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Proc Natl Acad Sci U S A. Aug 16, 2005; 102(33): 11740–11745.
Published online Aug 8, 2005. doi:  10.1073/pnas.0505171102
PMCID: PMC1187996
Developmental Biology

Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease

Abstract

Degenerative joint disease, also known as osteoarthritis, is the most common joint disorder in human beings. The molecular mechanism underlying this disease is not fully understood. Here, we report that disruption of mitogen-inducible gene 6 (Mig-6) in mice by homologous recombination leads to early onset degenerative joint disease, which is revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage, and the development of bony outgrowths or osteophyte formation within joint space. The osteophyte formation appears to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Absence of the Rag2 gene does not rescue the joint phenotype, excluding a role for the acquired immune system in the development of this disease. Our results provide insight into the mechanism of osteoarthritis by showing that loss of Mig-6 leads to early onset of this disease, implying that this gene or its pathway is important in normal joint maintenance. Because of the striking similarity of osteoarthritis in humans and mice, the Mig-6 mutant mouse should provide a useful animal model for studying the mechanism of this disease and for testing drugs or therapies for treating osteoarthritis.

Keywords: animal model, mutation, osteoarthritis, bony outgrowth, signaling pathway

Degenerative joint disease, or osteoarthritis, affects nearly 12% of the population between ages 25 and 74 in the United States (1) and greatly interferes with quality of life by causing acute and chronic pain and disability (2, 3). The characteristic features of this disease are joint pain, stiffness, joint enlargement and malalignment, damage of articular cartilage, and formation of osteophytes or bony outgrowths at the margin of the synovial and cartilage junctions (2, 3). Currently, therapy is directed toward controlling symptoms, and no disease-modifying or chondroprotective treatment is available (2, 3). In addition, the costs for pain relief medication are astronomical. Although several genetic and biomechanical factors, including heredity, obesity, injury and joint overuse, are thought to contribute to the development of osteoarthritis (2, 3), the molecular mechanism underlying this disease is still elusive.

Mitogen-inducible gene 6 (Mig-6), also known as gene 33, is an immediate early response gene that can be induced by many growth factors or stressful stimuli (4, 5). It has also been shown to act as a negative feedback inhibitor in EGF receptor signaling through direct interaction with EGF receptor family (57), and, similarly, the gene is induced by hepatocyte growth factor/scatter factor by means of the receptor tyrosine kinase Met (data not shown). To understand its role during mouse development and homeostasis, we generated Mig-6-deficient mice and demonstrated that Mig-6 is essential for normal joint maintenance and that loss of Mig-6 leads to early onset degenerative joint disease.

Materials and Methods

Mice and Genotyping. To generate Mig-6 knockout mice, we constructed a Mig-6 targeting vector by inserting a 5-kb genomic fragment upstream of exon 2 (shown as e2 in Fig. 1A) and a 3-kb genomic fragment downstream of exon4 (e4 in Fig. 1 A) into the pPNT vector (8) in front of and behind the PGK-NEO cassette, respectively (Fig. 1 A). ES clones were established by electroporation of linearized plasmid and selection in neomycin. Positive Mig-6+/ ES clones were screened by PCR and Southern blot analyses. Two independent clones were used to generate Mig-6 knockout mice. The primers used for PCR genotyping were forward primer p1 (5′-GACAATTTGAGCAACTTGACTTGG-3′), which is specific for the WT locus; reverse primer p2 (5′-GGTTACTTAGTTGTTGCAGGTAAG-3′), which is shared by both the WT and mutant locus; and the primer p3 (5′-CCTTCTATCGCCTTCTTGACG-3′), which is derived from the PGK-NEO cassette and is specific for the mutant locus.

Fig. 1.
Targeted disruption of the Mig-6 gene. (A) Diagram of strategy for targeting the Mig-6 locus. E, EcoRV; X, XhoI. (B) Southern blot analysis and PCR-based genotyping. A 7.8-kb fragment for the WT allele and 6-kb fragment for the mutant allele were detected ...

The Rag2 null mouse (9) was obtained from the Mouse Models of Human Cancer Consortium Repository at the National Cancer Institute (Frederick, MD). The primers used for genotyping Rag2 mice are as reported in ref. 10.

Northern Blot Analysis. Total RNAs were isolated from mouse tissues by homogenization in TRIzol reagent (Invitrogen). Twenty micrograms of each RNA sample was used for Northern blot analyses with mouse Mig-6 cDNA probe and β-actin probe.

