SEARCH

SEARCH BY CITATION

Abstract

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

Objective

To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissue and ligament cells from normal and osteoarthritis (OA)–affected knees.

Methods

Knee joints were obtained at autopsy (within 24–48 hours postmortem) from 13 donors with normal knees (mean ± SD age 36.9 ± 11.0 years), 16 donors with knee OA (age 79.7 ± 11.4 years), and 8 aging donors without knee OA (age 76.9 ± 12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative polymerase chain reaction. ACL-derived cells were used to study regulation of MKX expression by interleukin-1β (IL-1β). MKX was knocked down with small interfering RNA (siRNA) to analyze the function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells.

Results

The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry analysis showed that MKX-positive cells were significantly reduced in ACL tissue from OA donors, in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX expression and reduced expression of the ligament ECM genes COL1A1 and TNXB. In contrast, SOX9, a chondrocyte master transcription factor, was up-regulated by IL-1β treatment. Importantly, knockdown of MKX expression with siRNA up-regulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB was reduced.

Conclusion

Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints, and this may be mediated in part by IL-1β. MKX appears necessary to maintain the tissue-specific cellular differentiation status and ECM production in adult human tendons and ligaments.

Osteoarthritis (OA) is the most common musculoskeletal disease and is caused by age- or trauma-related changes in the homeostatic balance between anabolic and catabolic mechanisms ([1]). The main pathogenetic mechanisms in OA are cartilage degradation induced by excessive mechanical stress, age-related changes in cells and extracellular matrix (ECM) mediated by the production of ECM-degrading enzymes, and inflammatory cytokines ([2, 3]). While articular cartilage damage is central to the OA process, this process also involves all other joint tissues. The anterior cruciate ligament (ACL) is critical to the biomechanical stability and function of the knee joint. Traumatic ACL injury can lead to cartilage damage and OA. Aging-associated OA also leads to structural changes in the ACL, which can contribute to disease progression ([4, 5]). In this regard, we recently reported that the cartilage degradation and ACL degeneration in OA knee joints show parallel progression ([6]), suggesting the importance of molecular mechanisms of ACL degradation and regeneration in OA pathogenesis.

The ECM in the ACL consists predominantly of type I collagen, with small amounts of other collagens, proteoglycans, and other glycoproteins, including aggrecan, decorin, tenascins, and fibromodulin ([7, 8]). Collagen fibers provide tensile strength, and proteoglycans provide resistance to compression stress. ACL ruptures usually occur in the mid-substance of the femoral side ([6]).

Cell populations in the ACL are generally referred to as fibroblast-like cells, although they are heterogeneous and also include subsets of progenitor cells ([9, 10]). Degraded ACL is characterized by changes in cell organization, cell death, and proliferation and by abnormal differentiation, most notably chondrocyte-like cell morphology and gene expression ([6, 11]). Understanding of the mechanisms that govern survival, differentiation, and ECM production by ligament cells is essential to developing new concepts for pathogenesis and therapeutic approaches.

Mohawk (MKX) and scleraxis (SCX) are the 2 transcription factors with relative specificity for tendon/ligament. SCX is a helix-loop-helix transcription factor that regulates the differentiation of tendon/ligament progenitors during skeletal development ([12, 13]). However, its expression level is low in mature ligament and tendon cells, suggesting that it may not play a major role in mature tendon/ligament homeostasis. MKX is also important during tendon and ligament development ([14-16]). Mkx-deficient mice have hypoplastic tendons throughout the body and deficient type I collagen production in tendon cells ([14, 16]). Importantly, we have found that Mkx expression is maintained in mature tendon/ligament cells in mice ([14]), suggesting that it has a potential role in tendon/ligament tissue homeostasis and regeneration.

Based on these observations, we undertook the present investigation. We examined the expression patterns and function of MKX in human adult ligament and tendon tissue, in the context of OA pathogenesis.

MATERIALS AND METHODS

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

Tissue procurement and macroscopic and histologic analysis

Intact human knee joints were obtained at autopsy (within 24–48 hours postmortem) from 13 subjects with normal knees (OA grade 0 or I [see below]) who were age <60 years at the time of death (mean ± SD age 36.9 ± 11.0 years), 16 subjects with knee OA (grade II–IV) (age 79.7 ± 11.4 years), and 8 subjects who were aged (age ≥60 years) and had no history of OA and no or only minimal (grade 0 or I) cartilage degradation (age 76.9 ± 12.9 years) (Table 1). We obtained 18 ACLs from the normal knee group, 16 ACLs from the OA group, and 8 ACLs from the aging group. Seven ACLs from the normal group and 8 ACLs from the OA group were used for gene expression assays. Eight ACLs from each of the 3 groups were used for immunohistochemistry analysis. Six ACLs from the normal group were used for cell isolation and in vitro studies of MKX expression and function (Table 1). Tissue collection was approved by the Scripps Human Subjects Committee.

