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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

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.

Articular cartilage, the avascular connective tissue that covers diarthrodial joint surfaces, provides a low-friction surface that supports and distributes joint loads. Cartilage comprises a hydrated extracellular matrix (ECM) of proteoglycans and collagen, as well as chondrocytes, the cells responsible for maintaining the ECM. Chondrocyte metabolic activity is regulated in part by physical factors such as mechanical loading (1–3). The transduction of biomechanical stress to an intracellular signal in cartilage represents a tissue-specific form of mechanotransduction that may involve a number of biophysical as well as biochemical events. Secondary to compression of the ECM, changes in the mechanical environment of cartilage can alter the pericellular osmolarity (4–6), potentially initiating various intracellular signal transduction pathways (7–13).

The cartilage ECM is inherently charged due to the large concentration of negatively charged proteoglycans, predominantly aggrecan. This fixed charge attracts free cations (e.g., Na+, K+, Ca++), resulting in an increase in interstitial osmotic pressure that causes the tissue to retain water (14). Upon joint loading, water is exuded from the tissue and is reabsorbed when the tissue is no longer compressed (15). Thus, chondrocytes may experience acute as well as diurnal fluctuations in their osmotic environment as a result of normal joint loading, in addition to other mechanical stimuli such as cell deformation, fluid flow, and fluid pressure (16–18). Chondrocytes respond to such osmotic fluctuations with the initiation of intracellular signaling cascades and acute volume change (9, 10) followed by active volume regulation, which involves cytoskeletal filamentous actin restructuring as well as solute transport (8, 10, 19–22). In particular, osmotic stimulation elicits extracellular Ca++ influx, which is amplified by release from intracellular stores (9, 10, 12, 23, 24). The increase of intracellular Ca++ concentration may play a role in cell volume regulation, cell metabolism, and gene expression (25–27). Chondrocyte sensitivity to both mechanical and osmotic stress has been characterized at the cellular and tissue levels, although the molecular mechanisms by which these cells sense external osmotic changes are not fully understood (2).

One novel potential candidate involved in chondrocyte mechano-osmotic signal transduction is the Ca++-permeable, nonspecific cation channel transient receptor potential vanilloid 4 (TRPV4) (28–30). TRPV4 is a nonselective ion channel activated by numerous stimuli including hypo-osmolarity (31, 32), volume increase, warmth, and certain phorbol esters including 4α-phorbol 12,13-didecanoate (4α-PDD) (33). Trpv4–/– mice have impaired responses to both hyper- and hypo-osmotic noxious stimuli, and demonstrate impaired osmotic sensing in the central nervous system with subsequently blunted behavioral and neuroendocrine homeostatic defense mechanisms (34). However, many aspects of the involvement of TRPV4 in mammalian physiology are just emerging, such as its role in endothelial vascular functioning and in airways and lung (35, 36). TRPV4 is expressed in many tissues, including the central nervous system, sensory neurons, kidney, upper and lower airways, salivary gland epithelia, endothelia, testis, and bone (37, 38), yet it is highly expressed in articular cartilage, where the channel protein is localized to the outer cell membrane including the chondrocyte's cilium (39–41).

Recently we demonstrated that in porcine chondrocytes, Ca++ signaling via TRPV4 regulates volume recovery following hypo-osmotic stimulation (41). Furthermore, the inhibition of chondrocyte signaling and volume regulation evoked by exposure to interleukin-1 (IL-1) was restored by activation of TRPV4 with 4α-PDD. The expression of TRPV4 in the chondrocyte membrane (and critically in the primary cilium), as well as the potent effects of 4α-PDD on volume regulation of chondrocytes, provides strong support for the concept that TRPV4 plays a major role in the Ca++ response of chondrocytes to compression-induced changes in their osmotic environment in situ. In addition to its role in chondrocytes' response to osmotic challenge, TRPV4 has been implicated as a regulator of chondrogenic differentiation and shows expression patterns similar to those of the chondrogenic markers type II collagen and aggrecan (40). Furthermore, TRPV4-mediated Ca++ influx evoked by 4α-PDD up-regulates SOX9, an essential transcription factor for chondrocyte differentiation (40).