RT-PCR Analysis. First-strand cDNA was prepared from 1 μg of each RNA sample by using the Advantage RT-for-PCR Kit (Clontech) and used for PCR amplification. The primers for Mig-6 amplification were 5′-CAGAAGTTACATGGGATGAATGG-3′ and 5′-TGAACACAAACTGCGTGTCTCAC-3′. The primers for GAPDH amplification were 5′-TCCAGTATGACTCCACTCACG-3′ and 5′-ACAACCTGGTCCTCAGTGTAG-3′.

Preparation of Adult Skeleton. The method for preparation of the adult skeleton is described in ref. 11. Briefly, 4-month-old animals were killed, eviscerated, and immersed in 2% KOH for overnight. The carcasses were rinsed and stained in 1.9% KOH containing 0.04 g/liter Alizarin red S (Sigma) for 2 days and cleared in cleaning solution (consisting of 400 ml/liter white glycerin, 200 ml/liter benzyl alcohol, and 400 ml/liter 70% ethanol).

Histology and Immunohistochemistry (IHC). Mouse bone tissues were fixed in formalin or 4% paraformaldehyde, decalcified in formic acid bone decalcifier, and embedded in paraffin. Five-micrometer sections were prepared and stained with hematoxylin/eosin (H&E), Mason's trichrome to detect collagens, or safranin O to detect proteoglycans. Proliferating cell nuclear antigen (PCNA) or type II collagen was immunohistochemically detected by mouse monoclonal antibody against PCNA (Santa Cruz Biotechnology) or mouse monoclonal antibody directed against type II collagen (Chemicon International, Temecula, CA), respectively, by using a M.O.M. Kit (for detecting mouse primary antibodies on mouse tissue) and a peroxidase detection system (both from Vector Laboratories). For type II collagen IHC staining, sections were pretreated in Tris·HCl (pH 2.0) containing 1 mg/ml pepsin for 15 min at room temperature. For Von Kossa staining to detect calcium deposition, sections were prepared from nondecalcified bone tissues.

Results and Discussion

The Mig-6-deficient mice were generated by conventional gene targeting technology by replacing the entire coding region of Mig-6 with the PGK-NEO cassette (Fig. 1 A). The loss of WT alleles of Mig-6 was determined by Southern blot analysis and PCR-based genotyping (Fig. 1B). The lack of Mig-6 expression was confirmed in liver and thymus derived from homozygous mice by Northern blot analysis (Fig. 1C). Mig-6/ mice are viable, but we observe a 50% reduction of the homozygous litters, indicating that some embryonic lethality is associated with the loss of both alleles (data not shown).

Although Mig-6 is expressed at high levels in mouse liver and kidney (data not shown), we did not observe obvious pathological changes or defects in these tissues (data not shown). Surprisingly, we found that most of the Mig-6/ mice showed abnormal gait as early as 1 month of age. With time, we observed progressive enlargement and deformity of multiple joints in the Mig-6/ mice, especially the knees, ankles, and temporal-mandibular joints (TMJs) (Fig. 2 DF), but this was not observed in WT or heterozygous Mig-6 mice (Fig. 2 AC and data not shown). All Mig-6/ animals developed joint deformities and the majority died within 6 months, most likely due to TMJ ankylosis (Fig. 2F and data not shown), which resulted in inability to eat and/or drink water. Late-stage mutant mice were thin and appeared exhausted compared with their WT and heterozygous littermates.

Fig. 2.
Disruption of Mig-6 results in multiple joint deformities and fibrocartilagenous hyperplasia. (AF) Adult skeletons of 4-month-old mice were stained with Alizarin red and photographed under a microscope. Representative images of ankles (A and ...

We examined the joint deformities of Mig-6/ mice by preparing H&E sections from joints of mice at different ages and comparing them to Mig-6+/+ and Mig-6+/ mice. In the knee joint of Mig-6/ mice at ages from 1.5 to 6 months, we observed outgrowths of abnormal bony nodules within the joint space adjacent to the margin of the synovial and cartilage junctions, accompanied by joint space narrowing with time (Fig. 2 JL). Similar pathological changes were observed in the ankle joints of the mice as well as in the TMJ, neck, and other joints (data not shown). We also examined joint sections derived from younger Mig-6/ mice at ages of 12 and 20 days and observed no signs of structural abnormality (data not shown), showing that the bony nodules and the degenerative joint changes develop later. The abnormal nodules surrounded by spindle-shaped mesenchymal-like cells contain hyperplastic fibrocartilage with variable chondrocyte shapes, representing different stages of cartilage matrix development (Fig. 3A). The nodules are abundant in the cartilage matrix as determined by Mason's trichrome staining for collagen and safranin O staining for proteoglycan (Fig. 3A). Compared with the outer zones of the nodules, the inner zones have a higher density of proteoglycan produced by mature chondrocytes (Fig. 3A). The deeper zones of the nodules undergo endochondral ossification, and Von Kossa staining reveals calcium deposition in the osteoid matrix (Fig. 3A). This bony outgrowth has components that resemble osteophyte formation, a characteristic feature of human osteoarthritis (2, 3).