Table 1. ACL tissue and donor characteristics*
Group, ACL sample no.Age/sexTotal knee cartilage score (39–156)Cartilage grade (0–IV)Total ACL score (0–15)ACL histologic gradeExperiments applied
  1. Anterior cruciate ligaments (ACLs) were obtained postmortem from 13 subjects in the normal group (18 ACLs), 16 subjects in the osteoarthritis (OA) group (16 ACLs), and 8 subjects in the aging group (8 ACLs) (see Materials and Methods for explanation of groups). IHC = immunohistochemistry.

Normal      
128/F3900.5MildmRNA
250/F46I4MildmRNA
342/F42I2.5MildmRNA
445/F54I4MildmRNA
524/M3901.5MildmRNA
623/F40I2MildmRNA, IHC
723/F40I0.5MildmRNA, IHC, cell isolation
824/M3900NormalIHC
936/M43I0.5MildIHC
1042/F3900NormalIHC
1144/F42I0.5MildIHC
1248/F43I3.5MildIHC
1351/M44I0NormalIHC
1455/M53I1.5MildCell isolation
1529/M3900NormalCell isolation
1629/M3900NormalCell isolation
1749/F49I1.5MildCell isolation
1849/F43I1MildCell isolation
OA     
176/M75II5MildmRNA
278/M81III3MildmRNA
388/F83III7ModeratemRNA
462/F87III3.5MildmRNA
576/M84III3MildmRNA
688/F76II2MildmRNA
791/M71II7ModeratemRNA
894/M71II5SeveremRNA
964/F69II5MildIHC
1064/M101IV5MildIHC
1182/M65II6ModerateIHC
1285/F100IV12SevereIHC
1386/M77II8ModerateIHC
1490/F81III3MildIHC
1592/F81III6ModerateIHC
1692/F70II6ModerateIHC
Aging      
160/F42I3.5MildIHC
263/M45I1MildIHC
368/F45I4MildIHC
476/M57I4MildIHC
579/F54I1MildIHC
681/M53I0.5MildIHC
794/F54I2MildIHC
894/F58I3MildIHC

Articular cartilage in all knee compartments was graded macroscopically as described previously ([17]). Briefly, we established a detailed scoring method based on the International Cartilage Repair Society knee map ([18, 19]), by dividing the cartilage into 39 regions that were graded macroscopically using a modified Outerbridge scoring system ([20]). Each area was scored on a scale of 1–4, where 1 = intact surface, 2 = fibrillation, 3 = fissuring, and 4 = erosion. The total knee cartilage score could thus range from 39 (normal) to 156 (maximum severity) ([6]). Total knee cartilage scores were then translated into grades 0–IV, where grade 0 = normal (total score 39), grade I = minimal change (total score 40–58), grade II = mild change (total score 59–78), grade III = moderate change (total score 79–97), and grade IV = severe change (total score ≥98).

ACLs were examined macroscopically and histologically as described previously ([6]). Briefly, the ACL was resected at the insertion sites on the femur and tibia. Macroscopic appearance was graded as normal, abnormal (thinner than normal and sclerotic), or ruptured (complete disappearance of the ligament or persistence of only a few fibers) ([21]). For histologic analysis, the samples were immediately fixed, and each specimen was cut transversely at the proximal one-third of the ligament and longitudinally through the center of the ligament from the proximal one-third of the ACL substance and femur attachment site, where ACL tears frequently occur ([21, 22]). The ACL sections were stained with hematoxylin and eosin and graded histologically using a modification of previously described scoring systems ([6, 23]). The following features were examined and scored for each ligament: inflammation in the ACL substance, mucoid degeneration, chondroid metaplasia, cystic changes, and orientation of collagen fibers. The highest possible summed score for ligament degeneration (total ACL score) was 15.

