A. I. Cassady

Transgenic Mice Overexpressing Tartrate‐Resistant Acid Phosphatase Exhibit an Increased Rate of Bone Turnover

Tartrate‐resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate‐limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number–dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low‐level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue‐specific promoter.

Structure, function, and regulation of tartrate-resistant acid phosphatase

The tartrate-resistant acid phosphatases (TRAPs) are a class of metalloenzymes that catalyze the hydrolysis of various phosphate esters and anhydrides under acidic reaction conditions. Because the bound metal ions confer an intense color on these enzymes they are also known as purple acid phosphatases (PAPs). Resistance to inhibition by high concentrations of the competitive inhibitor L 1 tartrate distinguishes TRAP from acid phosphatases of lysosomal or prostatic origin present in many mammalian cells and tissues. TRAP enzymes have been isolated from many mammalian sources, including: bovine and rat spleen; the spleens of patients affected with hairy cell leukemia and Gaucher’s disease; human and rat bone; and human lungs and placenta. The TRAP purified from porcine allantoic fluid, which is also known as uteroferrin, was originally recognized as an abundant basic protein in uterine secretions induced by progesterone. The catalytic mechanism, structure, and properties of the iron center of porcine TRAP have been studied extensively by our group. Mammalian isolated TRAP enzymes all have similar physical properties, including a molecular weight of about 35 kDa, a basic isoelectric point (pI 7.6–9.5), and optimal enzyme activity at an acidic pH. The enzyme can be isolated as a single chain polypeptide, but a dimeric nicked form arises from posttranslational cleavage of the single chain enzyme. Cleavage occurs in an exposed loop that is conserved in all mammalian TRAP enzymes and leads to an increase in Vmax/kcat of the enzyme by an unknown mechanism. Several proteolytic enzymes are able to cleave the exposed loop, but only the cysteine proteinases papain and cathepsin B have been able to cause activation among several tested. Ljusberg et al. put forth the view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proenzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts. Mammalian TRAP enzymes are glycoproteins and, like most lysosomal enzymes, possess the mannose-6-phosphate lysosomal targeting sequence, which must presumably be cleaved or modified to permit secretion. TRAP isolated from allantoic fluid of the pig showed a single, unphosphorylated, high-mannose-type oligosaccharide composed of five or six mannose residues and two N-acetylglucosamine residues. In contrast, recombinant porcine TRAP secreted by Chinese hamster ovary (CHO) cells possessed N-linked, high-mannose oligosaccharide chains that were phosphorylated and could not be dephosphorylated by alkaline phosphatase treatment in vitro. This suggests that the uteroferrin oligosaccharide phosphates were not exposed, perhaps as a result of blocking by an N-acetylglucosamine residue. The glycoprotein structure of human bone TRAP was analyzed by lectin binding and, in agreement with the prior analysis of native uteroferrin, contained only N-linked high-mannose carbohydrates, implying that the native secreted protein is normally dephosphorylated. Analysis of TRAP activity present in electrophoretically separated human serum revealed two isoforms, termed 5a and 5b, with each isoform having a different pH optimum (5a: pH 4.9; 5b: pH 5.5–6.0). The carbohydrate content of the isoforms also differed with only isoform 5a containing sialic acid. TRAP contains two iron atoms at its active site, and the intense purple color of the enzyme results from a tyrosinate Fe(III) charge transfer. Reduction of the active site binuclear center to a mixed valency Fe(III)-Fe(II) form is required for activation and this corresponds to a shift in color from purple to pink. Further reduction or the presence of iron chelators can lead to reversible inactivation and formation of a colorless form of the enzyme. The enzyme is also inhibited noncompetitively by incubation with vanadate or simply following more extended incubation at 37°C. The latter case at least, produced a “yellowish” form of the enzyme. TRAP may become irreversibly inactivated by oxidation in the presence of ascorbate. The recent availability of monoclonal antibodies against TRAP has permitted the identification of an inactivated “yellowish” form of the enzyme as the major form in the circulation.

The Microphthalmia Transcription Factor Regulates Expression of the Tartrate‐Resistant Acid Phosphatase Gene During Terminal Differentiation of Osteoclasts

The defective terminal differentiation of osteoclasts in mice homozygous for the mi allele of the microphthalmia transcription factor (MITF) gene implies that MITF plays a critical role in regulating gene expression during osteoclast ontogeny. To begin addressing the role of this transcription factor in the osteoclast, target genes need to be identified. In the present work, several lines of evidence show that the gene encoding the enzyme tartrate‐resistant acid phosphatase (TRAP) is a target of MITF. Analysis of osteoclasts in vivo in the embryonic forelimb showed that MITF and TRAP RNA were coexpressed in a dynamic pattern during the process of endochondral ossification of long bone. Primary osteoclast‐like cells (OCLs) produced from mi/mi mutant mice expressed TRAP messenger RNA (mRNA) at 8‐fold lower levels than in OCLs derived from normal mice, indicating a direct link between MITF function and TRAP expression. The activity of mouse TRAP promoter‐reporter genes was assayed in the primary OCLs by DNA‐mediated transfection, and this activity was shown to depend on a conserved sequence (GGTCATGTGAG) located in the proximal promoter. Recombinant MITF protein recognized specifically this conserved sequence element Expression of a TRAP promoter–green fluorescent protein (GFP) transgene mimicked the expression of the endogenous TRAP gene during differentiation of osteoclast‐like cells, and the expression of the transgene was decreased 8‐fold when placed into the mutant mi/mi background. These results are consistent with a role for MITF in gene expression during terminal differentiation of the osteoclast and will allow osteoclast‐specific mechanisms of gene regulation to be studied in greater detail.

Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation.

The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation.

The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor

Microphthalmia transcription factor (MITF) regulates osteoclast function by controling the expression of genes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to receptor activator of nuclear factor-κB ligand (RANKL)-induced signaling. To identify novel MITF target genes, we have overexpressed MITF in the murine macrophage cell line RAW264.7 subclone 4 (RAW/C4) and examined the gene expression profile after sRANKL-stimulated osteoclastogenesis. Microarray analysis identified a set of genes superinduced by MITF overexpression, including Clcn7 (chloride channel 7) and Ostm1 (osteopetrosis-associated transmembrane protein 1). Using electrophoretic mobility shift assays, we identified two MITF-binding sites (M-boxes) in the Clcn7 promoter and a single M-box in the Ostm1 promoter. An anti-MITF antibody supershifted DNA-protein complexes for promoter sites in both genes, whereas MITF binding was abolished by mutation of these sites. The Clcn7 promoter was transactivated by coexpression of MITF in reporter gene assays. Mutation of one Clcn7 M-box prevented MITF transactivation, but mutation of the second MITF-binding site only reduced basal activity. Chromatin immunoprecipitation assays confirmed that the two Clcn7 MITF binding and responsive regions in vitro bind MITF in genomic DNA. The expression of Clcn7 is repressed in the dominant negative mutant Mitf mouse, mi/mi, indicating that the dysregulated bone resorption seen in these mice can be attributed in part to transcriptional repression of Clcn7. MITF regulation of the TRAP, cathepsin K, Clcn7, and Ostm1 genes, which are critical for osteoclast resorption, suggests that the role of MITF is more significant than previously perceived and that MITF may be a master regulator of osteoclast function and bone resorption.

Microphthalmia transcription factor regulates the expression of the novel osteoclast factor GPNMB

Microphthalmia transcription factor (MITF) regulates bone homeostasis by inducing expression of critical genes associated with osteoclast function. Gpnmb is a macrophage-enriched gene that has also been shown to be expressed in osteoblasts. Here, we have shown gpnmb to be highly induced in maturing murine osteoclasts. Microarray expression profile analysis identified gpnmb as a potential target of MITF in RAW264.7 cells, subclone C4 (RAW/C4), that overexpress this transcription factor. Electrophoretic mobility shift assays identified a MITF-binding site (M-box) in the gpnmb promoter that is conserved in different mammalian species. Anti-MITF antibody supershifted the DNA-MITF complex for the promoter site while MITF binding was abolished by mutation of this site. The gpnmb promoter was transactivated by co-expression of MITF in reporter gene assays while mutation of the gpnmb M-box prevented MITF transactivation. The induction of gpnmb expression during osteoclastogenesis was shown to exhibit similar kinetics to the known MITF targets, acp5 and clcn7. GPNMB expressed in RAW/C4 cells exhibited distinct subcellular distribution at different stages of osteoclast differentiation. At days 5 and 7, GPNMB protein co-localised with the osteoclast/macrophage lysosomal/endocytic marker MAC-3/LAMP-2, suggesting that GPNMB resides in the endocytic pathway of mature macrophages and is possibly targeted to the plasma membrane of bone-resorbing osteoclasts. The inclusion of gpnmb in the MITF regulon suggests a role for GPNMB in mature osteoclast function.

Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX‐2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half‐life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N‐terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.

Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS.

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow‐derived murine macrophages (BMMs) was examined using reverse transcriptase‐polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP‐1, a type III receptor PTP. Three mDEP‐1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl‐terminus and 3 UTR. The expression of mDEP‐1 was down‐regulated by CSF‐1 (macrophage colony‐stimulating factor) and up‐regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF‐1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP‐1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP‐1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP‐1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation. J. Leukoc. Biol. 64: 692–701; 1998.

Responses in vivo to purified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) implanted in a murine tibial defect model.

Effective bone biomaterials provide structural support for bone regeneration and elicit minimal inflammatory or toxic effects in vivo. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a bacterially derived biodegradable polymer that possesses suitable mechanical strength for use as a bone biomaterial and has a slow rate of degradation in biological environments. Our previous in vitro study showed that many PHBV preparations are contaminated with bacterial lipopolysaccharide, and we developed a purification procedure to substantially remove it. Here, we have evaluated the in vivo biocompatibility of PHBV purified by H(2)O(2) treatment and solvent extraction. We utilized a murine tibial defect model consisting of a hole drilled through the diameter of the tibial diaphysis into which nonporous cylindrical plugs of purified PHBV were implanted. The animals were sacrificed at 1 week and 4 weeks postsurgery, and tibiae were examined using histological staining. The PHBV implant induced a mild inflammatory response 1 week after injury, which persisted for 4 weeks. Granuloma type tissues formed only when the implant protruded into the overlaying tissue. Woven bone formation occurred adjacent to the implant, which gave rise to lamellar bone and stabilized the implant indicating that the PHBV did not affect this process. Our data validated the murine defect model and indicate that solid PHBV induces a mild tissue reaction with bone deposition adjacent to the implant with no fibrous tissue present at 4 weeks post surgery.