Fig. 3.
Mig-6/ mutant joints display multiple characteristic pathological features of osteoarthritis. (A) Bony outgrowth or osteophyte formation. Mason's trichrome staining for collagen (blue) or safranin O staining for proteoglycans (red) was ...

In addition to bony outgrowths, we observed other arthritic changes in Mig-6/ mutant joints, including degradation of articular cartilage, formation of subchondral cysts, synovial hyperplasia, and abnormally robust vascularization (Fig. 3 BE) (2, 3). In Mig-6/ mutant joints, the surfaces of the articular cartilage become rough and disorganized. Degradation of articular cartilage is observed at multiple regions across the joint, accompanied by signs of tissue regeneration (Fig. 3B). Subchondral cyst fomation, another characteristic feature in human osteoarthritis (2, 3), is also observed in the Mig-6–/– arthritic joints: Various sized subchondral cysts filled with fibroblast-like cells are present, and separated from the bony structures that form beneath the degraded articular cartilage (Fig. 3C). By comparison, the joints of age-matched Mig-6+/+ and Mig-6+/ mice do not show any evidence of these pathological changes (data not shown). Along with the articular cartilage degradation, synovial cells lining the joints of mutant mice are hyperplastic with multiple cell layers, compared with the thin layer of synovial cells found in normal joints (Fig. 3D). Accompanying the destructive and reconstructive remodeling activities in the mutant joints, we observe vascularization in regions that are normally avascular (Fig. 3E) (2, 3).

Articular cartilage provides not only a low-friction surface for joint movement but also flexibility for withstanding concussive forces applied to the joint. The cartilage matrices, of which proteoglycan and collagen are the two major components, are responsible for both tasks (12). In osteoarthritis, the density of proteoglycan is reduced within the articular cartilage because of disruption of a balance between degradation and production (13, 14). The hyaline cartilages of Mig-6+/+ and Mig-6+/ mice display intense proteoglycan staining throughout articulating surfaces, such as in the femur and patella (Fig. 4 D and E). However, we observed a lack of proteoglycan staining within some areas of these articular surfaces, especially in late stages of the Mig-6/ mutant joint destruction (Fig. 4F). Interestingly, beneath the cartilage, there is proteoglycan staining in the damaged joint (Fig. 4F). Proteoglycan staining in the joints of mutant mice in early stages of joint disease is similar to that in WT and heterozygous mice (Fig. 4 AC).

Fig. 4.
Changes of proteoglycan distribution are observed in articular cartilage of a late-stage Mig-6/ mutant joint. Safranin O staining was performed on knee joint sections prepared from litters of 1.5-month-old (AC) and 3-month-old ...

To determine which cells are responsible for the regeneration and formation of osteophyte in the Mig-6/ mouse joints, we performed immunohistochemical staining to identify proliferating cells in G1 and S phase by using antibody against PCNA. Interestingly, we found that the mesenchymal-like spindle-shaped cells at the outer zone of the osteophyte and in the region of cartilage repair were strongly positive for PCNA staining (Fig. 5). No PCNA-positive cells were observed in the inner zone of the osteophytes, suggesting that the spindle-shaped cells were proliferating and likely responsible for osteophyte formation. The mesenchymal progenitor cells have the capability to proliferate and differentiate into chondrocytes (15). We stained sections with antibody against mouse type II collagen that is synthesized by the mature chondrocytes but not the progenitor cells; nor is it found in terminally differentiated hypertrophic chondrocytes (13, 15). Only the layer of chondrocytes lying between the mesenchymal progenitor cells and the chondrocytes in the deeper zones were positive for type II collagen staining (Fig. 5), indicating that the bony outgrowths in the Mig-6/ mutant joints appear to be derived from proliferating mesenchymal progenitor cells that differentiate into chondrocytes.

Fig. 5.
Bony outgrowths are derived from proliferation of mesenchymal-like progenitor cells. Knee joint sections were prepared from a 3-month-old Mig-6/ mouse and immunohistochemically stained by anti-PCNA or anti-collagen type II antibodies. ...