Immunohistochemistry

Immunohistochemistry studies were performed to investigate the expression pattern of MKX in human ACL tissue. Paraffin-fixed samples were first deparaffinized in xylene substitute (Pro-Par Clearant; Anatech) and rehydrated in graded ethanol and water. For antigen retrieval, sections were incubated with 10% trypsin and kept at 37°C for 30 minutes. They were then washed with phosphate buffered saline and blocked with 10% normal goat serum for 30 minutes at room temperature. Rabbit anti-human MKX polyclonal antibody (1:1,000 dilution) (LS-C30267; LifeSpan Biosciences) was applied and incubated overnight at 4°C. After being washed again with phosphate buffered saline, sections were incubated with biotinylated goat anti-rabbit secondary antibody (1:200 dilution) or biotinylated goat anti-mouse secondary antibody (1:200 dilution) (both from Vector) for 30 minutes at room temperature, followed by incubation for 30 minutes using a Vectastain ABC-AP kit (Vector). Slides were washed, and sections were incubated with an alkaline phosphatase substrate (Vector) for 15 minutes. The slides were then rinsed in tap water and counterstained with hematoxylin.

For the quantification of cells that were positive for MKX, at least 6 images were randomly obtained under 40× magnification, showing the mid-substance of the ACL without severely degenerated regions with chondroid metaplasia or extensive mucoid degeneration. The total cell number and number of positive cells in dense collagenous tissue in each microscopic field were counted by 2 different readers.

RNA isolation and real-time reverse transcription–polymerase chain reaction

Total RNA was isolated from ACL tissue or cells by extracting the homogenate in TRIzol (Invitrogen). Quantitative polymerase chain reaction (qPCR) was performed using a LightCycler 480 (Roche Diagnostics) and TaqMan Gene Expression Assay probes for the transcription factors MKX (Hs00543190_m1), SCX (Hs03054634_ g1), and SOX9 (Hs00165814_m1); the ECM components aggrecan (ACAN) (Hs00202971_m1), α1 chain of types I, II, and III collagen (COL1A1 [Hs00164004_m1], COL2A1 [Hs00264051_m1], and COL3A1 [Hs00943809_m1]), decorin (DCN) (Hs00754870_s1), fibromodulin (FMOD) (Hs00157619_m1), tenomodulin (TNMD) (Hs00223332_m1), and tenascin-XB (TNXB) (Hs00372889_g1); matrix metalloproteinase 13 (MMP13) (Hs00233992_m1); and interleukin-6 (IL6) (Hs00985639_m1), according to the instructions of the manufacturer (Applied Biosystems). Gene expression levels were assessed relative to GAPDH (Hs99999905_m1).

Human ACL cell isolation

Human ACL–derived cells were isolated from the mid-substance of ACLs of normal donors and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) as previously described ([24]). Experiments with ACL-derived cells were performed at passage 1 or 2.

Treatment with interleukin-1β (IL-1β).

Human ACL–derived cells were plated in 12-well plates containing DMEM with 2% FCS and 1% penicillin/streptomycin. Cells were treated with recombinant human IL-1β (5 ng/ml; PeproTech) for 6 hours, and total RNA was isolated with TRIzol. Quantitative-PCR was performed with TaqMan Gene Expression Assays ([25]).

Small interfering RNA (siRNA) knockdown of MKX in ACL-derived cells

MKX-specific siRNA and negative control siRNA were purchased from Qiagen. ACL-derived cells were transfected with 100 nM siRNA using Lipofectamine 2000 according to the protocol of the manufacturer (Invitrogen). Cells were incubated for 48 hours after transfection and then harvested for qPCR and Western blot analyses.

Western blotting

Cell extracts were prepared with sodium dodecyl sulfate lysis buffer. Protein concentrations were measured with a BCA Protein Assay kit (Bio-Rad). Equal amounts of protein were applied. Western blot analysis was performed using anti-MKX antibody (1:5,000 dilution), anti–type I collagen antibody (1:5,000 dilution) (ab292; Abcam), and anti-SOX9 antibody (1:1,000 dilution) (AB5535; Millipore). Horseradish peroxidase–conjugated anti-rabbit IgG ECL antibody (NA9340; GE Healthcare) or anti-mouse IgG ECL antibody (NA9310; GE Healthcare) was used as secondary antibody (1:5,000 dilution; treatment for 1 hour).

Statistical analysis

Mean ± SEM or mean ± SD values were calculated. Wilcoxon's signed rank test was used to calculate the significance of differences in mean values between 2 groups, and differences among 3 groups were assessed by one-way analysis of variance. Post hoc comparisons were performed using Tukey's test. P values less than 0.05 were considered significant.