Of interest, very recently, in humans, gain-of-function mutations of Trpv4 have been linked to human brachyolmia, a skeletal deformation with short stature, scoliosis, and vertebrate and long bone abnormalities, in addition to another skeletal disorder, spondylometaphyseal dysplasia (42, 43). TRPV4 is also expressed in both osteoblasts and osteoclasts, and TRPV4-mediated influx of Ca++ regulates the differentiation of osteoclasts (44). Deletion of Trpv4 suppresses unloading-induced reduction in the mineral apposition rate and bone formation rate (45). These data suggest a possible role for TRPV4 in cartilage skeletal development and maintenance. Whether morphology and function of diarthrodial joints depend on the Trpv4 gene is currently not known.

In this study, we utilized Trpv4-null mice to investigate the role of Trpv4 in the in vivo development of spontaneous knee osteoarthritis as assessed histologically and by microfocal computed tomography (micro-CT). Additionally, we measured Ca++ signaling in response to osmotic load in a novel ex vivo preparation using condylar chondrocytes within the intact native cartilage of femoral condyles of wild-type and Trpv4-deficient mice. Ex vivo condylar femoral chondrocytes from Trpv4-null mice showed impaired Ca++ signaling in response to osmotic loading in situ. The absence of Trpv4 resulted in severe, progressive osteoarthritic changes, particularly in male mice, that were accompanied by significant increases in ossification of joint tissue, suggesting a chondroprotective role for this channel, possibly mediated by its Ca++ gating in response to hypotonicity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Generation of Trpv4-null (Trpv4–/–) mice.

All animal protocols were approved by the Duke University Institutional Animal Care and Use Committee. Trpv4-null mice were generated previously, based on the zygotic Cre/loxP-mediated excision of exon 12 of the Trpv4 gene, which codes for the pore-loop and adjacent transmembrane domains (34). In this model, Trpv4 is deleted from all tissues in the body. These mice were bred on the C57BL/6J background strain past the tenth generation and were genotyped by polymerase chain reaction following established protocols based on isolation of genomic DNA from tail biopsy samples. Once genotyped, Trpv4–/– and Trpv4+/+ mice were separated and maintained as separate colonies.

Immunolabeling for TRPV4.

Immunohistochemistry was utilized to confirm the absence of the TRPV4 protein in chondrocytes from Trpv4–/– mice. The anti-TRPV4 polyclonal antibody (46) was used at 6.4 μg/ml. For immunodetection, we used Alexa Fluor 595–conjugated goat anti-mouse IgG (Molecular Probes) diluted to 1:750 in the same solution used with the primary antibody.

Micro-CT imaging.

To examine changes in bone mineral density and structure, mouse limbs were imaged using 3-dimensional micro-CT. Eight wild-type and 8 Trpv4–/– animals (4 male and 4 female) were assigned to each of the 4-, 6-, 9-, or 12-month age groups. The final 12-month time point was chosen after considering that 80% of C57BL/6J mice survive to 24 months, and spontaneous knee osteoarthritis occurs in 39% of C57BL/6J mice at age 12–17 months (47). At each time point, 6 animals (3 male and 3 female) were weighed and euthanized. Skin was removed from the hind limbs, and the right legs were dislocated at the hip and detached from the body. All leg muscles were left intact, and the limbs were stored submerged in phosphate buffered saline, pH 7.4, in cryotubes at –80°C. Limbs were defrosted and then held at a physiologic joint angle in a plastic cassette while submerged in 10% neutral buffered formalin overnight. Once fixed, each limb was held vertically in a foam mold, and 2 limbs were carefully stacked on top of one another with their patellar tendons aligned vertically inside a formalin-filled scanning cylinder. The cylinder was then placed inside the micro-CT machine (microCT 40; Scanco Medical) for scanning. High-intensity medium-resolution (16 μm) scans in the transverse plane were performed on each sample.

To distinguish calcified from soft tissue, an image intensity threshold was set and used for all images in the study, using pilot limbs from wild-type and Trpv4–/– mice. A hydroxyapatite phantom was used to scale the image intensity to bone density in mg hydroxyapatite/cm3. Four regions of subchondral and trabecular bone were analyzed on each image: medial and lateral tibial plateaus and medial and lateral femoral condyles (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1). To examine differences in bone properties immediately below the cartilage layer (i.e., subchondral bone), this region was defined starting at the first transverse slice where the subchondral plate appeared in the center of the knee through to the twelfth slice either above or below, for femoral or tibial subchondral bone, respectively. To determine changes in trabecular bone properties, bone properties were determined in a region defined by the first transverse slice where trabecular bone appeared, through to the first slice above or below that where the growth plate was evident, for the femoral or tibial trabecular bone, respectively. Since the knee menisci in mice are ossified structures, we evaluated the properties of the calcified regions of the menisci, including the anterior and posterior horns of the medial and lateral menisci (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1).

