Tartrate-Resistant Acid Phosphatase Knockout Mice†
The authors have no conflict of interest.
TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of TRACP in vivo, we have generated mice in which the gene-encoding TRACP has been selectively disrupted by targeted homologous recombination in murine embryonic stem cells. Homozygous TRACP “knockout” mice have progressive foreshortening and deformity of the long bones and axial skeleton suggesting a role for TRACP in endochondral ossification. There is increased mineralization reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. These knockout mice also display an impairment of macrophage function with abnormal immunomodulatory cytokine responses. Superoxide formation and nitrite production were enhanced in stimulated macrophages lacking TRACP as was the secretion of the proinflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-12. TRACP knockout mice showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. The macrophages lacking TRACP showed an increase in tartrate-sensitive lysosomal acid phosphatase activity (LAP). The TRACP knockout mice were bred with mice lacking LAP. Mice lacking both TRACP and LAP had even shorter bones than the TRACP single knockouts. Osteopontin, identical to the T-cell cytokine η-1, accumulated adjacent to actively resorbing osteoclasts suggesting that both phosphatases are important for processing this protein. We propose that TRACP may be an important regulator of osteopontin/η-1 activity common to both the immune system and skeleton.
Studies in humans, mice, and rats have demonstrated that TRACP is widely distributed in normal tissues.(1–5) Both mRNA and protein are expressed abundantly in tissues including the spleen, liver, linings of the gastrointestinal tract, lung, thymus, and skin; however, phosphatase activity does not correlate with the amount of protein present.(6) TRACP activity is highest in bone, spleen, liver, thymus, and colon. Lower amounts of activity were measured in lung, stomach, skin, brain, and kidney. Traces of activity were measured in heart, testis, and muscle.
TRACP is synthesized as a latent proenzyme with low activity that is activated in vitro by the action of cysteine proteinases, resulting in a cleavage of the single chain 34 kDa form into a highly active two subunit form.(7) In tissues, these proteases are present in all sites where TRACP has been identified, and we therefore propose that this cleavage occurs in vivo, thus permitting the activation of latent TRACP at selected sites when required. Of particular interest is our recent demonstration that TRACP activity in cultured dendritic cells is low when the cells are immature, but on maturation, increases 5-fold, suggesting a potential role for TRACP in antigen processing and in immune responses.
To investigate the function and importance of TRACP in vivo, we have generated mice lacking TRACP by targeted gene disruption.(8) The phenotype of these mice implicates a role for TRACP in skeletal development and in the immune system.
TRACP activity was absent in the osteoclasts of bones derived from homozygous animals and reduced in heterozygous animals. The gross morphology of the bones was altered. The bones including those of the limbs and axial skeleton were overall fatter, shorter, and had thicker cortices than normal. A club-like deformity was observed in the distal femora and was associated with pyramidal malformation of the proximal tibias, which appeared to increase with age. The shape was abnormal with altered trabecular sculpting at the metaphyses. The epiphyseal growth plates were widened with delayed mineralization of cartilage. The columns of chondrocytes were disorganized, and the cells showed marked hypertrophy and hyperplasia, with increased retention of thicker metaphyseal trabeculae associated with reduced modeling and mineralization of the expanded cartilaginous matrix. Abundant TRACP-negative osteoclasts invaded the epiphyseal plates within the disorganized region of remodeling. These abnormalities indicate that TRACP has an essential role in endochondral ossification. Histomorphometry and mineralization density analysis by backscattered electron imaging demonstrated widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover.
Increased mineralization occurred in the long bones of older animals, reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. This was confirmed in the resorption pit assay in vitro where osteoclasts isolated from mutant mice resorb 26% less efficiently than wildtype littermates. However, this mild increased bone density could also have resulted from an alteration in the synthesis of the bone matrix proteins. This is currently under investigation.