In contrast to inflammatory arthritis (e.g., rheumatoid arthritis or infectious arthritis), osteoarthritis usually shows relatively few inflammatory cells infiltrating the affected joints (2, 3). Although no significant inflammatory cells were observed in Mig-6/ mutant joints, we frequently observed that the thymuses of these mice were larger than normal size (data not shown). To determine whether the immune system was playing a role in the development of the joint phenotype in Mig-6/ mice, we crossed these animals with Rag2-deficient mice (9) to generate mice deficient for both Mig-6 and Rag2. These mice displayed severe immune deficiency due to a failure of both mature B and T cell development (9), but this phenotype did not alter either the frequency or extent of joint phenotype of the Mig-6/ mouse (Fig. 6). Thus, the acquired immune system does not appear to play a role in this joint disorder.

Fig. 6.
Absence of Rag2 does not rescue the joint phenotype of Mig-6/ mice. (A) Crossing scheme for generating double-knockout mice. The litters derived from intercrossing Mig-6+/Rag2/ mice were used for analyzing ...

Many growth factors and cytokines have been shown to influence the pathogenesis of osteoarthritis (14), such as transforming growth factor β (TGF-β) (1622) and bone morphogenetic proteins (BMPs) (23). In addition, genetic predisposition to osteoarthritis has been linked to mutations in genes like COL2A1 (24). Here, we show that Mig-6-deficient mice display multiple joint defects. The phenotypes include joint deformities, degradation of articular cartilage, subchondral cyst formation, and bony outgrowths or osteophyte formation (Figs. (Figs.22 and and3).3). The pathological features are strikingly similar to human osteoarthritis. The most-affected joints are knee and ankle joints and TMJs, with less frequent occurrence in other joints (Fig. 2 and data not shown). The affected joints bear relatively high amounts of stress, which could be a major factor in developing this disorder. WT Mig-6 is expressed in the knee joint as determined by RT-PCR (Fig. 7). It has recently been shown that Mig-6 expression is increased in response to mechanical load, as well as in osteoarthritic cartilage of canine joints (25). Mechanical factors are thought to play an important role in the development and degeneration of articular cartilage by influencing expressions of many genes that are crucial for the processes of cell growth, vascularization, and ossification (26). We hypothesize that mechanical joint stress constitutively stimulates joint regeneration by inducing certain growth factors such as TGF-β, BMP, EGF, or hepatocyte growth factor/scatter factor and other cytokines that stimulate proliferation and differentiation of cells required for joint renewal. Under normal conditions, this regenerative activity is counterbalanced by a suppressor activity of Mig-6 that fine-tunes the extent of proliferation and renewal. Losing the suppressing function of Mig-6 causes overproliferation of mesenchymal progenitor cells, leading to an abnormal state of chondrogenic differentiation and bony outgrowths (Fig. 5). The profound osteoarthritic phenotype of Mig-6-deficient mice makes them a very useful model for (i) determining what factors in the Mig-6 signaling pathway are involved in osteoarthritis, (ii) understanding the molecular mechanism underlying this disease process, and (iii) testing drugs or therapies that may help to alleviate the symptoms or alter the disease progression of osteoarthritis.

Fig. 7.
Detection of Mig-6 expression in mouse joints. RT-PCR was performed by using total RNA prepared from a whole knee joint (lanes 1 and 2) or liver (lanes 3 and 4) derived from Mig-6+/+ (lanes 1 and 3) or Mig-6/ (lanes 2 and 4) mice. Arrows ...

Acknowledgments

We thank David Kingsley for critical reading of the manuscript; Bart Williams and Rick Hay for scientific suggestions; Matt VanBrocklin and Han-Mo Koo for technical help; James Resau, Bree Berghuis, and J. C. Goolsby for histological assistance; Kellie Sisson, Bryn Eagleson, Jason Martin, and the vivarium staff at the Van Andel Institute for helping with the mice; Michelle Reed and Troy Carrigan for manuscript preparation; and Theo Pretorius for assistance with the digital art. This work was supported by the Van Andel Foundation and Michigan Economic Development Corporation and Michigan Technology Tri-Corridor Grant MAMC 085P1000815.

Notes

Author contributions: Y.-W.Z. and G.F.V.W. designed research; Y.-W.Z., Y.S., N.L., P.J.S., R.T.B., and R.S. performed research; Y.-W.Z. and R.W.M. analyzed data; and Y.-W.Z. wrote the paper.

Abbreviations: H&E, hematoxylin/eosin; PCNA, proliferating cell nuclear antigen; TMJ, temporal-mandibular joint.

