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Integrin α1β1 protects against signs of post-traumatic osteoarthritis in the female murine knee partially via regulation of epidermal growth factor receptor signalling
Department of Radiology, Faculty of Medicine, University of Calgary, Calgary, AB, CanadaMcCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB, Canada
Address correspondence and reprint requests to: A.L. Clark, ANNU 342A, Department of Human Health and Nutritional Sciences, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada. Tel: 1-519-824-4120x52134; Fax: 1-519-763-5902.
Faculty of Kinesiology, University of Calgary, Calgary, AB, CanadaMcCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB, CanadaDepartment of Surgery, Faculty of Medicine, University of Calgary, Calgary, AB, Canada
To investigate the role of integrin α1β1 in the progression of post-traumatic osteoarthritis (PTOA), and elucidate the contribution of epidermal growth factor receptor (EGFR) signalling to the mechanism by which integrin α1β1 might control PTOA. We hypothesised that integrin α1β1 plays a protective role in the course of PTOA and that the effect of PTOA (e.g., synovitis, loss of cartilage and growth of osteophytes) would be exacerbated in mice lacking integrin α1β1 at every time point post destabilisation of medial meniscus (DMM).
Methods
DMM or sham surgery was performed on integrin α1-null and wild type (WT) mice and the progression of PTOA analysed at 2, 4, 8 and 12 weeks post-surgery (PS) using micro-computed tomography (microCT), histology, and immunohistochemistry. In addition, the effects of EGFR blockade were examined by treating the mice with the EGFR inhibitor erlotinib.
Results
Integrin α1-null female, but not male, mice showed earlier cartilage degradation post DMM surgery compared to WT controls. Furthermore, erlotinib treatment resulted in significantly less cartilage damage in integrin α1-null but not WT mice. Independent of genotype, erlotinib treatment significantly mitigated the effects of PTOA on many tissues of female mice including meniscal and fabella bone volume, subchondral bone thickness and density and cartilage degradation. In contrast, reduced EGFR signalling had little effect on signs of PTOA in male mice.
Conclusion
Integrin α1β1 protects against PTOA-induced cartilage degradation in female mice partially via the reduction of EGFR signalling. Furthermore, reduction of EGFR signalling protects against the development of PTOA in female, but not male mice.
Osteoarthritis (OA) is a degenerative joint disease that manifests as degraded articular cartilage, thickened subchondral bone, osteophyte growth and synovitis
High prevalence of osteoarthritis 14 years after an anterior cruciate ligament tear in male soccer players: a study of radiographic and patient relevant outcomes.
. In contrast to spontaneous OA (occurring with older age and without specific joint trauma) where diagnosis normally occurs with pain at endstage disease, the period of latency between injury and the development of PTOA offers an opportunity to intervene in the disease process before any signs or symptoms of OA are apparent. Despite this treatment window, there are still no effective strategies to delay or slow the progression of PTOA.
Integrins are heterodimeric transmembrane receptors that modulate the activation of growth factor receptors including transforming growth factor β (TGF-β) receptor (TGFβR) and epidermal growth factor receptor (EGFR)
Growth factor regulation of chondrocyte integrins. Differential effects of insulin-like growth factor 1 and transforming growth factor beta on alpha 1 beta 1 integrin expression and chondrocyte adhesion to type VI collagen.
on human chondrocytes. In knee cartilage, spontaneous OA results in increased integrin α1β1 expression before macroscopic damage to the cartilage occurs
. The mechanism(s) by which integrin α1β1 delays the signs of spontaneous OA and the role of integrin α1β1 in PTOA, are unknown. Two potential protective mechanisms may involve down-regulation of EGFR and/or TGFβR signalling pathways, based on convincing evidence that integrin α1β1 negatively regulates the activation of these two growth factor receptors
. Additionally, more severe spontaneous OA as a consequence of enhanced EGFR activation has been confirmed in mice with global or cartilage-specific mig-6 deletion
. In contrast, enhanced cartilage damage post destabilisation of the medial meniscus (DMM) has been reported in male mice with dampened EGFR signalling
, however an overabundance of TGF-β signalling in the mouse knee leads to ‘chondrophytes’, proteoglycan depletion and lesions in tibial cartilage indicative of mouse OA
. In light of these data, a degradative effect of elevated TGFβR-mediated signalling that contributes to the early OA experienced by integrin α1-null mice cannot be ruled out.