RESULTS

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

Gene expression differences between ACLs from normal knees and those from OA knees

To determine changes in gene expression patterns in ACLs from OA knees as compared to normal knees, we performed qPCR for the transcription factors MKX, SCX, and SOX9, the ECM components ACAN, COL1A1, COL2A1, COL3A1, DCN, FMOD, TNMD, and TNXB, and MMP13 (Figure 1). The expression of the transcription factor MKX was significantly reduced in the OA group. For SOX9, there was a trend toward increased expression in the OA group. Expression of SCX did not differ between the normal and OA ACLs. Expression levels of the ECM components COL1A1 and TNXB were significantly reduced, and that of MMP13 was significantly increased, in the OA group. The expression of ACAN, COL2A1, COL3A1, DCN, FMOD, and TNMD did not differ in ACLs from OA knees versus normal knees.

image

Figure 1. Differences in the expression of the transcription factors MKX, SCX, and SOX9 and of extracellular matrix (ECM)–related genes in anterior cruciate ligament (ACL) tissue from normal knee joints (n = 7) and osteoarthritic (OA) knee joints (n = 8). Gene expression in the mid-substance of human ACLs was determined by quantitative polymerase chain reaction. The expression of MKX, as well as that of COL1A1 and TNXB, was significantly reduced in the OA group; MMP13 expression was significantly increased in the OA group. Horizontal bars show the mean. ∗ = P < 0.05 versus normal group.

Download figure to PowerPoint

MKX expression pattern in human ACL tissue

Immunohistochemistry was used to examine the expression pattern of MKX on sagittal sections of human ACLs. Cells and collagen fibers from the normal group were smooth and uniformly arranged (Figures 2A–C), whereas ACLs from the OA group exhibited collagen fibril and cellular irregularity, angiogenesis, and areas with increased cell density (Figures 2D–F). In specimens from the aging group, collagen fibers were more frequently disorientated and cell density was lower, compared with those from the normal group (Figures 2G–I). MKX was robustly expressed by ACL cells in normal knees (mean ± SEM 77.8 ± 3.2%), and this was reduced in knees from the OA and aging groups (28.6 ± 3.6% and 49.6 ± 4.3%, respectively) (Figure 2J). In the OA group, collagen fibers were frequently disorientated, and fibroblast-like cells in disorientated fibers or small round cells were usually negative for MKX. In contrast, cells in uniformly arranged collagen fibers remained positive for MKX even in the OA and aging groups.

image

Figure 2. Mohawk (MKX) expression in anterior cruciate ligament (ACL) cells in situ. A–I, Representative staining of ACL from normal knees (A–C), osteoarthritic (OA) knees (D–F), and aging knees (G–I). Knee specimens were stained with hematoxylin and eosin (H&E) or with anti-MKX. In normal ACLs, a large majority of cells were MKX positive (B and C). Changes typically observed in the OA group included collagen fibril and cellular irregularity, angiogenesis, and increased cell density (D–F). MKX expression was decreased in cells in the ligament substance in both the OA group and the aging group (E, F, H, and I), although MKX-positive cells were present in perivascular areas in the OA specimens (E and F). Bars = 200 μm. J, Percentages of cells that were positive for MKX in each of the 3 groups. Values are the mean ± SEM (n = 8 per group). ∗∗ = P < 0.01.

Download figure to PowerPoint

In a previous study we observed disorientation of collagen fibers in 105 of 117 ACLs (89.7%) and chondroid metaplasia in 40 (34.2%). However, chondroid metaplasia was not observed in any of the 9 ACLs from normal knees of young donors in that study; disorientation of collagen fibers was observed in 6 of the 9 ([6]). In the present study, extensive chondroid metaplasia was observed in the OA group (n = 3), and the mean ± SD percentage of MKX-positive cells in these chondrocyte-like cells was 69.1 ± 4.7%.

Suppression of MKX gene expression in human ACL–derived cells by IL-1β.

To explore the premise that proinflammatory cytokines associated with ACL degeneration, such as IL-1β, may down-regulate MKX expression, primary cultures of ACL-derived cells from normal knees were treated with IL-1β and expression of MKX, SCX, SOX9, ACAN, COL1A1, COL2A1, COL3A1, DCN, FMOD, TNMD, TNXB, IL6, and MMP13 was analyzed. The expression levels of MKX and SCX were significantly reduced by IL-1β, while SOX9 was significantly increased (Figure 3A). IL-6 and MMP13 expression levels in ACL cells were significantly increased after IL-1β treatment (Figure 3B). Among ECM genes, the expression levels of COL1A1, COL3A1, and TNXB were reduced significantly after IL-1β treatment (Figure 3C).