All regions were outlined using a semiautomated contouring algorithm. Bone density and volume were evaluated for all contoured regions, and apparent bone density was calculated for trabecular regions. Femoral and tibial subchondral bone thickness was measured from the sagittal section in the center of the medial and lateral load-bearing regions of each hind limb. Three measurements across the section were made using image analysis software (LSM Image browser; Zeiss), and the average was reported. Analysis of variance (ANOVA) with Tukey's post hoc comparison was performed on all of the parametric morphologic bone data.

Histology.

After scanning, hind limbs were dissected free of muscle, leaving the knee capsule intact except for a small incision in the proximal patellar tendon. Hind limbs were decalcified and then dehydrated in increasing concentrations of ethanol solution at room temperature. Samples were embedded in paraffin, and 8-μm–thick histologic sections were cut throughout the entire knee width in the sagittal plane and stained with hematoxylin, fast green, and Safranin O. Sections were graded for osteoarthritic changes by 3 blinded graders using a modified histologic grading scheme (48). ANOVA with Tukey's post hoc comparison was performed on the total histologic grade data.

Fluorescence imaging of Ca++ in intact femora.

Specimen preparation.

Wild-type and Trpv4–/– mice (ages 4–5 months) were weighed and then euthanized. Immediately, hind limbs were dislocated from the body, and the femora were isolated and cleaned of muscle, ligament, and tendon tissue under a dissection microscope. The femora were frequently sprayed with medium, and fiber optic lighting was used to ensure that the femoral cartilage remained moist throughout. To confirm cell viability after dissection, preliminary studies were performed on isolated femora using a fluorescence viability assay (Invitrogen). After harvest, femora were submerged in medium (phenol red–free Dulbecco's modified Eagle's medium [Gibco BRL], 15 mM HEPES [Gibco BRL], pH 7.4) at 37°C and 5% CO2.

The femora were held between custom-built platens (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1) inside a heated perfusion chamber (Zeiss) on an inverted confocal laser scanning microscope (LSM 510; Zeiss). Femora were submerged in 2.5 ml of medium with the femoral condylar cartilage resting on the coverslip. The temperature of the medium within the chamber was maintained at 37°C (measured with a temperature probe positioned next to the femora) and varied <1°C throughout the 12 minutes of data collection.

Fluorescence imaging.

All femora were imaged on the day of isolation. Prior to imaging, the chondrocytes of the femora were loaded for 40 minutes with 2 visible light fluorescent Ca++ indicators: Fura-Red AM (60 μM; decreases fluorescence with increased Ca++) and Fluo-4 AM (16 μM; increases fluorescence with increased Ca++) (both from Invitrogen–Molecular Probes). Transient changes in intracellular Ca++ ion concentration ([Ca++]i) were measured using an adaptation of a previously described ratiometric imaging technique (fluorescence of Fluo-4 divided by that of Fura-Red) using a 40× objective lens (EC Plan Neofluar, numerical aperture 0.75; Zeiss) (49). This ratiometric approach serves to amplify the fluorescent signal and to correct for focal changes over the course of the test. The sample was excited using an argon ion laser (488 nm), and fluorescence emission was recorded at 505–550 nm (Fluo-4) and at greater than 650 nm (Fura-Red) (Figure 1). The pinholes of the confocal microscope were fully opened to allow collection of fluorescence from the entire cell. Nine scans were performed with the femora submerged in 300 mOsm medium before this medium was withdrawn and replaced by vehicular control, anisosmotic medium (200, 250, 300, 350, or 400 mOsm), or 10 μM 4α-PDD, in order to activate TRPV4 nonosmotically. Medium osmolarity was altered by adding distilled water or sucrose to medium and verified using a freezing point osmometer (Osmette A; Precision Systems). Sequential images (1,024 × 1,024) (Figures 1A–D) were recorded at a scan rate of 0.28 Hz for 12 minutes to measure relative [Ca++]i.

