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.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite biomaterials for bone tissue regeneration: in vitro performance assessed by osteoblast proliferation, osteoclast adhesion and resorption, and macrophage proinflammatory response.

The efficacy of composite materials for bone tissue engineering is dependent on the materials' ability to support bone regeneration whilst inducing a minimal inflammatory response. In this study we examined the in vitro osteogenic and inflammatory properties of poly(3-hydroxybutyrate-co-3-valerate) (PHBV) with various calcium phosphate-reinforcing phases: nano-sized hydroxyapatite (HA); submicron-sized calcined hydroxyapatite (cHA); and submicron-sized beta-tricalcium phosphate (beta-TCP), using bioassays of cultured osteoblasts, osteoclasts, and macrophages. Our study showed that the addition of a nano-sized reinforcing phase to PHBV, whilst improving osteogenic properties, also reduces the proinflammatory response. Proinflammatory responses of RAW264.7/ELAM-eGFP macrophages to PHBV were shown to be markedly reduced by the introduction of a reinforcing phase, with HA/PHBV composites having the lowest inflammatory response. Osteoclasts, whilst able to attach to all the materials, failed to form functional actin rings or resorption pits on any of the materials under investigation. Cultures of osteoblasts (MC3T3-E1) readily attached and mineralised on all the materials, with HA/PHBV inducing the highest levels of mineralization. The improved biological performance of HA/PHBV composites when compared with cHA/PHBV and beta-TCP/PHBV composites is most likely a result of the nano-sized reinforcing phase of HA/PHBV and the greater surface presentation of mineral in these composites. Our results provide a new strategy for improving the suitability of PHBV-based materials for bone tissue regeneration.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase.

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of approximately 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the approximately 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans.

Transgenic mice that express Cre recombinase in osteoclasts.

To study the physiological control of osteoclasts, the bone resorbing cells, we generated transgenic mice carrying the Cre recombinase gene driven by either the tartrate-resistant acid phosphatase (TRAP) or cathepsin K (Ctsk) promoters. TRAP-Cre and Ctsk-Cre transgenic mouse lines were characterized by breeding with LacZ ROSA 26 (R26R) reporter mice and immunohistochemistry for Cre recombinase. The Cre transgene was functional in all lines, with Cre-mediated recombination occurring primarily in the long bones, vertebrae, ribs, and calvaria. Histological analyses of the bones demonstrated that functional Cre protein was present in 1) osteoclasts (Ctsk-Cre); 2) osteoclasts, columnar proliferating, and hypertrophic chondrocytes (TRAP-Cre line 4); and 3) round proliferating chondrocytes (TRAP-Cre line 3). In conclusion, we generated transgenic mouse lines that will enable the deletion of floxed target genes in osteoclasts, which will be valuable tools for studying the regulation of osteoclast function.

Regulation of the murine TRACP gene promoter.

The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function.

Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation.

The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (TRAP) in osteoclasts. Several lines of evidence were consistent with this model. The combination of MITF and PU.1 synergistically activated the TRAP promoter in transient assays. This activation was dependent on intact binding sites for both factors in the TRAP promoter. MITF and PU.1 physically interacted when coexpressed in COS cells or in vitro when purified recombinant proteins were studied. The minimal regions of MITF and PU.1 required for the interaction were the basic-helix-loop-helix zipper domain and the Ets DNA binding domain, respectively. Significantly, mice heterozygous for both the mutant mi allele and a PU.1 null allele developed osteopetrosis early in life which resolved with age. The size and number of osteoclasts were not altered in the double heterozygous mutant mice, indicating that the defect lies in mature osteoclast function. Taken in total, the results afford an example of how lineage-specific gene regulation can be achieved by the combinatorial action of two broadly expressed transcription factors.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

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.

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.

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.

TFEC is a macrophage-restricted member of the microphthalmia-TFE subfamily of basic helix-loop-helix leucine zipper transcription factors.

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed with all other known MiT subfamily members (Mitf, TFE3, TFEB). Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU.1 under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.

Cloning and characterization of the murine genes for bHLH-ZIP transcription factors TFEC and TFEB reveal a common gene organization for all MiT subfamily members.

The microphthalmia-TFE (MiT) subfamily of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors, including TFE3, TFEB, TFEC, and Mitf, has been implicated in the regulation of tissue-specific gene expression in several cell lineages. In this report, we investigate the genomic organization and structural relatedness of MiT transcription factors. We characterized the gene for mTFEC, which covers a region of more than 50 kb and is composed of seven exons. Further, we cloned a cDNA for the murine TFEB homologue and characterized its genomic structure. The eight coding exons of mTFEB are distributed over a 6-kb region. A multiple alignment of amino acid sequences of known MiT subfamily members indicates undescribed, conserved N-terminal regions and common putative phosphorylation sites for TFE3, TFEB, and Mitf. Also, intron-exon borders for characterized MiT genes appear completely conserved. A new family member and closely related putative transcription factor in Caenorhabditis elegans was identified by database searches that show a similar genomic organization within the bHLH-ZIP region and the acidic domain. Evolutionary aspects and implications for structure-function relationships are discussed.

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.

Mapping and characterization of the eukaryotic early pregnancy factor/chaperonin 10 gene family.

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.

The rat pyruvate carboxylase gene structure. Alternate promoters generate multiple transcripts with the 5'-end heterogeneity.