The goal of this study was to examine the role of integrin α1β1 in the progression of PTOA. We hypothesised that integrin α1β1 plays a protective role in the course of PTOA following DMM and that the effect of PTOA would be exacerbated in mice lacking integrin α1β1. Furthermore, we administered erlotinib, an EGFR inhibitor approved by the Food and Drug Administration
and wild type (WT) mice, generated from pure BALB/c integrin α1-heterozygote breeding pairs, were crossed among themselves in order to produce 100 integrin α1-null and 100 WT pure BALB/c mice, 50 of each sex. At 13 ± 1 weeks of age DMM or sham microsurgery was performed on the left knee
. Twenty integrin α1-null and 20 WT mice, with equal numbers of surgery type (DMM/sham) and sex (male/female) were sacrificed at 2, 4, and 8 weeks post-surgery (PS). Forty integrin α1-null and 40 WT mice were sacrificed at 12 weeks PS.
Mice sacrificed at 12 weeks PS received 50 mg/kg/day erlotinib (Genentech, San Francisco, CA) suspended in 0.5% (w/v) hydroxypropyl methylcellulose (Sigma–Aldrich, St. Louis, MO) (Dow) and 0.1% (v/v) Tween 80 in distilled water or vehicle by oral gavage. Erlotinib hydrochloride (Tarceva®) is an EGFR inhibitor that reduces the severity of collagen-induced arthritis in mice
. The specificity/selectivity of erlotinib to EGFR is greater than that of the tyrosine kinases v-abl and c-Src and other kinase domains including that of the insulin receptor and insulin like growth factor 1 receptor, and erlotinib has been shown to inhibit EGFR phosphorylation in intact cells and in vivo in mice and humans
. Gavage began the day after surgery and continued until the day before sacrifice. Immediately after sacrifice, hindlimbs were removed, skinned and stored in phosphate buffered saline (Invitrogen) at −80°C until further processing. Contralateral knees of DMM joints were used as naïve controls.
Micro-computed tomography (microCT)
Hindlimbs were formalin fixed at a physiological joint angle and high intensity medium resolution (nominal isotropic 16 μm voxel size) microCT scans in the transverse plane were obtained (microCT 35; Scanco Medical, Brüttisellen, Switzerland). Calibration confirmation of the microCT machine was performed weekly
Calibration of Micro-CT Data for Quantifying Bone Mineral and Biomaterial Density and Microarchitecture. Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials.
and the calcified portions of the medial collateral ligament were analysed. Medial and lateral subchondral regions were defined from the first transverse slice where the subchondral bone appeared to the twelfth slice above or below, for femoral and tibial bone, respectively. Trabecular regions were defined as the first slice where trabeculae appeared through to the slice just prior to the appearance of the growth plate. A semi-automated contouring tool was used to define these regions of interest, and a custom written code identified bone on the microCT images using black/white contrast, and evaluated total volume, bone volume, bone volume fraction, bone mineral density, and tissue mineral density for all regions (IPL; Scanco Medical AG, Brüttisellen, Switzerland). Additionally, connectivity density, structure model index (SMI), trabecular thickness, and trabecular separation were evaluated for trabecular bone regions. Subchondral bone thickness was measured at three equidistant positions across the load bearing region of the knee using a distance transform algorithm
, and the average reported. The calcified medial collateral ligament (cMCL) was scored by finding the range of bone volume and tissue mineral density across all cMCL, then scoring each joint out of three for both parameters relative to that range (0 = no cMCL, 1 = bottom third, 2 = middle third, 3 = top third). The two scores were added together for a maximum score of six per joint.
Histology
Hindlimbs were dissected free of muscle (leaving the knee capsule intact) and decalcified. Coronal histological sections (8 μm) were stained with haematoxylin, fast green and safranin-O
Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: age-and sex-dependent progression of osteoarthritis in Trpv4-deficient mice.