image

Figure 3. Interleukin-1β (IL-1β)–induced changes in gene expression in human anterior cruciate ligament (ACL)–derived cells. ACL cells from normal donors were treated with IL-1β (5 ng/ml) for 6 hours, and expression of the transcription factors MKX, SCX, and SOX9 (A), of IL6 and MMP13 (B), and of extracellular matrix (ECM)–related genes (C) was determined by quantitative polymerase chain reaction. IL-1β stimulation significantly reduced MKX and SCX expression and increased SOX9 expression. IL6 and MMP13 were also increased significantly by IL-1β stimulation. Among ECM-related genes, COL1A1, COL3A1, and TNXB were reduced significantly after IL-1β treatment, whereas expression of COL2A1, DCN, FMOD, TNMD, and ACAN was unchanged. Values are the mean ± SEM (n = 12 different preparations from 6 different donors per group). ∗ = P < 0.05; ∗∗ = P < 0.01, versus no IL-1β treatment.

Download figure to PowerPoint

Knockdown of MKX expression in human ACL–derived cells

To investigate the function of MKX in regulating ECM genes and other transcription factors, ACL-derived cells were transfected with either control siRNA or siRNA specific for MKX (siMKX). After transfection with siMKX, the expression of MKX was reduced to ∼30% of control (Figure 4A). As a result, SOX9 expression was significantly increased, while the expression of SCX was unaffected. Inflammation-related genes such as IL6 and MMP13 were also unaffected in ACL-derived cells transected with siMKX (Figure 4B). Among the ECM-related genes we examined, only TNXB was significantly affected, showing a decrease in expression after siMKX treatment (Figure 4C).

image

Figure 4. Effects of MKX-specific small interfering RNA (siMKX) on gene and protein expression in human anterior cruciate ligament–derived cells. Cells from normal donors in primary culture were treated with siMKX for 48 hours. A–C, Expression of the transcription factors MKX, SCX, and SOX9 (A), of IL6 and MMP13 (B), and of extracellular matrix–related genes (C) was determined by quantitative polymerase chain reaction. Treatment with siMKX significantly increased the expression of SOX9, while expression of MKX and of TNXB was significantly reduced. Knockdown of MKX had no significant effect on the expression of the other genes studied. D and E, Cell lysates were prepared for Western blotting analysis of the protein levels of Mohawk (MKX), type I collagen, and SOX9, and the results were quantified by densitometry using ImageJ software (National Institutes of Health). Levels of MKX and type I collagen protein were reduced by siMKX treatment, while SOX9 protein levels were significantly increased. Values are the mean ± SEM (n = 12 different preparations from 6 different donors per group). ∗ = P < 0.05; ∗∗ = P < 0.01.

Download figure to PowerPoint

In addition, we examined protein-level expression of MKX, SOX9, and type I collagen in ACL-derived cells, using Western blot analysis. Levels of SOX9 protein were significantly elevated by siMKX. Type I collagen levels, in contrast, were significantly reduced (Figures 4D and E).

DISCUSSION

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

Mkx has recently been identified as a tendon/ligament specific transcription factor regulating expression of tendon/ligament ECM genes, including those for α1 chain of type I collagen, tenomodulin, and/or fibromodulin, during skeletal development in mice ([14, 16]); however, MKX expression and function in human tendon/ligament cells have not been elucidated. In this study, we demonstrated that MKX is expressed in adult human ligaments and its expression is clearly decreased in ligaments from patients with OA, correlating deficient expression of important ECM genes such as COL1A1 and TNXB.

The relationship between ACL degeneration and OA has been examined in earlier studies ([6, 26-28]). Typically, degenerative changes in the ACL start with collagen fiber disorientation, followed by mucoid degeneration, inflammatory cell infiltration, and/or neovascularization. IL-1β is among the critical proinflammatory cytokines involved in cartilage degradation during OA pathogenesis, as well as in ACL degeneration ([29, 30]). IL-1β stimulation in human tendon–derived cells significantly reduces expression of the genes for types I and III collagen, tenomodulin, and SCX and promotes expression of genes associated with ACL degeneration, such as those for aggrecanase, cyclooxygenase 2, matrix metalloproteinases 1, 3, and 13, ADAMTS-4, and IL-6 ([31, 32]). Herein we have shown that MKX is also down-regulated by IL-1β stimulation in human ligament cells. In addition, the down-regulation of MKX by siRNA in human ACL–derived cells reduces the expression of TNXB. These results support the notion that down-regulation of MKX is involved in ligament degeneration in OA and that MKX maintains ligament function and prevents degeneration.