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Figure 1. A–C, Fluorescence imaging of intracellular Ca++ ion concentration showing different channels for A, Fluo-4, B, Fura-Red, and C, the composite ratio image. D, Enlargement of a portion of the composite ratio image to demonstrate resolution. (Original magnification × 40.) E–G, Percentage of chondrocytes from E, wild-type and F,Trpv4–/– mouse femoral condyles responding with single or multiple calcium signals as a function of final osmolarity (starting osmolarity 300 mOsm [control] for all cases) or G, as a function of the presence of 4α-phorbol 12,13-didecanoate (4α-PDD) or its absence (control). ∗∗ = P < 0.01; ∗ = P < 0.05 versus control, by chi-square test.

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Statistical analysis.

The ratiometric fluorescence was normalized to the average value over the first 9 scans for each individual cell. A positive [Ca++]i response was then defined as an increase in normalized ratiometric fluorescence greater than 2 SD over the background noise, with both fluorescent indicators responding. The percentage of cells responding with single or multiple [Ca++]i oscillations was examined. The magnitude, duration, and time to each [Ca++]i peak were measured together with the time between [Ca++]i peaks and the rise time of each peak. The chi-square test was applied to the nonparametric [Ca++]i signaling data, and ANOVA with Tukey's post hoc comparison was performed on the parametric data describing the [Ca++]i peak characteristics.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Immunohistochemistry confirmed the absence of the TRPV4 protein in chondrocytes from Trpv4–/– mice (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1). The mice used in this study did not demonstrate any overt signs of altered gait or other signs of altered joint function during regular routine observations of the animals in their cages. Immediately after mice were euthanized, the knees showed no differences in laxity, stiffness, or range of motion. Male mice were heavier than female mice (mean ± SD 35.3 ± 7.4 grams versus 29.9 ± 4.3 grams), and older mice were heavier than younger mice (mean ± SD 33.0 ± 5.5 grams for 12-month-old females, 27.3 ± 2.3 grams for 4-month-old females, 42.2 ± 12.0 grams for 12-month-old males, 31.5 ± 2.1 grams for 4-month-old males). There were no significant differences in body weight between wild-type and knockout mice (mean ± SD 29.5 ± 4.1 grams for wild-type females, 30.3 ± 4.5 grams for Trpv4–/– females, 35.4 ± 9.4 grams for wild-type males, 35.2 ± 4.3 grams for Trpv4–/– males).

Micro-CT imaging findings.

Calcification and ossification.

Micro-CT imaging was performed to determine changes in bone architecture and bone mineral density with age (Figure 2). Enlargement of sesamoid bones and increased calcifications in the knee were qualitatively observed. These included the patellar tendon, both anterior and posterior to the patella, and both sesamoid bones posterior to the femoral condyles (Figure 2). Increased calcification of the growth plate was also observed (Figure 2).

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Figure 2. Representative frontal (left) and sagittal (right) microfocal computed tomography views of the intact knee of male wild-type (A and B) and Trpv4–/– (C and D) mice at ages 4 months (A and C) or 12 months (B and D). Significant enlargement of the calcified regions of the menisci was observed, as well as expansion of the patellar and condylar sesamoid bones (arrows), in Trpv4–/– mice at age 12 months.

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At 4, 6, and 9 months, the calcified meniscal regions of both male and female wild-type and Trpv4–/– mice had similar volume, with the anterior regions being larger than posterior areas (Figures 3A and C). While menisci generally ossify with age in rodents, at 12 months there was a profound increase in calcified volume in all meniscal regions of Trpv4–/– mice (Figure 2), with this effect being exacerbated in male compared with female mice (Figure 3A). On average, the volume of calcified meniscus was larger in Trpv4–/– mice than in wild-type mice in both anterior locations and in the posterior portion of the medial meniscus (Figure 3C). In addition, the density of these calcified portions of the menisci increased linearly with time and in a similar manner in wild-type and Trpv4–/– mice (Figure 3B).

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Figure 3. A and B, Calcified meniscal bone volume (A) and density (B) as a function of age, sex, and genotype. C, Calcified meniscal bone volume as a function of site (lateral [Lat], medial [Med]), position (anterior, posterior), and genotype. Vertical bars represent 95% confidence intervals. a = significant genotype difference between equivalent data points; b = significant difference from all 4-month data points; c = significant difference from 6-month equivalent data points; d = significant difference from all other data points; e = significant difference from posterior equivalent data points. (P values less than 0.05 considered significant). HA = hydroxyapatite.

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Subchondral bone.