. Eight sections at 60–70 μm intervals in the centre of each contact region were imaged (Zeiss AxioCam ICc1; Jena, Germany) and scored using the Osteoarthritis Research Society International grading scale
. The sum and maximum histological scores across the eight sections per joint at each of the four regions were calculated separately. Synovitis was scored based on the enlargement of the synovial lining cell layer (0 = 1–2 cells thick, 1 = 2–4 cells thick, 2 = 4–9 cells thick, 3 = Thickness ≥10 cells, modified from Ref.
) at all four regions of the joint. The scores of all four regions were summed and reported.
Immunohistochemistry
Sections were incubated with Proteinase K (20 μg/ml in TE Buffer, pH 8.0) followed by 3% hydrogen peroxidase and blocking with 5% goat serum. Primary antibodies against pSmad2/3 Ser465/467 (1:50, Cell Signaling Technology Inc., Danvers, MA), pSmad1/5/8 Ser187 (1:50, ab73211, Abcam, Toronto, ON) or pEGFR Y1092 (1:50, ab40815, Abcam, Toronto, ON) were incubated at 4°C overnight. A secondary horseradish peroxidase labelled anti-rabbit polymer (Dako, Burlington, ON) and 3,3′-diaminobenzidine developer (Sigma–Aldrich, Oakville, ON) were sequentially applied. Slides were counterstained and mounted.
One slide from the central contact region of three animals was imaged on a Nikon Eclipse E400 (Nikon, Mississauga, ON) upright microscope. For the pEGFR stained sections, slides were imaged from female and male, integrin α1-null and WT, erlotinib and vehicle treated 12 weeks PS mice. For pSmad2/3 and pSmad1/5/8 stained sections, slides were imaged from male, integrin α1-null and WT, 2, 4, 8 and 12 weeks PS mice. Images were graded by two experienced, blinded graders.
Statistical analysis
Statistica (StatSoft Inc., Tulsa, OK) was used for all statistical analysis and significance defined as P < 0.05. All histological cartilage, morphological bone, and immunohistochemical data were analysed using Multivariate analysis of variance (MANOVA) with all applicable independent categorical variables (time PS (2, 4, 8, and 12) weeks), sex (male/female), genotype (α1-null/WT), surgery type (DMM/sham), drug (erlotinib/vehicle), bone (femur/tibia), site (anterior/posterior), and compartment (medial/lateral) and a continuous predictor of mass. The Fisher LSD post hoc test was performed to determine significance. Statistical analyses of cMCL and synovitis data were performed using chi-square and t-tests.
Results
Animals
Male mice weighed approximately 5 g more than female mice and had consistent mass across groups at surgery [Supplementary Fig. 1(A)]. Female integrin α1-null mice in the 2 and 4 weeks PS group were 2 g heavier than the equivalent mice assigned to later time points, and female erlotinib treated WT mice weighed 4 g less than their vehicle treated counterparts [Supplementary Fig. 1(A)]. All mice in the 8 weeks PS group gained significantly more mass during the study compared to mice assigned to all other time points, and in WT mice of both sexes an increase in mass was evident in the 4 week compared to 2 weeks PS group [Supplementary Fig. 1(B)]. Interestingly, there was minimal change in mass in all 12 and 12E group mice, suggesting that the weight gained at other time points was due to food consumption rather than skeletal growth [Supplementary Fig. 1(B)].
Subchondral bone
Subchondral bone was thicker at 8 and 12 weeks PS in DMM compared to sham and contralateral joints in both femur and tibia, but only in the medial compartment [Fig. 1, Fig. 2]. Interestingly, the thickened subchondral bone after surgery was diminished in erlotinib treated females in both the femur and tibia [Fig. 2(A) and (B)] but increased in males in the femur [Fig. 2(A)]. In addition to thickness, DMM surgery significantly affected subchondral bone tissue mineral density and bone volume (Supplementary Fig. 2) with both parameters being larger in DMM compared to sham and contralateral joints in the medial femur. Erlotinib treatment diminished the increase in tissue mineral density with surgery in both sham and contralateral knees but the decrease did not reach statistical significance in DMM joints [Supplementary Fig. 2(A)]. Finally, subchondral bone volume was larger in the medial than the lateral compartment, and erlotinib treatment decreased this parameter in the medial compartment but increased it in the lateral compartment, but only in tibia [Supplementary Fig. 2(C) and (D)].
