexual dimorphism in knee OA development and presentation as it is framed by two contrasting paradigms: biomechanics and biology. Methods: A comprehensive search of databases was conducted including, but not limited to, MEDLINE via Ovid, PubMed, and Google Scholar. Keywords including osteoarthritis, sex differences, and/or sexual dimorphism were searched in combination with knee biomechanics, ACL, joint malalignment, estrogen, chondrocyte signal(l)ing, growth factor and integrin(s). Results: The biomechanical approach has identified sex differences in joint malalignment, bone shape, gait, and lower limb muscle strength leading to altered load transmission, as well as increased knee laxity in women predisposing them to joint injury. The biological approach has largely focused on the influence of estrogen receptor signaling on the maintenance of joint tissues. Preliminary work identifying sexual dimorphism in chondrocyte signaling pathways involving growth factors and collagen receptors has been reported in addition to more systemic levels of inflammatory cytokines and metabolites. Conclusion: Understanding the true etiology of OA is crucial for developing effective, individualized treatment in the age of personalised medicine. A shift from a 'one size fits all' mentality towards an individualized approach for therapeutic treatment must begin with the acknowledgment of sex differences in the biomechanical and biological factors underlying the onset and development of OA.

Investigating how combined multifidus injury and facet joint compression influence changes in surrounding muscles and facet degeneration in the rat.

PURPOSE: To examine whether unilateral multifidus damage could promote degeneration at the L5-6 facet joint (FJ) and compensatory changes in lumbo-pelvic muscles in rats. METHODS: 12 facet clamp, 12 facet sham and 7 control rats were studied. Facet clamp and sham animals had the left L5-6 FJ exposed, and the clamp group had a mild compressive clamp applied using hemostatic forceps to model post-traumatic arthritis. Both groups then had the left multifidus detached from the L1-L6 spinous processes. Animals were euthanized 28 days post-surgery. Muscle mass and fascicle length were evaluated bilaterally for the paraspinal muscles, gluteal muscles and biceps femoris. Intra-muscular collagen of the paraspinal muscles was measured histologically. FJ transverse plane angles were measured from micro-computed tomography scans. L5-6 FJ degeneration was evaluated through the 24-point OARSI scale. RESULTS: Differences, compared to control, were observed in the detached multifidus from both facet clamp and sham groups; namely decreased mass and fascicle length and increased collagen content. However, no between group differences were found for any other muscle. Further, mild FJ degeneration was more prevalent in the groups that had experienced multifidus injury but was not exacerbated by the mild compressive clamping of the FJ. CONCLUSION: Unilateral multifidus injury with or without FJ compressive clamping does not have a clear impact on the characteristics of surrounding spinal musculature within 28 days post-surgery in rats. Mild FJ degeneration was present in some animals from all three groups, and the impact of multifidus injury on this degeneration is inconclusive.

The Anatomical Distribution of Mechanoreceptors in Mouse Hind Paw Skin and the Influence of Integrin α1ß1 on Meissner-Like Corpuscle Density in the Footpads.

Afferent neurons and their mechanoreceptors provide critical sensory feedback for gait. The anatomical distribution and density of afferents and mechanoreceptors influence sensory feedback, as does mechanoreceptor function. Electrophysiological studies of hind paw skin reveal the different types of afferent responses and their receptive fields, however, the anatomical distribution of mechanoreceptor endings is unknown. Also, the role of integrin α1ß1 in mechanoreceptor function is unclear, though it is expressed by keratinocytes in the stratum basale where it is likely involved in a variety of mechanotransduction pathways and ion channel functionalities. For example, it has been shown that integrin α1ß1 is necessary for the function of TRPV4 that is highly expressed by afferent units. The purpose of this study, therefore, was to determine and compare the distribution of mechanoreceptors across the hind paw skin and the footfall patterns of itga1-null and wild type mice. The itga1-null mouse is lacking the integrin α1 subunit, which binds exclusively to the ß1 subunit, thus rendering integrin α1ß1 nonfunctional while leaving the numerous other pairings of the ß1 subunit undisturbed. Intact hind paws were processed, serially sectioned, and stained to visualize mechanoreceptors. Footfall patterns were analyzed as a first step in correlating mechanoreceptor distribution and functionality. Merkel cells and Meissner-like corpuscles were present, however, Ruffini endings and Pacinian corpuscles were not observed. Meissner-like corpuscles were located exclusively in the glabrous skin of the footpads and digit tips, however, Merkel cells were found throughout hairy and glabrous skin. The increased density of Merkel cells and Meissner-like corpuscles in footpads 1 and 3 and Meissner-like corpuscles in footpad 4 suggests their role in anteroposterior balance, while Meissner-like corpuscle concentrations in digits 2 and 5 support their role in mediolateral balance. Finally, a larger density of Meissner-like corpuscles in footpads 3 and 4 in male itga1-null mice compared to wild type controls paves the way for future site-specific single fiber in vivo recordings to provide insight into the role of integrin α1ß1 in tactile mechanotransduction.