Male Trpv4–/– mice had larger subchondral bone volume compared with their wild-type counterparts at 12 months (Figure 4A). This increase in volume could not be fully accounted for by an increase in subchondral thickness and thus includes expansion in the medial/lateral and anterior/posterior directions (Figure 2). In contrast, subchondral bone volume was similar at all time points in female Trpv4–/– and wild-type mice (Figure 4A). On average, subchondral bone volume was larger in medial than in lateral sites (except for the tibial plateau in Trpv4–/– mice) and in femoral condyles than in tibial plateaus (except for the lateral aspect in Trpv4–/– mice) (Figure 4C). Subchondral bone density increased in a linear manner with time in both female and male Trpv4–/– and wild-type mice (Figure 4B). Additionally, subchondral condylar regions were more dense than their plateau counterparts (data not shown).

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Figure 4. A, B, and D, Subchondral bone volume (A), subchondral bone density (B), and trabecular bone density (D) as a function of age, sex, and genotype. C and E, Subchondral bone volume (C) and trabecular bone density (E) as a function of site (lateral [Lat], medial [Med]), bone (femoral condyle/tibial plateau), and genotype. Vertical bars represent 95% confidence intervals. a = significant genotype difference between equivalent data points; b = significant difference from females at equivalent data points; c = significant difference from all 4-month data points; d = significant difference from 4-month equivalent data points; e = significant difference from all other data points except 4- and 9-month equivalents; f = significant difference from tibial equivalent data points; g = significant difference from medial equivalent data points. (P values less than 0.05 considered significant). HA = hydroxyapatite.

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Trabecular bone.

Trabecular bone density increased in a linear manner with time in both female and male Trpv4–/– and wild-type mice (Figure 4D). Trabecular bone density was greater in wild-type mice than in Trpv4–/– mice in all regions except the medial tibial plateau and was greater in lateral femoral condylar regions than in lateral tibial plateau regions in both wild-type and Trpv4–/– mice (Figure 4E). In wild-type and Trpv4–/– mice, trabecular bone density of the tibial plateau was greater on the medial than on the lateral side (Figure 4E). Trabecular bone apparent density was greater in all condylar regions than in tibial plateau regions and greater on the medial than on the lateral side except for the femoral condyle in wild-type mice (data not shown). There was no consistent relationship between trabecular bone apparent density and age in male or female Trpv4–/– or wild-type mice (data not shown).

Histologic findings.

By qualitative and semiquantitative histologic measures, knockout mice showed age-and sex-related increases in joint abnormalities associated with osteoarthritic degeneration. At 12 months, male Trpv4–/– mice had higher histologic scores than all other mice (Figure 5A). These mice demonstrated severe end-stage osteoarthritic pathology including complete cartilage erosion exposing subchondral bone on both femoral and tibial articulating surfaces (Figure 6). Furthermore, menisci were considerably enlarged throughout the joints. At 9 months, male Trpv4–/– mice had increased histologic scores compared with their wild-type counterparts, with all 4-month-old mice, and with their female counterparts (Figure 5A). Cartilage was fibrillated and demonstrated proteoglycan depletion (images not shown). In complete contrast, there was no significant difference in histologic scores between female Trpv4–/– mice and wild-type mice at any time point (Figure 5A). On average (pooled sexes), Trpv4–/– mice had higher histologic scores than wild-type mice at femoral and tibial sites on both the lateral and medial sides of the knee, although this effect was principally due to the differences in the male mice (Figure 5B). These scores were higher for the tibial than for the femoral articulating surfaces in both genotypes (Figure 5B).

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Figure 5. Total histologic score as a function of age, sex, and genotype (A) and as a function of site (lateral [Lat], medial [Med]), bone (femoral condyle/tibial plateau), and genotype (B). Vertical bars represent 95% confidence intervals. a = significant genotype difference between equivalent data points; b = significant difference from females at equivalent data points; c = significant difference from all 4-month data points; d = significant difference from all other data points; e = significant difference from tibial equivalent data points. (P values less than 0.05 considered significant).

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Figure 6. Representative sagittal histologic sections through the central region of the medial condyle of male wild-type (AD) and Trpv4–/– (EH) mice at ages 4 months (A, B, E, and F) or 12 months (C, D, G, and H). Sections were stained with hematoxylin, fast green, and Safranin O (original magnification × 40 in A, C, E, and G[bar = 500 μm]; × 200 in B, D, F, and H[bar = 100 μm]). Note the enlarged menisci, thickened subchondral bone, and severe loss of articular cartilage (arrow) in Trpv4–/– mice at age 12 months.

