Elucidating the role of Transient Receptor Potential Channel Vanilloid 4 (TRPV4) mutation on channel activity and chondrocyte function in metatropic dysplasia

Metatropic dysplasia (MD) is a severe skeletal dysplasia that results in a significant impact on quality of life in patients diagnosed with disease. MD is characterized by striking skeletal changes which include general shortening of long bones, widened metaphysis, and halberd shaped pelvis. Over time, patients display wedging of vertebral bodies (platyspondyly), and progressive kyphoscoliosis. These spinal changes result in reduced spinal integrity with cervical vertebrae instability, spinal stenosis and a reduced respiratory capacity. Mutations in non-selective cation channel, Transient Receptor Potential Channel Vanilloid 4 (TRPV4) have been found to cause MD. TRPV4 associated channelopathies also include peripheral neuropathies and other skeletal dysplasias that range in clinical severity and symptomatic phenotype. As a result, causation, classification and development of TRPV4 channelopathies are very complex.;TRPV4 channel activity is integral in the Ca2+ mediated response to a large variety of sensory stimulation from the extracellular environment. TRPV4 activity can also be regulated by changes in temperature, nociception and natural and synthetic ligands, such as arachidonic acid metabolites and phorbol esters. It is most classically studied as a regulator of Ca 2+ response to changes in osmotic and mechanical forces. The spatial, temporal and intensive properties of [Ca2+]i as well as spontaneous Ca2+ oscillations can have a profound effect on cell behavior. In the musculoskeletal system, TRPV4 Ca2+ signaling regulates the chondrogenic response to osmotic signals and anabolic chondrogenic response under mechanical loads. TRPV4 also plays a chondroprotective role in normal articular cartilage and is imperative to maintaining joint health. Loss of TRPV4 often results in osteoarthritis.;Whereas most information pertains to normal TRPV4 physiology and signaling, the effect of TRPV4 mutation in terms on skeletal development has only recently been studied. Histological analysis of growth plates from lethal variants of MD revealed a highly unorganized growth plate with reduced hypertrophic regions and delayed endochondral ossification, suggesting that mutational TRPV4 activity disrupts ordered chondrogenesis. Expression of TRPV4 mutations in numerous cell types alludes to gain-of function activity that results in increased basal and stimulated intracellular Ca 2+ ([Ca2+]i) activity, as well as constitutively open channel gating. However, most studies have been performed in non-endogenous cell types that are either transfected or transformed.;To better understand channel properties directly affected by disease, we studied the effect of two TRPV4 mutations, p.Pro799Leu and p.Gly800Asp on Ca2+ channel activity and chondrocyte function in cells directly isolated from patients with metatropic dysplasia. P.Pro799Leu is considered a mutational hotspot and there is a high prevalence of this specific mutation in disease, whereas p.Gly800Asp is a novel severe mutation identified by our group. We hypothesize that MD mutations will exhibit increased basal and stimulated [Ca2+]i activity that results in improper terminal differentiation of chondrocytes. We further hypothesize that this increase in channel activity is from a direct result of alteration with C-terminal regulatory domains that occur within and immediately prior to mutational occurrence.;We observed temperature-dependent [Ca2+]i oscillations in both intact and MD chondrocytes; however, MD mutations exhibited increased peak magnitudes of [Ca2+]i during oscillations. We have also found increased baseline [Ca2+]i in MD primary cells, as well as increased [Ca2+]i response to either hypotonic swelling or the TRVP4-specific chemical agonist, GSK1016790A. Oscillations and stimulation responses could be blocked with TRPV4-specific antagonist, GSK205. Analysis of [Ca2+] i response kinetics reveals that MD chondrocytes have significantly increased frequency of temperature-sensitive oscillations, as well as increased magnitude and duration of [Ca2+]i responses to given stimuli. Additionally, duration of the response of the novel p.Gly800Asp mutation to stimulation was significantly greater than the p.Pro799Leu mutation. The altered [Ca2+]i activity caused by these TRPV4 mutations indicates that this region of the channel is essential for proper [Ca2+]i regulation and that these mutations result in a gain of Ca2+ channel activity.;TRPV4 activity has been shown to mimic the expression pattern of chondrogenic markers, Col2a1 and aggrecan, and modulate Sox9 promotor activity. These data suggest that TRPV4 is an essential regulator of chondrogenesis. We postulate that MD mutations would result in increased proliferation and reduced terminal differentiation compared to intact chondrocytes. Basally, the proliferative capacity of MD chondrocytes was unaltered compared to WT, yet both p.Pro799Leu and p.Gly800Asp chondrocytes display altered chondrogenic differentiation compared to intact chondrocytes. Proteoglycan production was dysregulated in both p.Pro799Leu and p.Gly800Asp chondrocytes with both an increase and decrease in proteoglycan production observed, respectively.;Expression of chondrogenic markers after bead culture mirrored the effect observed in micromass proteoglycan production. Differential gene expression was observed in that p.Pro799Leu chondrocytes upregulated proliferative chondrocyte makers, COL2A1, AGGRECAN and SOX9 whereas p.Gly800Asp chondrocytes were downregulated in all chondrogenic markers. While the mutational effect appeared to result in opposing dysregulation, both MD chondrocytes p.Pro799Leu and p.Gly800Asp mutations exhibited decreased markers of terminal hypertrophy, COL10A1 and RUNX. Therefore, while the mechanism behind regulation of TRPV4 in chondrogenesis appears differential between, p.Pro799Leu and p.Gly800Asp, both mutations result in a disruption of normal chondrogenesis.;Mutations of p.Pro799Leu and p.Gly800Asp occur directly in or prior to important regulatory domains within the C-terminal end of TRPV4. In this region, TRPV4 has been shown to interact with cytoskeletal proteins, MAP7, actin and tubulin as well as proteins involved in Ca2+ sensing, including calmodulin (CaM). We have found that similar to what was observed during chondrogenesis, dysregulation of interaction with C-terminal binding partners occurs differentially between the two mutations compared to intact chondrocytes. Actin regulation and binding was lost in p.Pro799Leu chondrocytes; however, microtubule binding was increased in p.Gly800Asp chondrocytes. Depolymerization of actin with Cytochalasin D results in a reduction in basal and stimulated Ca2+ activity that was not observed in p.Pro799Leu chondrocytes. Additionally, we observed increased CaM binding in p.Pro799Leu that was not observed in p.Gly800Asp or intact chondrocytes. Abolishment of microtubules with Vincristine results in a loss of response to HTS; however, it had no effect on basal TRPV4 activity. These data suggest a role for both actin and tubulin in the regulation of TRPV4 activity that seems to be stimulation and mutation-dependent.;In this study, we present differential modes of regulation for two metatropic dysplasia-causing mutations in the C-terminal end of the protein, p.Pro799Leu and p.Gly800Asp. Overall, our findings indicate the complex regulation of TRPV4 activity in terms of Ca2+ signal and downstream chondrogenic effects. We also present several possible modes of TRPV4 channel regulation through interaction with both cytoskeletal proteins and calmodulin. Understanding the mechanisms of TRPV4 regulation presents multiple avenues of interest for not only skeletal dysplasia causing mutations, but for future study into the maintenance of articular cartilage. In the case of osteoarthritis, the variety of signaling mechanisms we've studied provides several different approaches that could be used to target TRPV4 as a possible therapeutic for the promotion of overall joint health.