Scientific Report 2007

Molecular and Experimental Medicine

Division of Arthritis Research

Adult Stem Cells in Articular Cartilage

S. Grogan, M. Lotz

Cartilage injury, limited repair capacity, and aging-associated changes in cartilage cells and extracellular matrix are major risk factors for osteoarthritis, the most prevalent joint disease. Mesenchymal stem cells (MSCs) have been studied extensively for potential therapeutic use in tissue engineering to repair cartilage injury. Recent findings suggest that MSCs are present in mature human articular cartilage. The role of resident MSCs in cartilage repair is unknown. Cells expressing MSC markers are prominent in the clusters of proliferating cells characteristic of osteoarthritic cartilage. These cells express high levels of inflammatory mediators and markers of aberrant chondrocyte differentiation, suggesting that their activation contributes to the pathogenesis of arthritis.

Our understanding of chondrogenesis and the molecular mechanisms that regulate MSC differentiation is incomplete. Identification of factors that repress and activate chondrogenesis may lead to the development of techniques and technologies that will allow use of MSCs for tissue repair and for the treatment of osteoarthritis.

Published data and our preliminary findings strongly suggest that the Notch signaling pathway is a critical mediator of differentiation toward the chondrocytic lineage. We have shown that Notch and its downstream targets Hes-1 and Hey-1 regulate chondrogenesis through novel interactions with Sox9, the principal prochondrogeneic transcription factor.

Mechanotransduction in Chondrocytes

D. Haudenschild, D. D'Lima, M. Lotz

Mechanical forces regulate chondrocyte proliferation, survival, differentiation, gene expression, and biosynthetic responses. The type and duration of mechanical stimulation determine the outcome of the cellular responses, which in their extreme manifestations can range from cell proliferation to cell death and from matrix formation to matrix destruction. We measured the actin reorganization that occurs in response to dynamic compression of agarose-embedded chondrocytes, tested whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression, and investigated whether dynamic compression alters the intracellular localization of Rho kinase and actin-remodeling proteins in chondrocytes.

Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling and caused a significant increase in punctate actin structures. Rho kinase activity was required for these cytoskeletal changes; dynamic compression in the presence of Rho kinase inhibitor did not induce punctate actin structures. Dynamic compression increased the amount of phosphorylated Rho kinase, as shown by immunofluorescence confocal microscopy. The genes for the chemokine CCL20 and inducible nitric oxide synthase were the ones most highly upregulated by dynamic compression, and this response was reduced by the Rho kinase inhibitor hydroxyfasudil.

In conclusion, we found that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we developed a method to measure these changes. Furthermore, we showed that Rho kinase activity is required for compression-induced actin reorganization and gene expression.

GLUT1 and Chondrocyte Homeostasis

A. Shikhman, D. Brinson, M. Lotz

Articular cartilage is an avascular tissue that receives its nutrients and oxygen by diffusion from blood vessels in the subchondral bone and from synovial fluid. Energy generation in cartilage strongly depends on glucose supply. Transmembrane transport of glucose is facilitated by a group of highly specialized glucose transporter proteins termed GLUTs. Human articular chondrocytes express at least 6 different GLUTs, including GLUT1, GLUT3, GLUT6, GLUT8, GLUT10, and GLUT11.

GLUT1 is the most abundant glucose transporter in human articular chondrocytes. Expression of GLUT1 is increased in cartilage affected by osteoarthritis and in chondrocytes activated by cytokines and growth factors in vitro. Inhibition of GLUT1 mRNA and protein expression with specific small interfering RNA inhibits IL-1–induced production of nitric oxide, suppresses growth factor–stimulated transmembrane thymidine transport and chondrocyte proliferation, and enhances growth factor–induced production of hyaluronan and expression of hyaluronan synthase type 2.

The effects of GLUT1 on chondrocyte proliferation and thymidine transport are mediated via AMP-activated protein kinase. However, GLUT1-regulated hyaluronan synthesis does not depend on this kinase.

To determine the role of GLUT1 in the initiation of intracellular signaling, we studied specific plasma membrane proteins with binding affinity for GLUT1. Mass spectrometry of chondrocyte lysates immunoprecipitated with antibodies to GLUT1 revealed that annexin II was the main plasma membrane protein that reproducibly coprecipitated with GLUT1. In addition, using Western immunoblotting with antibodies to annexin II, we found that annexin II was present in GLUT1 coprecipitates. Immunoprecipitation of annexin II resulted in coprecipitation of GLUT1. Interaction between GLUT1 and annexin II was also shown by confocal microscopy. The interactions between GLUT1 and annexin II depend on interactions between their oligosaccharide side chains.

