Biogenesis of tartrate-resistant acid phosphatase isoforms 5a and 5b in cell and animal models

Sammanfattning: Tartrate-resistant acid phosphatase (TRAP) is an iron-containing binuclear acid metallohydrolase that belongs to the purple acid phosphatase (PAP) family. It is a glycoprotein translated as a monomeric protein but is commonly isolated from tissues as a proteolytically processed dimeric form with increased enzyme activity. In human serum, the enzyme exists as two isoforms, monomeric TRAP 5a and proteolytically-processed TRAP 5b. Traditionally, the enzyme has been used as a marker for osteoclasts and participates in bone resorption. Activated macrophages, dendritic cells, certain neurons and epithelial cells as well as several cancer cell lines and tissues have also been shown to express TRAP. TRAP 5a and 5b isoforms are compartmentalized differently by different cell types and their serum levels correlate independently with different human diseases, consistent with the notion that the TRAP isoforms exert different functions. The aims of the present investigation were to characterize biogeneration of these two isoforms through proteolytic cleavage and differential intracellular sorting, and their differential expression in physiological and pathological settings. Cathepsin K (Ctsk) is an essential cysteine proteinase in bone matrix degradation by osteoclasts. The enzyme can efficiently activate TRAP through proteolytic processing in vitro and these two proteins co-localize in osteoclasts. Analysis of Ctsk-deficient mice revealed that absence of Ctsk was associated with increased abundance of monomeric TRAP in osteoclasts of long bones as well as calvaria. In long bones, this increase was more pronounced in osteoclasts of the distal compared to the proximal part of the metaphyseal trabeculae, suggesting a site-dependent role for Ctsk in TRAP processing in vivo and highlighting functional differences between bone-resorbing osteoclasts within the trabecular metaphyseal bone. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of Ctsk. Absence of Ctsk was also associated with altered intracellular distribution and secretion of TRAP, suggesting a novel role for Ctsk in the regulation of intracellular trafficking in osteoclasts. Biogenetic relationship between TRAP 5a and 5b was further studied in a stably transfected TRAP-overexpressing breast cancer epithelial cell line. The study demonstrated that distinct monomeric TRAP populations are diverted early in the secretory pathway either giving rise to a secreted, monomeric TRAP 5a or to an intracellular, proteolytically processed TRAP 5b. In support of different functional roles for TRAP 5a and 5b, the majority of the intracellular monomeric TRAP was destined for secretion as TRAP 5a, while a minor portion provided the putative precursor for intracellular TRAP 5b. Examination of breast cancer bone metastases revealed that TRAP 5a was the predominant form expressed by metastatic cancer cells, whereas tumor-associated macrophages expressed the proteolytically processed TRAP 5b. Moreover, a previously unstudied TRAP 5a variant was identified in cancer cells, which may have functional and diagnostic implications.