Fig. 1Representative microCT images of left knees from male and female integrin α1-null and WT mice at 2 and 12 weeks PS that underwent either DMM or sham surgery. Mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle
daily. A, B, D, F, G, I, K, L, N, P, Q, S Cortical bone at the femoral condyles and tibial plateaus is rendered transparent to show the analysed trabecular regions. Trabecular bone is shaded blue, subchondral bone pink, and ossified menisci green. C, E, H, J, M, O, R, T Frontal cross-section taken in the middle of femoral condyle/tibial plateau contact region shaded with a thickness map with thicker regions shaded with hot colours located further right of the spectrum scale. Note the medial displacement of medial meniscus in DMM joints (A, B, D, K, L, N), larger medial meniscus and cMCL at 12 weeks PS in DMM knees (B, L) with this effect lessened with erlotinib treatment in female (D) but worsened in male mice (N), and thicker subchondral bone in DMM (C, E, M, O) compared to sham (H, J, R, T) joints.
Fig. 2Subchondral bone remodelling in knees of integrin α1-null and WT mice at 2, 4, 8, and 12 weeks post DMM or sham surgery as a function of (A, B) sex and bone or (C, D) sex and compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle
daily. Data points represent mean n ≥ 9 with 95% confidence interval. a Different from 2, 4, and 12E weeks PS equivalent (P < 0.01). b Different from all time points PS equivalent (P < 0.05). c Different from 2 and 12E weeks PS equivalent (P < 0.05). d Different from 2 and 4 weeks PS equivalent (P < 0.01). e Different from 12 weeks PS (P < 0.05). f Different from 2, 4, and 8 weeks PS (P < 0.05). * Different from time-matched counterpart(s) (P < 0.05). Note increased thickness with DMM in both femur (A) and tibia (B) but only in the medial compartment (C), with this diminished in erlotinib treated females but not males (A–C).
Calcified meniscal bone volume was larger in DMM joints at 8 and 12 weeks PS compared to sham and contralateral joints at both anterior and posterior sites [Fig. 3(A) and (B)] but only in the medial compartment [Fig. 1, Fig. 3]. Erlotinib treatment diminished this surgery effect in females at both anterior and posterior sites but had no affect or increased the surgery effect in males at the posterior and anterior sites, respectively [Fig. 1, Fig. 3]. When female and male data were combined, a significant drug effect was observed in the medial but not the lateral compartment of the knee [Fig. 3(C)].
Fig. 3Meniscal bone remodelling in knees of integrin α1-null and WT mice at 2, 4, 8, and 12 weeks post DMM or sham surgery as a function of (A, B) site and sex or (C) compartment. The contralateral knees (contra) of DMM mice served as a naïve control. Mice in the 12 week group were administered erlotinib (12E) or vehicle
daily. Data points represent mean (A, Bn ≥ 9, Cn ≥ 19), with 95% confidence interval. a Different from all time points equivalent (P < 0.05). b Different from 2 and 4 weeks PS equivalent (P < 0.01). * Different from time-matched counterpart(s) (P < 0.05). Note (A, B) the increase in meniscal bone volume with DMM in both anterior and posterior sites but (C) only in the medial compartment and (A, B) the reduction of this with erlotinib treatment in females but not males.
More than twice as many females had a cMCL compared to males, but the severity of calcification (as measured by bone volume and density) was similar between sexes (Supplementary Table 1). In a similar manner, approximately twice as many DMM joints had a cMCL compared to sham and contralateral joints, but with similar severity across surgery groups [Fig. 1(B), (G), (L), (Q) and Supplementary Table 1]. The effect of surgery on increased frequency of cMCL was seen at 8 weeks PS (Supplementary Table 1). No genotype or drug effects on cMCL were observed (Supplementary Table 1).