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Osmotically induced Ca++ signaling in chondrocytes.

The technique developed in this study allowed us to measure changes in [Ca++]i in chondrocytes that were maintained in their fully intact femoral condylar cartilage matrix environment. Experiments demonstrated a significant effect of hypo-osmolarity on [Ca++]i in wild-type mice but not in Trpv4/ mice (Figures 1E and F). Consistent with this, 4α-PDD evoked [Ca++]i transients in wild-type mice but not in Trpv4/ mice (Figure 1G). In contrast, hyperosmolarity had no effect on [Ca++]i in preparations from either wild-type or Trpv4/ mice (Figures 1E and F).

The Ca++ responses in wild-type mice were characteristically different for osmolarity compared with 4α-PDD stimuli. Peak magnitudes of [Ca++]i were larger in magnitude and longer in duration with hypo-osmotic stimulation compared with 4α-PDD exposure (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1). The latency between and rise time of [Ca++]i peaks were both longer for osmotic compared with 4α-PDD stimulation; in contrast, time to peak was similar for hypo-osmotic and 4α-PDD exposure (further information is available at http://ortho.duhs.duke.edu/modules/obl_pubs/index.php?id=1).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The findings of this study show that Trpv4–/– mice spontaneously develop osteoarthritis at a younger age and to a more severe extent than their wild-type controls. Trpv4+/+ and Trpv4–/– mice were indistinguishable at age 4 months by nearly every micro-CT and histologic parameter measured, suggesting that TRPV4 expression is not necessary for skeletal development of the knee. However, significant deleterious changes were observed by 9 months, and by age 12 months, Trpv4–/– mice exhibited erosions of the articular cartilage penetrating down through the subchondral bone. Furthermore, Trpv4–/– mice exhibited increased bone mass and overgrown, calcified menisci that completely surrounded the tibiofemoral compartments. Accompanying these changes was a loss of osmotically activated Ca++ signaling in chondrocytes of Trpv4–/– mice. These unexpected findings suggest that the mechanically and osmotically sensitive TRP channel, TRPV4, functions in a critical role in cartilage maintenance and “chondroprotection,” presumably via its role as a mediator of osmotically activated Ca++ conductance in chondrocytes.

Another surprising result of our investigation was that the bony changes and osteoarthritic degeneration in Trpv4–/– mice were more pronounced in males than in females. This finding is consistent with the majority of the reports of spontaneous osteoarthritis in mice (50) as well as with reports of murine osteoarthritis models based on surgical intervention (51) or intraarticular injection (52). In all of these models, joint degeneration was found to be increased in male mice compared with female mice. Our present findings extend this concept to a genetically encoded model of cartilage degeneration, namely, the deletion of Trpv4. In contrast, the incidence and severity of osteoarthritis in humans are significantly higher in women, particularly after menopause, than in men (53–55). Little is currently known regarding the sex-specific expression of TRPV4. A recent study has shown that hyponatremia in humans is associated with a Trpv4 polymorphism, but only in men (56). Furthermore, progesterone has been shown to regulate the expression of TRPV4 in human airway and mammary gland epithelial cells as well as in vascular smooth muscle cells (57). The exposure of these cells to progesterone decreased TRPV4 expression at both the gene and protein levels, thus diminishing 4α-PDD–induced Ca++ signaling. The mechanisms responsible, be they gene or channel regulatory, for the sexual dimorphism of the chondroprotective role of Trpv4 remain to be determined, and future studies will address the obvious and pertinent questions, namely, whether the Trpv4 gene and/or the TRPV4 channel function are regulated by gonadal steroids.

In addition to cartilage and bone, TRPV4 is present in many tissues throughout the body, including the central nervous system, sensory neurons, kidney, upper and lower airways, salivary gland epithelia, endothelia, and testis. The Trpv4–/– model used in this study is not tissue specific, and therefore the changes observed in this study may also involve the loss of TRPV4 from other tissues. In future studies, conditional knockout models specific to the cartilage or bone may provide further insight into the specific role of TRPV4 in cartilage or bone.