LYSOSOMAL ACID PHOSPHATASE/TRACP MICE
The genetically distinct lysosomal acid phosphatase (LAP) is ubiquitously expressed. Mice deficient in LAP have lysosomal storage in podocytes and tubular epithelial cells of the kidney as well as in microglia, ependymal cells, and astroglia within the central nervous system.(9) Abnormalities in bone structure were apparent in mice older than 15 months, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. LAP knockout mice had higher than normal levels of TRACP activity, suggesting that LAP and TRACP may have overlapping functions. LAP-deficient mice were bred with TRACP-deficient mice to create mice lacking both LAP and TRACP.(10)
LAP/TRACP double knockout mice are characterized by progressive hepatosplenomegaly, gait disturbances, and even shorter limb bones than were seen in the TRACP single knockouts. Histologically these animals displayed an excessive lysosomal storage in the macrophages of the liver, spleen, bone marrow, and kidney, and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of TRACP- and LAP/TRACP-deficient mice. Vacuoles in the osteoclasts from TRACP and LAP/TRACP knockout mice contained mineral crystallites as well as osteopontin, suggesting that TRACP is important for the processing of this protein. Extracts from these mice were unable to dephosphorylate recombinant osteopontin in vitro. Isolated osteoclasts from the LAP/TRACPs showed a small reduction in resorption compared with osteoclasts deficient in only TRACP.
MACROPHAGE PHENOTYPE OF TRACP KNOCKOUT MICE
To investigate a putative function for TRACP in macrophages, we investigated pro-inflammatory responses and systemic microbial clearance in knockout mice compared with age- and gender-matched congenic wildtype mice.(11) Phorbol 12-myristate 13-acetate-stimulated and interferon-γ-induced superoxide formation was enhanced in peritoneal macrophages lacking TRACP. Nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-γ was also increased, and the secretion of the proinflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-12 was significantly greater when stimulated with LPS with or without addition of either TNF-α or IFN-γ. The activity of tartrate-sensitive (lysosomal) acid phosphatase was increased in macrophages from knockout mice. Evidence of impaired macrophage function in vivo was obtained in TRACP knockout mice, which showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates had a reduced population of macrophages. As peritoneal macrophages and neutrophils lacking TRACP were able to phagocytose and kill S. aureus normally in vitro, TRACP may influence recruitment of macrophages to sites of microbial invasion. These studies show that TRACP participates in the inflammatory response of the macrophage and influences effector signaling pathways in innate immunity.
TRACP AND DENDRITIC CELLS
We have demonstrated widespread expression of TRACP mRNA and protein in 18-day normal mouse embryos and in adult murine and human tissues.(4–6) The cellular distribution of TRACP mRNA and enzyme antigen in skin and the gastrointestinal tract tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. To confirm its putative localization in dendritic cells, isolated bone marrow-derived dendritic cells were matured in culture. These costained strongly for TRACP protein and the CD80 antigen. Blood-derived human DCs were assayed specifically for TRACP activity using an immunoassay. As the cells matured, TRACP activity in the cells increased 5-fold, suggesting a role for TRACP in antigen presentation. We are currently investigating the function of TRACP in dendritic cells using the TRACP knockout mice. It has recently been reported that osteopontin is abundantly expressed in lesions of rats with multiple sclerosis where it appears to regulate T helper cell-1-mediated demyelinating disease.(12) Because TRACP is able to dephosphorylate osteopontin, it may be involved in the signaling of chronic T-cell-mediated demyelinating diseases. The report by Lang et al. of TRACP expression in rat nervous tissue strongly supports this.(13)
TRACP is a protein expressed by osteoclasts, macrophages, and dendritic cells that can be activated by proteolytic cleavage. The knockout mouse studies have demonstrated that TRACP is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption. The macrophages from these mice displayed an increased capacity to secrete free radicals and disturbed proinflammatory cytokine secretory profiles, suggesting a role for TRACP in innate immunity.
The LAP/TRACP mice have shown that TRACP has a role in the degradation of osteopontin. Osteopontin is a protein with a wide tissue distribution like TRACP and a variety of functions. It is identical to the T-cell cytokine η-1, which interacts with macrophages to induce inflammatory responses. η-1-deficient mice fail to clear pathogens and have defective IL-12 and delayed hypersensitivity responses.(14) η-1 is secreted in phosphorylated and non-phosphorylated forms, and dephosphorylation abolishes η-1-mediated IL-12 release. Because the IL-12 inflammatory pathway is enhanced in TRACP-deficient macrophages, we propose that TRACP may be an important regulator of η-1 (osteopontin) activity common to both the immune system and skeleton. Our studies showing that TRACP is expressed by dendritic cells are fully compatible with a central role of this enzyme in pleiotropic defense processes.
We thank the Arthritis Research Campaign for financial support.