Kirsch Lab: Projects

Project: The Role of Annexins in Chondrocyte Differentiation Events During Development and Pathology

During endochondral ossification long bones are first cartilaginous. During this process, chondrocytes in the growth plate undergo a series of differentiation events, including proliferation, hypertrophy, terminal differentiation, mineralization, and programmed cell death (apoptosis). Eventually apoptotic chondrocytes and mineralized cartilage are replaced by bone. Once the long bones have reached their final size, most of the cartilage is replaced by bone except for a small rim of cartilage—the articular cartilage—that plays a crucial role in normal joint function. Destruction of articular cartilage is associated with osteoarthritis and rheumatoid arthritis.


 Annexin VI expression in human normal, moderately osteoarthritic, and severely osteoarthritic cartilage.

Our laboratory is interested in how terminal differentiation, mineralization, and apoptotic events in chondrocytes are regulated during normal development and under pathological conditions such as osteoarthritis. We have shown that retinoic acid (vitamin A) regulates terminal differentiation, mineralization, and apoptotic events in growth plate chondrocytes. Treatment of growth plate chondrocytes with retinoic acid leads to calcium channel formation of annexins II, V, and VI. Annexins are cytoplasmic proteins, which in the presence of calcium bind to acidic phospholipids. Our studies have characterized the mechanisms leading to membrane binding and channel formation of annexins II, V, and VI and the consequences of alterations of calcium homeostasis mediated by these annexins in chondrocyte differentiation. These studies have demonstrated that blocking annexin channels inhibits terminal differentiation events of growth plate chondrocytes. Interestingly, these annexins bind to extracellular matrix molecules and cytoskeletal proteins. Therefore, these studies test the hypothesis that annexins act as mechanosensitive calcium channels controlling chondrocyte differentiation.


Annexin II and V expression in human normal, moderately osteoarthritic, and severely osteoarthritic cartilage.

Our laboratory was the first to show that articular chondrocytes in osteoarthritic cartilage undergo similar terminal differentiation events as growth plate chondrocytes. Whereas these events are crucial for endochondral bone formation, they lead to cartilage destruction when occurring in articular chondrocytes. Annexins II, V, and VI are highly expressed by articular chondrocytes in osteoarthritic cartilage, but not in healthy articular cartilage. In addition, we detected other terminal differentiation markers in osteoarthritic cartilage. Therefore, we hypothesize that articular chondrocytes lose their phenotype in osteoarthritis and undergo similar differentiation events as growth plate chondrocytes, leading to terminal differentiation, mineralization, and apoptosis. Furthermore, we propose that annexins are crucial regulators of these differentiation events during development and pathology and that interfering with annexin functions may provide novel therapeutic targets to stop or slow down the progression of osteoarthritis.

Project: The Role of the Progressive Ankylosis Gene (ank) in Physiological and Pathological Mineralization

Physiological mineralization is important for bone development and for the proper function of bones. Mineralization has to be restricted to certain sites, however, as uncontrolled or pathological mineralization can have severe adverse consequences. For example, mineralization of articular cartilage leads to its destruction, while mineralization of cardiovascular tissues leads to morbidity or even death.


Proposed model of how Ank regulates physiological and pathological mineralization. Normal articular chondrocytes express low amounts of Ank, which in turn transports intracellular pyrophosphate (PPi) to the extracellular milieu. Extracellular pyrophosphate acts as an inhibitor of mineralization. In osteoarthritic cartilage, Ank protein expression is highly upregulated. Upregulated Ank expression results in an increased extracellular pyrophosphate concentration. In the absence of alkaline phosphatase, increased extracellular pyrophosphate results in calcium pyrophosphate crystal (CPPD) deposition in articular cartilage. If articular chondrocytes or growth plate chondrocytes express alkaline phosphatase, then extracellular pyrophosphate is hydrolyzed to inorganic phosphate (Pi), resulting in basic calcium phosphate (BCP) crystal deposition. Furthermore, inorganic phosphate is transported back into the cell, where it acts as a signaling molecule stimulating terminal differentiation and mineralization events.