Fabella
Medial but not the lateral fabellae were larger in DMM compared to sham and contralateral joints at all time points PS [Supplementary Fig. 3(A)]. Bone volume fraction was also larger in DMM compared to sham joints at 4, 8 and 12 weeks PS and in DMM compared to contralateral joints at 4 and 12 weeks PS [Supplementary Fig. 3(B)]. Erlotinib treatment significantly reduced both lateral and medial fabella bone volume and bone volume fraction, with this effect being most consistent in DMM compared to sham and contralateral joints [Supplementary Fig. 3(A) and (B)].
The lateral and medial fabellae were significantly larger, but less dense in male compared to female mice at all time points [Supplementary Fig. 3(C) and (D)]. A 4 week delay in the increase of tissue mineral density in males compared to females was also observed in both lateral and medial fabellae [Supplementary Fig. 3(D)]. The effect of Erlotinib treatment was sex dependent, decreasing both bone volume and tissue mineral density in females but not males [Supplementary Fig. 3(C) and (D)].
Trabecular bone
Surgery did not significantly affect any of the trabecular bone parameters. However, erlotinib treatment consistently altered multiple parameters of trabecular bone in female, but not male mice [Fig. 4]. Tissue mineral density in both femur and tibia [Fig. 4(A) and (B)], and bone volume fraction [Fig. 4(C)] and trabecular thickness [Fig. 4(D)] in the femur, were all decreased in female mice receiving erlotinib, in contrast to femoral connectivity density [Fig. 4(E)] which was increased. Additionally, there was a notable 4 week delay in the increase of femoral tissue mineral density [Fig. 4(A)], bone volume fraction [Fig. 4(C)], and trabecular thickness [Fig. 4(D)] in male compared to female mice. In general, tissue mineral density [Fig. 4(A) and (B)], bone volume fraction [Fig. 4(C)], and trabecular thickness [Fig. 4(D)] were larger and connectivity density [Fig. 4(E)] smaller in females compared to males.
Fig. 4Trabecular bone remodelling in knees of integrin α1-null and WT mice at 2, 4, 8, and 12 weeks PS as a function of sex, compartment and bone. Mice in the 12 week group were administered erlotinib (12E) or vehicle
daily. Data points represent mean (n ≥ 19) with 95% confidence interval. a Different from 2 weeks PS equivalent (P < 0.05). b Different from all other points (P < 0.01). c Different from 8 weeks PS equivalent (P < 0.05). d Different from all other points except 8 weeks PS equivalent (P < 0.01). e Different from 2 and 12E weeks PS equivalent (P < 0.05). f Different from 12 and 12E PS equivalent. * Different from time-matched counterpart(s) (P < 0.05). Note the significant effect of erlotinib in females but not males on all parameters and a delayed response in males compared to females in some femoral parameters.
Bone volume fraction was larger [Supplementary Fig. 4(B)], and trabecular separation smaller [Supplementary Fig. 4(D)] in the tibiae of WT compared to integrin α1-null mice at 2 and 4 weeks PS. Erlotinib treatment reduced femoral and tibial bone volume fraction [Supplementary Fig. 4(A) and (B)] in female but not male mice independent of genotype, however erlotinib reduced tibial trabecular spacing [Supplementary Fig. 4(D)] only in WT female (but not male) mice.
Cartilage
In male mice of both genotypes, increased cartilage damage was seen as early as 4 weeks PS in the medial compartment of both femur and tibia of DMM but not sham or contralateral knees [Fig. 5, Fig. 6, Supplementary Fig. 5(A) and (B) and Supplementary Table 2]. Interestingly, this surgery effect was delayed until 8 weeks PS in integrin α1-null females and 12 weeks PS in WT females [Fig. 5, Fig. 6, Supplementary Fig. 5(C) and (D) and Supplementary Table 2]. Despite this contrasting time of onset in cartilage degradation in male and female mice, the severities of both summed and maximum cartilage degeneration at 12 weeks PS were similar between the two sexes [Fig. 6, Supplementary Fig. 5 and Supplementary Table 2]. Importantly, erlotinib treatment decreased maximum histology score in both sexes (Supplementary Figs. 5 and 6 and Supplementary Table 2) and summed histology score in females only [Fig. 6 and Supplementary Table 2] primarily in the medial femoral condyle of integrin α1-null but not in WT mice.
