Role of tartrate-resistant acid phosphatase in bone remodeling

Tartrate-resistant acid phosphatase is a metalloenzyme that exists as two isoforms: the monomeric TRAP 5a and the proteolytically cleaved TRAP 5b, responsible for phosphatase activity, which is highly expressed in osteoclasts (OCs). TRAP 5b has been used as a serum marker of bone resorption, as it correlates with the absolute number of OCs and with resorption markers such as CTX-I. Despite being used as biomarker for bone metabolic diseases, little is known about the role of TRAP isoforms in OCs and thus bone remodeling (the process of bone degradation by osteoclasts, and bone formation by osteoblasts). Therefore, this thesis aimed to investigate the role of TRAP isoforms in bone remodeling.

To enable the investigation of TRAP 5a and 5b we (1) developed a sandwich TRAP 5a/5b ELISA for the quantification of human TRAP isoforms. This ELISA was then used for (2) evaluating the expression and secretion pattern of TRAP 5a and 5b in healthy individuals and during OC differentiation. Additionally, we used the ELISA to (3) investigated if TRAP protein levels correlate to osteoarthritis (OA) or rheumatoid arthritis (RA). Here we correlated the phosphorylation status of the known TRAP in vivo substrate, osteopontin, to the TRAP isoforms. (4) Using a competitive inhibitor for TRAP 5b, we studied the role of TRAP in OCs differentiation. Lastly, we (5) developed a high throughput system to identify a subclone in a murine cell line that is a more homogeneous and stable OC precursors that could be used as a screening tool for OC biology studies.

A double TRAP 5a/5b sandwich ELISA was developed and designed as a two-step process. Using the ELISA, we showed that in vitro cultures of OCs secrete not only TRAP 5b but also TRAP 5a and that both isoforms were present intracellular establishing that 5b can also be formed intracellular in OCs. Correlation between TRAP 5a and 5b indicated a dependence between TRAP 5a and formation of 5b. There was a positive correlation in both serum from healthy men, and media from in vitro OC cultures of not only 5b but also TRAP 5a with CTX-I further suggesting that TRAP 5a also originates partly from OCs. Measurement of TRAP 5a and 5b in synovial fluid from OA and RA patients revealed a correlation between low TRAP 5b/ TRAP 5a ratio and phosphorylated osteopontin. This suggested that synovial fluid from RA patients contained an insufficient amount of TRAP 5b increasing levels of phosphorylated OPN leading to a higher OC activation and bone destruction. Inhibition of TRAP 5b using the competitive inhibitor, 5-phenylnicotinic acid, decreased the number of OCs formed and the expression of several OC markers. However, some OCs were able to fuse and resorb bone.

In this thesis we show that measurement of TRAP isoforms protein is an important tool in research and possibly also in diagnostic to understand the biological implications of TRAP 5a and 5b in OCs, which may lead to therapeutic targeting of certain isoforms for inflammatory and metabolic bone diseases. We further show that TRAP is involved in the bone remodeling process in OCs and defects in TRAP may cause alterations in OCs function and differentiation.

List of scientific papers

I. Laia Mira-Pascual#, Christina Patlaka#, Suchita Desai, Staffan Paulie, Tuomas Näreoja, Pernilla Lång, Göran Andersson. A novel sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b protein reveals that both isoforms are secreted by differentiating osteoclasts and correlate to the Type I collagen degradation marker CTX-I in vivo and in vitro. Calcif Tissue Int. (2019). #Equal contribution.
https://doi.org/10.1007/s00223-019-00618-w

II. Jani Luukkonen, Laia Mira-Pascual, Christina Patlaka, Pernilla Lång, Sanna Turunen, Jussi Halleen, Tomi Nousiainen, Maarit Valkealahti, Juha Tuukkanen, Göran Andersson, Petri Lehenkari. Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio. PLos One. (2016). 12(8):e0182904.
https://doi.org/10.1371/journal.pone.0182904

III. Laia Mira-Pascual, Anh N Tran, Göran Andersson, Tuomas Näreoja, Pernilla Lång. A subclone of RAW264.7 forms osteoclast-like cells capable of bone resorption faster than RAW264.7 through increased de novo expression and nuclear translocation of NFATc1. [Submitted]

IV. Laia Mira-Pascual, Suchita Desai, Anh N. Tran, Christina Patlaka, Pernilla Lång, Angela Casini, Barbro Melgert, Ulf Lerner, Petra Henning, Tuomas Näreoja, Göran Andersson. Tartrate-resistant acid phosphatase activity promotes RANKL-stimulated osteoclast differentiation in vitro. [Manuscript]