Taken together with recently published reports (41–45), including our own on the role of TRPV4 in human and porcine articular chondrocytes, this study reveals a critical role for the Trpv4 gene in skeletal function. Our present analysis of mice with genetically encoded deletion of Trpv4 indicates that the knee shows progressive degeneration in the absence of Trpv4, with the first morphologic signs at age 9 months and more severe cartilage loss at age 1 year. Of note, these observed differences between Trpv4–/– mice and wild-type controls were significant only in male mice.

In order to gather explanatory evidence at the level of the chondrocyte, we conducted Ca++ imaging in response to tonicity stimuli in chondrocytes in an ex vivo preparation using intact femoral condyles from wild-type and Trpv4–/– mice. In the absence of Trpv4, chondrocytes did not show an increased response to hypotonicity (i.e., they were osmotically nonresponsive). Under normal circumstances, the ability to respond to mechanical and/or osmotic stimuli constitutes a hallmark of chondrocyte physiology; thus, our basic insight of chondrocytes' nonresponsiveness to hypotonicity in the absence of Trpv4 could likely be a partial mechanistic explanation for the impaired homeostatic balance in the cartilage of Trpv4-null mice. Lack of plasticity in response to hypo-osmotic stress translates to a lack of Ca++ influx into chondrocytes, where it is needed in cellular volume homeostasis specifically to regulate increased cell volume. In this regard, our recent work with primary chondrocytes from porcine knee joints demonstrated that activation of TRPV4 is sufficient to rescue defective regulatory volume decrease evoked by IL-1 receptor signaling (41), a reductionist molecular model of the joint's inflammatory injury response in early osteoarthritis. Thus, TRPV4-mediated Ca++ influx into chondrocytes underlies a chondroprotective role. In other words, for genetically encoded absence of Trpv4, over the animal's lifetime an osmotic-sensing incompetent chondrocyte will likely be incompetent at maintaining tissue homeostasis as well, given the importance of mechano-osmotic signaling in the normal maintenance of cartilage physiology (2).

In addition, our Ca++ measurements demonstrated that stimulation of TRPV4 using either hypo-osmotic loading or the synthetic agent 4α-PDD led to contrasting characteristics in the resulting Ca++ transients. Calcium peaks were larger in magnitude and longer in duration in hypo-osmosis compared with 4α-PDD experiments. Further, the latency between and rise time of calcium peaks were both longer with osmotic compared with 4α-PDD stimulation. While these differences could perhaps be due to differences in the equivalent “dose” of hypo-osmotic stress as compared with 4α-PDD in activating TRPV4 in chondrocytes, the data rather suggest that different characteristics of the [Ca++]i response depend on the TRPV4-specific stimulus modality. With regard to possible costimulated signaling pathways in response to hypotonicity in chondrocytes, our result of a complete absence of [Ca++]i response in Trpv4-null mice provides evidence of the predominant position of TRPV4 channels in such a signal transduction hierarchy, namely, that TRPV4 is absolutely necessary for Ca++ to enter chondrocytes in response to hypotonicity.

In conclusion, we have shown that the genetically encoded absence of Trpv4 results in early and severe development of spontaneous osteoarthritis in male mice. This pathology appears throughout the joint and is first observed in articular cartilage at 9 months. At the cellular level, TRPV4 is necessary for murine chondrocytes to transduce hypotonicity into Ca++ transients in intact femora. Together, these observations suggest a “chondroprotective” role for TRPV4 in addition to its significant roles in homeostasis and maintenance of articular cartilage, all in a sexually dimorphic manner with male animals more susceptible to the arthritogenic sequelae of Trpv4 deletion. An understanding of the role of TRPV4 and other ion channels in the response of chondrocytes to mechanical stimuli will provide new insights into the growth, development, and pathology of articular cartilage, as well as the potential for influencing cartilage regeneration in the context of tissue engineering.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Guilak had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Clark, Votta, Kumar, Liedtke, Guilak.

Acquisition of data. Clark, Liedtke.

Analysis and interpretation of data. Clark, Kumar, Liedtke, Guilak.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The authors would like to thank Steve Johnson for exceptional help with all animal procedures, Greg Meyers for assistance with histologic sectioning, staining, and photography, Eric Mansfield for assistance with the Ca++ data analysis, Tim Griffin, Bridgette Furman, and Holly Leddy for assistance with histologic grading, and Sukhee Lee and Holly Leddy for TRPV4 immunohistochemistry.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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ART_27624_sm_suppfigures.pdf503KSupplementary Figure

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