References

1. Lawrence, R. C., Hochberg, M. C., Kelsey, J. L., McDuffie, F. C., Medsger, T. A., Jr., Felts, W. R. & Shulman, L. E. (1989) J. Rheumatol. 16, 427–441. [PubMed]
2. Koopman, W. J. (1997) in Arthritis and Allied Conditions: A Textbook of Rheumatology (Williams & Wilkins, Baltimore), 13th Ed., Vol. 2.
3. Resnick, D. (2002) Diagnosis of Bone and Joint Disorders (Saunders, Philadelphia), 4th Ed., Vol. 2.
4. Makkinje, A., Quinn, D. A., Chen, A., Cadilla, C. L., Force, T., Bonventre, J. V. & Kyriakis, J. M. (2000) J. Biol. Chem. 275, 17838–17847. [PMC free article] [PubMed]
5. Fiorentino, L., Pertica, C., Fiorini, M., Talora, C., Crescenzi, M., Castellani, L., Alema, S., Benedetti, P. & Segatto, O. (2000) Mol. Cell. Biol. 20, 7735–7750. [PMC free article] [PubMed]
6. Anastasi, S., Fiorentino, L., Fiorini, M., Fraioli, R., Sala, G., Castellani, L., Alema, S., Alimandi, M. & Segatto, O. (2003) Oncogene 22, 4221–4234. [PubMed]
7. Xu, D., Makkinje, A. & Kyriakis, J. M. (2005) J. Biol. Chem. 280, 2924–2933. [PubMed]
8. Tybulewicz, V. L. J., Crawford, C. E., Jackson, P. K., Bronson, R. T. & Mulligan R. C. (1991) Cell 65, 1153–1163. [PubMed]
9. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M. & Alt, F. W. (1992) Cell 68, 855–867. [PubMed]
10. Corazza, N., Eichenberger, S. Eugster, H. & Mueller, C. (1999) J. Exp. Med. 190, 1479–1491. [PMC free article] [PubMed]
11. Selby, P. B. (1987) Stain Technol. 62, 143–146. [PubMed]
12. Hamerman, D. (1989) N. Engl. J. Med. 320, 1322–1330. [PubMed]
13. Sandell, L. J. & Aigner, T. (2001) Arthritis Res. 3, 107–113. [PMC free article] [PubMed]
14. Rowan, A. D. (2001) Expert Rev. Mol. Med. 2001, 1–20. [PubMed]
15. Cancedda, R., Descalzi Cancedda, F. & Castagnola, P. (1995) Int. Rev. Cytol. 159, 265–358. [PubMed]
16. Hulth, A., Johnell, O., Miyazono, K., Lindberg, L., Heinegard, D. & Heldin, C. H. (1996) J. Orthop. Res. 14, 547–553. [PubMed]
17. van Beuningen, H. M., Glansbeek, H. L., van der Kraan, P. M. & van den Berg, W. B. (2000) Osteoarthritis Cartilage 8, 25–33. [PubMed]
18. Allen, J. B., Manthey, C. L., Hand, A. R., Ohura, K., Ellingsworth, L. & Wahl, S. M. (1990) J. Exp. Med. 171, 231–247. [PMC free article] [PubMed]
19. Bakker, A. C., van de Loo, F. A., van Beuningen, H. M., Sime, P., van Lent, P. L., van der Kraan, P. M., Richards, C. D. & van den Berg, W. B. (2001) Osteoarthritis Cartilage 9, 128–136. [PubMed]
20. Scharstuhl, A., Glansbeek, H. L., van Beuningen, H. M., Vitters, E. L., van der Kraan, P. M. & van den Berg, W. B. (2002) J. Immunol. 169, 507–514. [PubMed]
21. Serra, R., Johnson, M., Filvaroff, E. H., LaBorde, J., Sheehan, D. M., Derynck, R. & Moses, H. L. (1997) J. Cell Biol. 139, 541–552. [PMC free article] [PubMed]
22. Yang, X., Chen, L., Xu, X., Li, C., Huang, C. & Deng, C. X. (2001) J. Cell Biol. 153, 35–46. [PMC free article] [PubMed]
23. Rountree, R. B., Schoor, M., Chen, H., Marks, M. E., Harley, V., Mishina, Y. & Kingsley, D. M. (2004) PLoS Biol. 2, 1815–1827.
24. Aigner, T. & Dudhia, J. (2003) Curr. Opin. Rheumatol. 15, 634–640. [PubMed]
25. Mateescu, R. G., Todhunter, R. J., Lust, G. & Burton-Wurster, N. (2005) Biochem. Biophys. Res. Commun. 332, 482–486. [PubMed]
26. Carter, D. R., Beaupre, G. S., Wong, M., Smith, R. L., Andriacchi, T. P. & Schurman, D. J. (2004) Clin. Orthop. Relat. Res. 427, Suppl., S69–S77. [PubMed]

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