Androgens may regulate the male skeleton directly through a stimulation of androgen receptors or indirectly through aromatization of androgens into estrogen and, thereafter, through stimulation of estrogen receptors (ERs). The relative importance of ER subtypes in the regulation of the male skeleton was studied in ER␣-knockout (ERKO), ER␤-knockout (BERKO), and double ER␣͞␤-knockout (DERKO) mice. ERKO and DERKO, but not BERKO, demonstrated decreased longitudinal as well as radial skeletal growth associated with decreased serum levels of insulin-like growth factor I. Therefore, ER␣, but not ER␤, mediates important effects of estrogen in the skeleton of male mice during growth and maturation.

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TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site ( M AMMALIAN TARTRATE-RESISTANT purple acid phosphatase (TRACP/PAP) is an iron-containing, cationic glycoprotein with a molecular weight of around 35 kDa. Although the enzyme is translated as a single polypeptide, the protein isolated from different tissues commonly exist as a disulfide-linked two-subunit structure with an N-terminal fragment of 20 -23 kDa joined to the 16-to 17-kDa C-terminal part.(1) It now appears clear that the two-subunit form is generated by proteolytic excision of a loop domain protruding between helix 5 and 6 at the surface of the molecule, and that its removal leads to a significant increase in the enzymatic activity of the molecule at pH Ͼ5. Besides serine proteases (e.g., trypsin and chymotrypsin), several cysteine proteinases of the cathepsin family have been shown to cleave in the loop region, with the latter proteases leading to markedly higher enzyme activation. (3,4) We have focused our interest on the cysteine proteinases as potential physiological regulators of TRACP enzyme activation in osteoclasts and macrophages, primarily because members of this family have been implicated in resorption of bone as well as in lysosomal protein degradation. Interestingly, both cathepsin K and L are highly efficient activators of the latent monomeric TRACP in vitro.(5,6) Cathepsins B, H, and S were much less effective, and MMP 2 and 9 were completely ineffective.(5) Of the two cathepsins, only cathepsin K is expressed to a significant extent in osteoclasts, while cathepsin L predominate in other types of macrophages.(7) That cathepsin K is involved in processing of TRACP in bone was shown by use of cathepsin K knockout mice, where an increased content of monomeric TRACP was recovered from their bones.(5) Thus, members of the cathepsin family can process TRACP to a more active enzyme, and we propose that cathepsin K fulfills this role in osteoclasts while in other TRACP expressing cells of the macrophage lineage cleavage can be accomplished by cathepsin L.

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The activation sequence of clasts (the designation clast was used because ultrastructurally in this tissue, it is not always possible to differentiate between chondroclasts sitting on cartilage and osteoclasts sitting on bone matrix) was studied in vivo using the healing of low-phosphate, vitamin D-deficiency rickets as a model system. Thus, the bones of 7-week-old rachitic animals were analyzed with a combination of morphological, biochemical, and molecular biological methods at 48 and 72 h, respectively, after change to normal food. A quantitative ultrastructural analysis showed that the number of clast profiles exhibiting the characteristic polarized features of actively resorbing cells, i.e., ruffled borders and clear zones, had reached normal levels after 48 h. By combining the data with quantitative analyses by the immunogold technique, we demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing). In contrast, the levels of tartrate-resistant acid phosphatase (TRAP) both between ruffles and in the outside matrix adjoining the ruffled border were low in polarized clasts both in rickets and at the early (48 h) healing time-point, but were increased at the latest (72 h) healing time-point. Interestingly, expression of TRAP and the cathepsin K at the mRNA level, as well as protein expression and the activity of TRAP, were not different during the healing sequence. Although the two enzymes are confined to the same clast populations, their secretion during the resorption process is apparently differentially regulated: cathepsin K secretion is coupled to ruffled border formation in clasts, whereas TRAP is secreted at a later stage during the resorption sequence, suggesting a role for secreted TRAP as a modulator of resorptive activity.

Focusing on resorption processes, we have extended our previous studies on chondroclasts and osteoclasts in normally developing tissues, using a model of nutritionally induced vitamin D-deficiency rickets. To analyze the resorption process, we investigated the matrix-resorbing cells in this modified and poorly mineralized tissue regarding morphological features and expression of tartrate-resistant acid phosphatase (TRAP) at the subcellular level. Our goal was to test the hypotheses that initiation of resorption is impaired with unmineralized matrix, and that such alterations involve changes in the subcellullar distribution of TRAP, implicating a role for this enzyme in the resorption process. Our results reveal distinctly different morphological appearances of clast-like cells in rickets compared with normal osteoclasts and chondroclasts. Ordinary resorption structures of osteoclasts and chondroclasts at the cell-matrix border, i.e., ruffled borders and clear zones, are profoundly altered in favor of a less well-defined intermediate zone. TRAP distribution at the subcellullar level is also clearly different from that in osteoclasts and chondroclasts from normal rodents, with impaired secretion; consequently, the enzyme is unable to function in the matrix outside the ruffled border. Our ultrastructural observations demonstrate that in rickets, the clasts are incapable of degrading the poorly mineralized cartilage and bone efficiently. Rachitic clasts seem to be recruited to the matrix surface and interaction between cell and matrix is also initiated, but definitive resorption structures at the cell-matrix border are not normally developed. Whether resorption is inhibited by the mere lack of mineral or mineral-associated proteins, or by other mechanisms remains to be settled.

Bone remodeling is a central event in the maintenance of skeletal tissue, and involves cycles of resorption followed by the formation of bone tissue. The activity of osteoclasts and osteoblasts during these cycles is tightly regulated by systemic and local factors coupling the action of these cells. Tartrate-resistant acid phosphatase (TRAP) is predominantly expressed in bone by osteoclasts but has also been detected in osteoblasts and osteocytes. Moreover, TRAP can stimulate the differentiation of mesenchymal lineage cells, i.e. progenitors of osteoblasts and adipocytes. In order to further explore the effects of TRAP on bone turnover, the structural and molecular phenotypes of osteoclasts and osteoblasts were assessed in TRAP-overexpressing transgenic mice. Transgenic mice of both sexes display increased cortical bone mineral content and density, which cannot be accounted for by decreased bone resorption since osteoclast numbers and resorptive activity do not differ from wild-type mice. Examination of the osteoblast phenotype revealed that markers of bone formation, i.e. procollagen type I N-terminal propeptides, and osteoblast lineage markers as well as the TRAP 1B mRNA transcript are increased in TRAP-overexpressing mice. Expression of the osteoclast-selective TRAP 1C mRNA is not increased in TRAP transgenic mice. Elevated expression of TRAP mRNA and protein were detected in osteoblasts, osteocytes and in the bone matrix of TRAP transgenic mice, suggesting that TRAP overexpression in osteoblast lineage cells is associated with increased cortical bone mineral content and density. The data presented here support the hypothesis that TRAP overexpression in the osteoblastic cell lineage stimulates the differentiation and/or activation of these cells.