Transcriptional Regulation of Cartilage Development

T. Ito, N. Taniguchi, M. Tsuda, H. Asahara

Chondrogenesis and cartilage development are tightly regulated processes in which multipotential mesenchymal stem cells differentiate into chondrocytes to form cartilage. This process is initiated by commitment to the chondrogenic lineage and condensation of the stem cells, followed by differentiation of the cells into chondrocytes, a change associated with cartilage-specific gene expression. Such activity is regulated transcriptionally both spatially and temporally, such that transcription factors have dynamic expression patterns during chondrogenic differentiation. Subsequently, chondrocytes proliferate and secrete a cartilage-specific matrix to form the cartilage anlagen. We examined 2 molecules, coactivator-associated arginine methyltransferase 1 (CARM1) and high mobility group box 1 protein (HMGB1), as critical regulators of endochondral ossification.

Cartilage development is regulated by the transcription factor Sox9, but the molecular mechanisms that underlie this activity remain unclear. We found that CARM1 regulates chondrocyte proliferation via arginine methylation of Sox9. Mice lacking the gene for CARM1 had delayed endochondral ossification. Conversely, cartilage development in CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. These results establish a role for CARM1 as an important regulator of cell proliferation during development.

HMGB1 has dual roles. First, as a nuclear factor, it alters chromatin formation and regulates gene expression. Second, extracellular HMGB1 released from damaged cells acts as a cytokine and chemoattractant. However, the role of extracellular HMGB1 in physiologic conditions has not been fully understood. We discovered that mice lacking the gene for HMGB1 have severely impaired endochondral ossification during embryogenesis. HMGB1 is secreted from cultures of cartilage, and the protein is specifically located in the cytosol of hypertrophic chondrocytes. Recombinant HMGB1 promotes osteoclast migration as well as endothelial cell migration, suggesting that extracellular HMGB1 regulates endochondral ossification as a chemoattractant, at least in part. Taken together, these data provide evidence for a critical role of HMGB1 in skeletal development.

Publications

Davis, D.K., Goltz, D.H., Fithian, D.C., D'Lima, D. Anatomical posterior cruciate ligament transplantation: a biomechanical analysis. Am. J. Sports Med. 34:1126, 2006.

D'Lima, D.D., Patil, S., Steklov, N., Chien, S., Colwell, C.W., Jr. In vivo moments and shear after total knee arthroplast. J. Biomech., in press.

D'Lima, D.D., Patil, S., Steklov, N., Slamin, J.E., Colwell, C.W., Jr. Tibial forces measured in vivo after total knee arthroplasty. J. Arthroplasty 21:255, 2006.

Gordon, A.C., D'Lima, D.D., Colwell, C.W., Jr. Highly cross-linked polyethylene in total hip arthroplasty. J. Am. Acad. Orthop. Surg. 14:511, 2006.

Hall, J., Copp, S.N., Adelson, W.S., D'Lima, D.D., Colwell, C.W., Jr. Extensor mechanism function in single radius vs multiradius femoral components for total knee arthroplasty. J. Arthroplasty, in press.

Hardwick, M.E., Pulido, P.A., D'Lima, D.D., Colwell, C.W., Jr. e-Knee: the electronic prosthesis. Orthop. Nurs. 25:326, 2006.

Hiraoka, K., Grogan, S., Olee, T., Lotz, M. Mesenchymal progenitor cells in adult human articular cartilage. Biorheology 43:447, 2006.

Patil, S., D'Lima, D.D., Fait, J.M., Colwell, C.W., Jr. Improving tibial component coronal alignment during total knee arthroplasty with use of a tibial planing device. J. Bone Joint Surg. Am. 89:381, 2007.

Tam, H.K., Srivastava, A., Colwell, C.W., Jr., D'Lima, D.D. In vitro model of full-thickness cartilage defect healing. J. Orthop. Res., in press.

Taniguchi, N., Yoshida, K., Ito, T., Tsuda, M., Mishima, Y., Furumatsu, T., Ronfani, L., Abeyama, K., Kawahara, K., Komiya, S., Maruyama, I., Lotz, M., Bianchi, M.E., Asahara, H. The stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. Mol. Cell. Biol., in press.

Temple, M.M., Bae, W.C., Chen, M.Q., Lotz, M., Amiel, D., Coutts, R.D., Sah, R.L. Age- and site-associated biomechanical weakening of human articular cartilage of the femoral condyle. Osteoarthritis Cartilage, in press.

Zhao, D., Banks, S.A., D'Lima, D.D., Colwell, C.W., Jr., Fregly, B.J. In vivo medial and lateral tibial loads during dynamic and high flexion activities. J. Orthop. Res. 25:593, 2007.

Zhao, D., Banks, S.A., Mitchell, K.H., D'Lima, D.D., Colwell, C.W., Jr., Fregly, B.J. Correlation between the knee adduction torque and medial contact force for a variety of gait patterns. J. Orthop. Res. 25:789, 2007.