Tartrate resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in subsets of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomemlar mesangial cells of the kidney and pancreatic acinar cells. Despite thirty years of research, the function of TRAP in vivo remains unclear. It is implicated in bone resorption, but also may function in immune defence and iron transport. This thesis set out to investigate two aspects of TRAP function. The potential for TRAP to function in mouse embryonic development was investigated through the localisation of TRAP mRNA and TRAP enzyme activity. The function of TRAP in osteoclast-mediated bone resorption was investigated by characterising the phenotype of the TRAP over-expressing mouse. Prior to this thesis little was known about the mechanisms controlling tissue-specific expression of the TRAP gene. The last section of this thesis investigates the regulatory mechanisms controlling TRAP expression at the transcriptional level.
The pig TRAP homologue, uteroferrin, is implicated in mediating trans-placental iron transport in the developing pig. It is not known if TRAP performs a similar role in the developing mouse. This thesis describes for the first time the localisation of TRAP mRNA and TRAP enzyme activity in the extra-embryonic tissues of the developing mouse embryo. TRAP enzyme activity was restricted to the cells of the visceral endoderm, parietal endoderm and the trophoblast cells from 6.5 days post coitum (dpc) to 10.5 dpc. Together these cells function as an early placenta prior to the formation of the mature hemochorial placenta at 9.5-10.5 dpc. It is proposed that TRAP may function in macromolecule degradation in these cells or alternatively TRAP may provide iron to the fetus for use in hemopoiesis.
The TRAP over-expressing mice are mildly osteoporotic with reduced trabecular bone area but increased bone formation rate to presumably compensate for increased osteoclast activity. This thesis further characterises the phenotype of the TRAP over-expressing mouse. Neonatal calvarial calcium release assays demonstrated that the TRAP-SV40E mice have a 20% increase in net calcium release. TRAP enzyme activity was elevated in the TRAP-SV40E mice serum and tissue extracts including kidney, liver, lung, and spleen. Surprisingly no difference in TRAP enzyme activity was observed between the wild type and TRAP-SV40E protein extracts from adult bone tissue. The mild differences in the resorption activity of the TRAP-SV40E osteoclasts and the absence of elevated TRAP activity in the TRAP-SV40E bone environment may hold the key to understanding the mild bone pathology observed in these animals. The apparent increase in TRAP protein levels in primary neonatal TRAP-SV40E osteoclasts suggests that although the protein is being expressed at elevated levels, mechanisms to either inhibit or inactivate the excess TRAP enzyme activity may be in place. Although even a slight increase in osteoclast activity may result in an eventual net bone loss, the elevation in osteoblast activity, as demonstrated in this study by an increase in total serum alkaline phosphatase activity, is likely to minimise the impact of any increase in osteoclast resorption activity. The observations noted in this study are consistent with the results obtained previously using bone histomorphometry analysis.
Finally this thesis describes the identification and characterisation of two novel TRAP mRNAs that differ in their 5’UTR sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'UTRs represent alternative first exons located upstream of the known 5'UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'UTR (Exon lA) is restricted to adult bone and spleen tissue. Exon IB is expressed primarily in tissues containing TRAP-positive non-haematopoietic cells. The known TRAP 5'UTR (Exon IC) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon IC promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon IC transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression pattems for each of the TRAP 5'UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue and cell restricted promoters.