To understand the mechanisms regulating biomineralization, our laboratory is conducting experiments to determine the role of Ank, a protein that transports intracellular pyrophosphate to the extracellular milieu in normal and pathological mineralization of cartilage and other tissues. Pyrophosphate is hydrolyzed to inorganic phosphate by alkaline phosphatase, an enzyme present in hypertrophic chondrocytes and bone cells. Mutations in the human ank gene result in hypermineralization and diseases such as craniometaphyseal dysplasia and chondrocalcinosis. Our goal is to determine how Ank regulates extracellular pyrophosphate concentration and subsequent mineralization.

So far we have determined that expression of ank in articular chondrocytes that do not express alkaline phosphatase results in calcium pyrophosphate crystals, whereas expression of ank in articular chondrocytes that express alkaline phosphatase results in basic calcium phosphate crystal deposition. Interestingly, both forms of crystals are found in patients with osteoarthritis, and these crystals are main contributors to cartilage destruction during osteoarthritis. More importantly, our research provides evidence that inorganic phosphate not only is a component of basic calcium phosphate crystals but is transported back into the cell where it acts as a signaling molecule stimulating terminal differentiation and mineralization events of chondrocytes during development and pathology.

Project: The Role of the Progressive Ankylosis Gene (ank) in Mesenchymal Stem Cell Commitment Toward a Specific Cell Lineage

Embryonic and adult stem cells are multipotential and can differentiate into multiple cell lineages. Skeletal tissue cells are derived from mesenchymal stem cells, which can differentiate along several lineages, including fibroblasts, muscle cells (myoblasts), fat cells (adipocytes), cartilage cells (chondrocytes), and bone cells (osteoblasts). To use stem cells for tissue-engineered repair, it is important to understand the mechanisms involved in them along the path to a particular cell lineage.

Our lab has found evidence that the progressive ankylsosis gene (ank) is required for the commitment of mesenchymal stem cells along the osteoblast lineage. We have shown that loss of Ank function results in delayed differentiation of preosteoblastic cells toward immature osteoblasts and a differentiation of these preosteoblastic cells along the chondrocytic lineage. Loss of Ank function in these preosteoblastic cells was found to result in a marked decrease the expression of osterix—a key transcription factor required for the differentiation of preosteoblastic cells into immature osteoblasts. Loss of osterix results in a total loss of bone formation. Therefore, Ank appears to be upstream of osterix and regulates its expression and ultimately the differentiation of precursor cells along the osteoblastic lineage.


Bone histomorphometric analysis of tibiae of 2- and 4-month-old ank/ank mice and wild-type littermates. (A) Hematoxylin and eosin staining of sections from the proximal tibiae of 4-month-old ank/ank mice (ank/ank; a, b) and wild-type littermates (WT; c, d). Bar = 200 µm. Panels b and d show higher magnification of the area outlined in panels a and c. Bar = 50 µm.(B-E) Quantitative histomorphometry showing significantly lower bone volume/total volume, trabecular number, and increased trabecular separation in 2- and 4-month-old ank/ank mice compared to wild-type littermates. Trabecular thickness was significantly decreased in 4-month-old ank/ank mice but not in 2-month-old ank/ank mice (*p < .01 vs. wild-type littermates; n = 5 per genotype group).

Our current studies focus on how Ank regulates precursor cell commitment toward a specific cell lineage. We have found that transgenic mice that have a complete loss of Ank function exhibit an osteopenic phenotype in long bones as well as severe pathologic mineralization in joints, intervertebral discs, and aortas; these animals die by 4 month of age. Interestingly, heterozygous mutations of ank in humans result in a similar, slightly milder version of the phenotype in homozygous mice. These studies, which promise to enhance our understanding of the mechanisms regulating early osteoblastogenesis, suggest that Ank regulates bone formation not only during development but also during bone remodeling and therefore may be a novel candidate gene important in the development of osteoporosis, a disease that is characterized by loss of bone mass and strength and that often results in bone fractures from even minor trauma. Understanding the factors and mechanisms regulating cell lineage commitment is moreover crucial for the use of stem cells in tissue repair.