Fig. 5Representative frontal histology images of left knees from male and female integrin α1-null and WT mice at 2, 4, 8 or 12 weeks PS. Knees underwent either DMM or sham surgery and mice in the 12 weeks PS group were administered erlotinib (12E) or vehicle
daily. Sections were stained with haematoxylin, fast green, and safranin-O. Scale bar represents 500 μm. Note the thinner and more fibrillated cartilage in the medial compartment (arrows) beginning at 4 weeks PS in male integrin α1-null and WT DMM knees with this effect delayed until 8 weeks PS in female integrin α1-null mice and 12 weeks PS in female WT mice. Erlotinib treatment alleviated cartilage damage in both male and female integrin α1-null but not WT mice.
Fig. 6Summed histology OA score (out of 48) of knees of WT and integrin α1-null mice at 2, 4, 8, and 12 weeks post DMM or sham surgery as a function of sex, genotype, and region. Mice in the 12 week group were administered erlotinib (12E) or vehicle
daily. MFC Medial Femoral Condyle. MFC Medial Femoral Condyle. MTP Medial tibial plateau. LFC Lateral femoral condyle. LTP Lateral tibial plateau. Data points represent mean (n ≥ 4). 95% confidence intervals are provided in Supplementary Table 2. a Different from 2 and 4 weeks PS equivalent (P < 0.05). b Different from 2, 12, and 12E weeks PS equivalent (P < 0.01). c Different from 2, 4, and 12E weeks PS equivalent (P < 0.05). d Different from all points of equivalent region except 12E (P < 0.01). e Different from all points of equivalent region (P < 0.05). * Different from time-matched medial compartment (P < 0.05). Note the significant effect of DMM in the medial compartment, the erlotinib effect in integrin αl-null but not WT mice, and a delayed response in females compared to males and in WT females compared to integrin α1-null females.
The frequency of synovitis was four times higher in DMM compared to sham and contralateral joints, and was observed at all time points PS (Table I, Supplementary Fig. 7). The number of contralateral and sham joints with synovitis were similar. Furthermore, synovitis was present in more integrin α1-null compared to WT mice, though did not reach statistical significance (Table I). Sex and erlotinib had no effect on the frequency of synovitis, and the maximum severity of synovitis was similar across all groups (Table I).
Table IThe frequency and severity of synovitis in murine knees as a function of time, sex, genotype, surgery to DMM, and erlotinib treatment. Synovitis was scored at all four regions (medial and lateral tibial plateau and femoral condyle) of the joint and summed. The maximum score per joint on this scale is 12. Note the four fold increase in the frequency of synovitis in the DMM compared to sham and contralateral joints, and the increased frequency of synovitis in α1-null compared to the WT mice. Significant differences: § (P = 0.001)
Table IThe frequency and severity of synovitis in murine knees as a function of time, sex, genotype, surgery to DMM, and erlotinib treatment. Synovitis was scored at all four regions (medial and lateral tibial plateau and femoral condyle) of the joint and summed. The maximum score per joint on this scale is 12. Note the four fold increase in the frequency of synovitis in the DMM compared to sham and contralateral joints, and the increased frequency of synovitis in α1-null compared to the WT mice. Significant differences: § (P = 0.001)
Immunohistochemistry
A smaller proportion of chondrocytes immuno-stained positively for phosphorylated EGFR in female compared to male cartilage, regardless of genotype, site, or drug (data not shown). In addition, a larger proportion of femoral compared to tibial chondrocytes immuno-stained positively for pSmad2/3 and pSmad1/5/8 (data not shown). Interestingly, the proportion of chondrocytes immuno-stained positively for pSmad1/5/8 decreased with time PS and was significantly reduced at 12 compared to 2 weeks PS mice of both genotypes [Fig. 7 and Supplementary Fig. 8]. In contrast, a higher proportion of chondrocytes were immuno-stained positively for pSmad2/3 in 8 compared to 2 weeks PS, but only in WT murine cartilage [Fig. 7 and Supplementary Fig. 8]. Staining for pSmad2/3 and pSmad1/5/8 was evenly distributed throughout cartilage depth rather than confined to any particular zone (Supplementary Fig. 8).
