Transgenic Overexpression of Tartrate-Resistant Acid Phosphatase Is Associated with Induction of Osteoblast Gene Expression and Increased Cortical Bone Mineral Content and Density

Abstract

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.

Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes

“…This may indicate that the technique is not sensitive enough to detect small amounts of protein along these pathways and indirectly supports the assumption that the observed labeling of TRAP is localized to secretory lysosomes where newly synthesized protein gets stored and thus concentrated to a degree that allows detection. TRAP gene expression in osteoblasts and osteocytes is well documented by RT-PCR (Kogawa et al 2013 ; Qing et al 2012 ) and in situ hybridization (Gradin et al 2012 ; Nakano et al 2004 ). Moreover, osteocytes in both cancellous and cortical bone express loop-TRAP/mTRAP (Solberg et al 2014 ), and it is therefore not likely that the TRAP + vesicles contain TRAP with another origin than from within the osteoblasts and osteocytes themselves.…”

“…The isoform TRAP 5b shows different biological abilities in bone, e.g., dephosphorylation of osteopontin (OPN) and integrin-binding sialoprotein (IBSP) (Ek-Rylander and Andersson 2010 ; Ek-Rylander et al 1994 ), dephosphorylation of Man-6-P recognition marker on lysosomal proteins (Bresciani and Von Figura 1996 ; Sun et al 2008 ) and generation of reactive oxygen species for bone matrix degradation (Halleen et al 1999 , 2003 ; Vaaraniemi et al 2004 ). On the other hand, the monomeric TRAP isoform 5a exhibits growth factor-like properties for mesenchymal cells, e.g., pre-adipocytes (Lang et al 2008 ; Patlaka et al 2014 ) and pre-osteoblasts (Gradin et al 2012 ). TRAP gene expression and enzyme activity seems to be most abundant in bone tissue, and young rats express more TRAP mRNA and TRAP enzyme activity than adults (Ek-Rylander et al 1991 ; Lang and Andersson 2005 ).…”

“…TRAP 5b is mainly found in osteoclasts and has been advocated as a serum marker for osteoclast number. However, also other bone cells express TRAP in vivo; hypertrophic chondrocytes in the rat epiphysis (Hessle et al 2013 ), osteoblasts in the metaphysis and in the endosteal and periosteal compartments of diaphyseal rat bone (Bianco et al 1988 ; Bonucci et al 2001 ; Gradin et al 2012 ; Mocetti et al 2000 ; Yamamoto and Nagai 1998 ) as well as osteocytes in cancellous and cortical rat bone near bone surface or bone resorption sites (Bianco et al 1988 ; Nakano et al 2004 ). The function of TRAP in these cells has been debated and recently increased levels of TRAP gene expression and enzyme activity were demonstrated in osteocytes with active osteocytic osteolysis (Kogawa et al 2013 ; Qing et al 2012 ).…”

CD109 Plays a Role in Osteoclastogenesis

“…Cells were cultured for 4 days, with a change of cell culture medium and sRANKL supplementation on the second day of incubation. At day 4, cells were gently washed twice with pre-warmed PBS (phosphate buffered saline), fixed with 4% paraformaldehyde (PFA) and stained for tartrate-resistant acid phosphatase (TRACP), which is considered a chemical marker of osteoclasts [30], [31]. Briefly, the fixed cells were incubated in a solution of naphthol AS-TR phosphate disodium salt and fast red TR salt (Sigma-Aldrich) in 0.2 M acetate buffer (pH 5.2) containing 100 mM sodium tartrate (Sigma-Aldrich) for 15 min at 37°C.…”

Osteoblast Differentiation and Bone Matrix FormationIn VivoandIn Vitro

“…The site contains residual material secreted by osteoclasts, including tartrate-resistant acid phosphatase. There is direct evidence that tartrate resistant acid phosphatase is osteoinductive, 104 but the utility of making conditioned artificial calcified templates for osteoblast attachment has not been investigated fully. Many resorbable substrates have been tested, often with added growth factors.…”