Development of Novel Models for Studying Osteoclasts

University dissertation from Division of Molecular Medicine and Gene Therapy

Abstract: Popular Abstract in English My PhD thesis has focused on increasing the understanding of the osteoclasts, the cells responsible for degradation of bone, by studying the rare hereditary disease autosomal recessive osteopetrosis (ARO). ARO is present already at birth and is caused by mutations affecting the osteoclasts ability to degrade bone. Non-functional osteoclasts lead to dramatic increases in bone thickness affecting bone marrow, blood vessels, and nerves due to having smaller cavities in the skeleton. Despite the increase in bone mass, the bones are weak and brittle because of how the bones are molded during growth, and fractures often occur. In almost half the cases, the disease is caused by mutations in the gene called TCIRG1, which encodes a protein important in the degradation of calcium in bone. As osteoclasts come from blood stem cells, the disease can be corrected by bone marrow transplantation, replacing sick stem cells with healthy cells from a donor. However, this treatment is risky and is highly dependent on a matching donor. Without bone marrow transplantation the disease is fatal within five years of birth. Under normal circumstances the osteoclast mediated bone degradation process leads to release of signals that direct new bone formation by specialized cells called osteoblasts. In ARO patients these osteoclast derived signals are maintained despite the lack of bone degradation, suggesting that simply the osteoclasts being present may be enough for bone formation. A great effort has been put into identifying these osteoclast derived signals as they may lead to new treatments for patients with too little bone, such as osteoporosis. In the first two papers we developed an adult mouse model with osteopetrosis to investigate how signals from non-functional osteoclasts affected bone formation. We induced osteopetrosis by transplanting stem cells from osteopetrotic mice, with defects in the mouse version of TCIRG1, into normal adult mice. The healthy osteoclasts are thereby replaced by non-functional osteoclasts and the mice develop a mild form of osteopetrosis. In paper I we found that these “osteoclast-rich” osteopetrotic mice have increased bone mass and surprisingly increased bone strength compared to control mice. In Paper II we compared the osteoclast-rich model to an osteoclast-poor model. For this we instead used stem cells from a mouse model which has a mutational defect in a gene crucial for osteoclast development. Comparing these two types of osteopetrosis in mice, we found that bone formation was increased in osteoclast-rich osteopetrotic mice compared to their osteoclast-poor counterparts. These findings suggest that blocking bone degradation, while keeping osteoclasts, maintains the signals directing bone formation. A molecule blocking the osteoclasts ability to degrade bone but without removing the cell itself, may be interesting as potential treatment for low bone mass diseases such as osteoporosis. In paper III we used different ways of modifying osteoclast function to investigate how the release of signals from osteoclasts, directing bone formation is affected by these changes in various osteoclast related functions. We also used osteoclasts from ARO patients to show that signals are still being released directing bone formation despite their lack of bone degradation. The studies indicate that the signals are both dependent on how old the osteoclast is, and what type of bone the osteoclast is degrading. Blood stem cells can renew themselves and produce all the different kinds of blood cells in the blood throughout a lifetime. By correcting these cells one can change the function of all osteoclasts that are to be made. The principle of gene therapy is to insert the normal functional gene into a cell having dysfunctional gene. This may result in the patient’s ability to produce the correct functional protein themselves. In Paper IV we used a modified HIV-virus as a tool to insert the correct functioning TCIRG1 gene into human ARO blood stem cells. We found that the corrected cells expressed the functional protein and that function was almost fully restored in these osteoclasts, as could be measured by the release of degradation products such as calcium and collagen fragments. In summary these studies have increased the understanding of ARO and the osteoclast related defects causing this disease as well as taken a step towards gene therapy as a treatment for this ARO. The experiments have also shed light on the important regulation undertaken by the osteoclasts in the maintenance of bone, and findings suggest that targeting the osteoclasts ability to degrade bone without removing the osteoclasts may be a novel target for treatment of bone turnover diseases.