Fig. 7Percent of chondrocytes from male integrin α1-null and WT mice immuno-stained positively for phosphorylated (A) Smad1/5/8 (pSmad1/5/8) or (B) Smad2/3 (pSmad2/3) at 2, 4, 8, and 12 weeks PS. Data points represent mean (n = 3) with 95% confidence interval. a Different from all points of equivalent genotype (P < 0.05). b Different from 2 weeks PS equivalent (P < 0.05). c Different from time-matched counterpart (P < 0.05). d Different from all points (P < 0.05). Note the reduced staining of pSmad1/5/8 at 12 compared to 2 weeks PS in both genotypes and the higher staining of pSmad2/3 at 8 compared to 2 weeks PS, only in WT cartilage.
The goal of this study was to examine the protective role of integrin α1β1 in the progression of PTOA and to test whether dampening EGFR signalling is one mechanism by which integrin α1β1 offers that protection. As hypothesised, integrin α1-null mice showed earlier cartilage degradation post DMM surgery compared to WT female, but not male, mice. Additionally, we observed a trend towards a higher frequency of synovitis in integrin α1-null compared to WT mice. Surprisingly however, all of the effects of PTOA on ossified tissues were independent of genotype. This mildly protective influence of integrin α1β1 on the progression of PTOA contrasts with its more extensive role, albeit still protective, in spontaneous OA that includes cartilage degradation, calcified meniscal volume and the thickness and density of subchondral bone
. As we hypothesised, the majority of genetic modifications in mice have the same effect on both spontaneous OA and PTOA, however some demonstrate a diminished effect on one subtype of OA compared to the other as observed in integrin α1-null mice, or even an opposite effect
. This diminished protective role of integrin α1β1 against PTOA compared with spontaneous OA may be due to differences between these OA subtypes, including knee trauma, mechanical instability, and timeframe
In cartilage where the effects of integrin α1β1 on PTOA were most apparent, dampening EGFR signalling resulted in significantly less cartilage damage in integrin α1-null but not WT mice, despite the potential confounding influence of less mass at surgery for erlotinib treated vs control WT mice. This evidence supports our hypothesis that the dampening of EGFR signalling by integrin α1β1 may be one mechanism by which integrin α1β1 protects against OA. Further in vitro studies comparing EGFR signalling in integrin α1-null and WT chondrocytes may provide additional support for this hypothesis and shed light on the specific pathways downstream of EGFR that influence OA progression. In addition to EGFR, integrin α1β1 also dampens TGFβR signalling
. Here we show similar Smad1/5/8 activation in integrin α1-null and WT chondrocytes decreasing with time post DMM surgery. In contrast, Smad2/3 activation is higher in integrin α1-null compared to WT chondrocytes at 2 weeks PS and remains consistent with time in integrin α1-null chondrocytes, but increases out to 8 weeks PS in WT chondrocytes. Therefore, heightened TGFβR signalling in the cartilage of integrin α1-null mice, and a differential response of Smad2/3 activation to DMM surgery may also play a role in the earlier cartilage degradation seen in integrin α1-null compared to WT mice post DMM surgery.
Independent of genotype, dampening EGFR signalling by erlotinib treatment mitigated the effects of PTOA on many tissues of female mice including meniscal and fabella bone volume, subchondral bone thickness and density and cartilage degradation. In contrast, dampened EGFR signalling had little effect on signs of PTOA in male mice, and in some instances actually made them worse. Zhang et al. (2014) showed that the down-regulation of EGFR signalling via genetic modification or administration of gefitinib enhanced cartilage destruction post DMM surgery in male mice
. This study, together with our results suggest that the dampening of EGFR signalling plays a sex dependent role in the development of PTOA in mice, protecting against PTOA in females but having no effect or worsening the signs of PTOA in males. Importantly, this sex dependence was not evident in the development of spontaneous OA in mig-6 deficient mice
. These studies, together with our result suggest that the sex dependent role of EGFR signalling in the development of OA may be influenced by the method of dampening EGFR signalling, the model of OA used and the species studied.