As previously reported ([14, 16]), Mkx regulates type I collagen during tendon/ligament development in mice. In the present study, MKX knockdown in human ACL–derived cells did not significantly reduce the expression of COL1A1 messenger RNA (mRNA). However, Western blot analysis indicated that type I collagen protein expression was reduced by siMKX treatment. This discrepancy between mRNA and protein expression could be caused by an indirect effect of MKX knockdown via reduced TNXB expression. Tenascin-XB is a member of the tenascin family of ECM glycoproteins that contribute to matrix structure and regulate collagen fibrillogenesis via direct binding with type I collagen ([33]). Studies of cultured dermal fibroblasts showed that fibroblasts from Tnxb−/− mice failed to deposit type I collagen into cell-associated matrix, although type I collagen synthesis by cells from Tnxb−/− and wild-type mice was similar ([34]).

Interestingly, expression of the chondrogenic transcription factor SOX9 in human ACL–derived cells was increased with both IL-1β stimulation and MKX siRNA knockdown. Further, immunohistochemical analysis of human ACL tissue demonstrated that SOX9-positive cells were increased in ACLs from OA knees compared with normal ACLs. In this regard, we and others have previously demonstrated chondroid metaplasia in ACLs from knees with cartilage degeneration but not in ACLs with normal cartilage ([6, 10, 35]). Several reports have indicated that ACL-derived cells have high chondrogenic capacity and that SOX9 drives the differentiation from tenocyte to chondrocyte ([10, 36]).

It thus appears that ligament/tendon may contain a substantial proportion of progenitor cells. In OA, progenitor cells are activated, abnormally expressing SOX9 and differentiating to chondrocyte-like cells ([28, 37]). Although these abnormal cells in degenerated ACLs are frequently positive for SOX9, other chondrogenic markers such as type II collagen or aggrecanase are not expressed by all of these cells. In a recent study, we demonstrated that type II collagen or aggrecanase was present only in a subset of cells, even in areas with chondroid metaplasia ([37]). This is consistent with our in vitro observations in the present study, in which increased SOX9 expression was not associated with increased expression of other chondrogenic markers such as COL2A1 and ACAN. Moreover, other studies on chondrogenesis of human mesenchymal stem cells have indicated that the increasing SOX9 expression precedes expression COL2A1 or ACAN ([38]).

In this study we also sought to distinguish between changes that are related to normal aging and OA-related changes. We studied ACLs from donors age ≥60 years with no history of OA and minimal articular cartilage changes seen on macroscopic examination of the knees. The mean age of the normal aging group was similar to that of the OA group. We found that MKX levels differed between the 2 non-OA groups, with significantly lower levels in the aging group. Thus, there is an evident effect of aging on suppression of MKX. MKX concentrations were even lower in ACLs from OA-affected knees, indicating that OA-related mechanisms also contribute to the changes in MKX. The observation that IL-1β suppressed MKX in ACL-derived cells suggests a potential mechanism for this.

In conclusion, this study demonstrates that MKX is expressed in cells from normal adult human ACLs and that its expression is reduced in ACLs from OA-affected joints. In vitro, IL-1β suppresses MKX and enhances SOX9. These observations support the novel concept that loss of MKX, driven in part by proinflammatory cytokines such as IL-1β, leads to abnormal chondrocyte-like differentiation of ligament cells and production of ECM with deficient biomechanical properties (Figure 5). Correction of abnormal MKX expression could be pursued as a new approach to address tissue repair after injury and during chronic processes such as OA.

image

Figure 5. Hypothesis on the role of MKX in ligament homeostasis and degeneration. MKX maintains ligament function and prevents degeneration via regulation of TNXB and SOX9. Reduced expression of MKX in degenerated ligaments, mediated by proinflammatory cytokines, leads to abnormal differentiation and extracellular matrix production.

Download figure to PowerPoint

AUTHOR CONTRIBUTIONS

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

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. Asahara 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. Nakahara, Ozaki, Lotz, Asahara.

Acquisition of data. Nakahara, Hasegawa, Otabe, Ayabe, Matsukawa.

Analysis and interpretation of data. Nakahara, Hasegawa, Otabe, Ayabe, Matsukawa, Onizuka, Ito, Lotz, Asahara.

Acknowledgments

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

We thank Lilo Creighton for assistance with the histologic analysis.

REFERENCES

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