Serum Metabolite Profiles Are Altered by Erlotinib Treatment and the Integrin α1-Null Genotype but Not by Post-Traumatic Osteoarthritis.

The risk of developing post-traumatic osteoarthritis (PTOA) following joint injury is high. Furthering our understanding of the molecular mechanisms underlying PTOA and/or identifying novel biomarkers for early detection may help to improve treatment outcomes. Increased expression of integrin α1ß1 and inhibition of epidermal growth factor receptor (EGFR) signaling protect the knee from spontaneous OA; however, the impact of the integrin α1ß1/EGFR axis on PTOA is currently unknown. We sought to determine metabolic changes in serum samples collected from wild-type and integrin α1-null mice that underwent surgery to destabilize the medial meniscus and were treated with the EGFR inhibitor erlotinib. Following (1)H nuclear magnetic resonance spectroscopy, we generated multivariate statistical models that distinguished between the metabolic profiles of erlotinib- versus vehicle-treated mice and the integrin α1-null versus wild-type mouse genotype. Our results show the sex-dependent effects of erlotinib treatment and highlight glutamine as a metabolite that counteracts this treatment. Furthermore, we identified a set of metabolites associated with increased reactive oxygen species production, susceptibility to OA, and regulation of TRP channels in α1-null mice. Our study indicates that systemic pharmacological and genetic factors have a greater effect on serum metabolic profiles than site-specific factors such as surgery.

Integrin α1ß1 participates in chondrocyte transduction of osmotic stress.

BACKGROUND/PURPOSE: The goal of this study was to determine the role of the collagen binding receptor integrin α1ß1 in regulating osmotically induced [Ca(2+)]i transients in chondrocytes. METHOD: The [Ca(2+)]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice. RESULTS/INTERPRETATION: Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca(2+)]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca(2+)]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1ß1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1ß1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4.

Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: age- and sex-dependent progression of osteoarthritis in Trpv4-deficient mice.

OBJECTIVE: Mechanical loading significantly influences the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(++)-permeable ion channel that is highly expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli. The goal of this study was to determine the role of Trpv4 in the structure of the mouse knee joint and to determine whether Trpv4(-/-) mice exhibit altered Ca(++) signaling in response to osmotic challenge. METHODS: Knee joints of Trpv4(-/-) mice were examined histologically and by microfocal computed tomography for osteoarthritic changes and bone structure at ages 4, 6, 9, and 12 months. Fluorescence imaging was used to quantify chondrocytic Ca(++) signaling within intact femoral cartilage in response to osmotic stimuli. RESULTS: Deletion of Trpv4 resulted in severe osteoarthritic changes, including cartilage fibrillation, eburnation, and loss of proteoglycans, that were dependent on age and male sex. Subchondral bone volume and calcified meniscal volume were greatly increased, again in male mice. Chondrocytes from Trpv4(+/+) mice demonstrated significant Ca(++) responses to hypo-osmotic stress but not to hyperosmotic stress. The response to hypo-osmotic stress or to the TRPV4 agonist 4α-phorbol 12,13-didecanoate was eliminated in Trpv4(-/-) mice. CONCLUSION: Deletion of Trpv4 leads to a lack of osmotically induced Ca(++) signaling in articular chondrocytes, accompanied by progressive, sex-dependent increases in bone density and osteoarthritic joint degeneration. These findings suggest a critical role for TRPV4-mediated Ca(++) signaling in the maintenance of joint health and normal skeletal structure.

Osteoarthritis: what we have been missing in the patellofemoral joint.

Patellofemoral osteoarthritis is common clinically and often independent of tibiofemoral disease. Intriguingly, the patella demonstrates more severe degeneration earlier in the disease process compared with the juxtaposed femoral groove. Here, we consider three hypotheses influencing this disparity and thus discover crucial insights into the etiology of osteoarthritis.