Interestingly, oestrogen receptors are known to be involved in ligand-independent activation of EGFR in human breast cancer cells
Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer.
. Although both male and female growth plate chondrocytes express oestrogen receptors, the mRNA and protein levels of oestrogen receptors are higher in chondrocytes from female compared to male mice
Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors.
. Based on this evidence, we hypothesise that the sex dependent effect of erlotinib treatment on PTOA in our study is due to higher ligand-independent activation of EGFR by oestrogen receptors in female compared to male mice. Further studies are required to test this hypothesis in mouse chondrocytes and osteocytes.
In addition to dampening the signs of PTOA, erlotinib treatment also lessened changes in numerous trabecular, fabella, and subchondral bone parameters that occurred independently of DMM surgery. This result was also sex dependent, evident in female but not male mice, and suggests that EGFR signalling can also influence bone remodelling processes that occur due to normal skeletal development. In support of this finding, it was previously reported that administration of gefitinib into 1-month-old rats produced significant defects in endochondral ossification which highlights the critical role of EGFR signalling in the remodelling of growth plate cartilage during skeletal development
. Although this influence has not been confirmed in other cell types (chondrocytes, osteocytes, synoviocytes), care must be taken when interpreting the erlotinib-related changes observed in this study.
We have performed DMM surgery on integrin α1-null mice for the first time, observing effects as early as 4 weeks PS in both soft and calcified tissues of DMM compared to sham and contralateral joints. As expected, these signs were primarily found in the medial compartment of the knee and the changes conform to the characteristic signs of OA observed after DMM surgery
. The delay of cartilage damage in females compared to males PS agrees with others who have reported that male hormones aggravate and female hormones protect against the effects of DMM surgery up to 8 weeks PS
. Despite this initial protection, our results suggest that by 12 weeks PS females experience a similar severity of cartilage damage to their male counterparts. Interestingly, twice as many females compared to males developed a calcified MCL, and the increase in meniscal bone volume seen post DMM was larger in female compared to male mice. Together, these results highlight the importance of considering sex dependent responses and changes in both soft and bony tissues of the knee when using the DMM model of PTOA in mice.
In conclusion, we have shown that integrin α1β1 protects female mice against PTOA-induced cartilage degradation up to 8 weeks post DMM surgery partially via the dampening of EGFR signalling. We also showed that reduced EGFR signalling plays a sex dependent role in the development of PTOA in cartilage, bone and meniscal tissues in mice, protecting against PTOA in females but having no effect or worsening the signs of PTOA in males. Further studies are needed in order to examine the influence of interplay between sex specific hormones and EGFR in the sex dependent role of EGFR signalling in the responses of knee tissues to PTOA.
Author contributions
All authors made substantial contributions to the design, acquisition of data, and/or analysis and interpretation of data, and were involved in drafting or critically revising the article for important intellectual content. All authors approved the final version to be published. Dr. Clark had full access to all of the data in this study and takes responsibility for both the integrity and accuracy of the data and data analysis.
Study conception and design: Pozzi, Clark.
Acquisition of data: Shin, Clark.
Analysis and interpretation of data: Shin, Pozzi, Boyd, Clark.
Conflict of interest
None of the authors declare any conflict of interest.
Funding & support
This work was supported by Canadian Institutes of Health Research MOP 136800 (AC), Canadian Institutes of Health Research Studentship (SS), Alberta OA Team Studentship (SS), NSERC CREATE Incentive Award (SS), University of Calgary Faculty of Kinesiology (SS), the U.S. Department of Veterans Affairs Merit Reviews 1I01BX002025-01 (AP), and the U.S. Department of Health and Human Services, National Institutes of Health grants DK095761 (AP). The erlotinib hydrochloride (Tarceva®) was provided by Genentech, Inc. and Astellas Pharma, Inc.
Acknowledgements
The authors would like to thank Carin Pihl and Dawn Martin for expert assistance in all animal procedures, Britta Jorgenson for microCT expertise, Hakan Kadir and Charlie Shin for gavage assistance, Erica Floreani, Lisa Milo and Hakan Kadir for microCT and histology assistance, Dilene Mugenzi for histology assistance.
Appendix A. Supplementary data
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