Pyruvate carboxylase (EC 6.4.1.1) is a biotin-containing enzyme that plays an important role in gluconeogenesis and lipogenesis. Here we report the structural organization of the rat pyruvate carboxylase gene, which spans over 40 kilobases and is composed of 19 coding exons and 4 5'-untranslated region exons. From this data, it is clear that alternative splicing of the primary transcripts from two promoters is responsible for the occurrence of the multiple mRNA species previously reported (Jitrapakdee, S., Walker, M. E., and Wallace, J. C. (1996) Biochem. Biophys. Res. Commun. 223, 695-700). The proximal promoter, which is active in gluconeogenic and lipogenic tissues, contains no TATA or CAAT boxes but includes a sequence that is typical of a housekeeping initiator protein 1 box while the distal promoter contains three CAAT boxes and multiple Sp1 binding sites. Several potential transcription factor binding sites are found in both promoters. A series of 5'-nested deletion constructs of both promoters were fused to a firefly luciferase reporter plasmid and transiently expressed in COS-1 cells. The results show that the 153 and 187 base pairs, preceding the transcription start sites of the proximal and distal promoters, respectively, are required for basal transcription. Insulin selectively inhibits the expression of the proximal promoter-luciferase reporter gene by 50% but not the distal promoter in COS-1 cells, suggesting the presence of an insulin-responsive element in the proximal promoter. A half-maximal effect was found at approximately 1 nM insulin.

The human early pregnancy factor/chaperonin 10 gene family.

cDNA clones corresponding to the sequence for human early pregnancy factor were isolated from a human melanoma library and hybridized to DNA digested with four restriction enzymes obtained from twelve different subjects. Up to 20 cross hybridizing bands were observed. When hybridized to metaphase spreads from four different humans, significant signals were present in nine locations, on eight different chromosome arms. These results suggest that the early pregnancy factor gene is a member of a large gene family. The coding sequence for early pregnancy factor has a high degree of homology with the sequence for human chaperonin 10, and the gene family described here should contain the genes for both of these proteins.

Cloning, sequencing and expression of rat liver pyruvate carboxylase.

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated M(r) of 129848. This is flanked by a 5' untranslated region of 66 bp and a 3' untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249-10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

Isolation and characterization of the genes encoding mouse and human type-5 acid phosphatase.

The gene (mT5AP) encoding murine type-5 acid phosphatase has been isolated and completely sequenced while the gene (hT5AP) encoding human T5AP has been partly sequenced. The murine gene spans 4 kb and contains five exons. Exon 1 is completely non-coding and exon 2 starts with the initiation codon in both mT5AP and hT5AP. The positions of the intron/exon boundaries are completely conserved between mT5AP and hT5AP, but are distinct from the gene encoding the related porcine protein, uteroferrin (Utf). There is strong homology at both the nucleotide (nt) and amino acid (aa) levels between the inferred mouse cDNA and the sequences of rat T5AP and hT5AP, and pig Utf. The mT5AP and hT5AP genes were found to have multiple transcription start points (tsp) by primer extension analysis, consistent with the absence of a consensus TATA box. The sequences for the 5'-flanking regions of mT5AP and hT5AP were determined to -1.6 and -1.0 kb, respectively, relative to the tsp. A 2-kb segment of the mT5AP 5' flanking region linked to a luciferase-encoding reporter gene (Luc) was sufficient to direct tissue-specific transcription in the mouse macrophage cell line, RAW264. Significant sequence similarity between the mT5AP and hT5AP promoters is restricted to the most proximal 200 bp, which also resembles the porcine Utf gene, and a 300-bp segment 700 bp upstream. A progesterone-response element is present only in the mouse promoter and the estrogen- and iron-response elements described previously in the pig gene are absent from both the mouse and human genes. These differences may result in distinctive regulation of T5AP and Utf expression.

The resistance of macrophage-like tumour cell lines to growth inhibition by lipopolysaccharide and pertussis toxin.

The process of tumorigenesis is frequently associated with resistance to growth inhibition by physiological regulators of normal cells. Murine macrophage-like cell lines BAC1.2F5, RAW264, J774.1A and PU5/1.8 were resistant to growth inhibition by bacterial lipopolysaccharide (LPS) and pertussis toxin, agents that blocked growth of primary bone marrow-derived macrophages (BMDM) in the presence of macrophage colony-stimulating factor (CSF-1). The resistance of the CSF-1-dependent cell line BAC1.2F5 to growth inhibition by pertussis toxin argues against the possibility that pertussis toxin-sensitive G proteins are essential for the pathway of growth stimulation by CSF-1. Conversely, these data add further weight to the argument that LPS mediates some of its biological activities by mimicking the action of pertussis toxin and inhibiting G protein function. The resistance of cell lines to LPS and pertussis toxin was not correlated with any alteration in the expression of mRNA encoding any of three pertussis-toxin sensitive G protein alpha subunits. The pattern of G protein expression was consistent between primary cells and tumour cells, suggesting that this is a differentiation marker. In particular, Gi alpha 2 mRNA was expressed at remarkably high levels in all of the cells. The specificity of LPS resistance was investigated by studying down-regulation of CSF-1 binding and induction of protooncogene c-fos and tumour necrosis factor (TNF) mRNA. BAC1.2F5 cells were LPS-resistant in each of these assays. In CSF-1 binding, RAW264 and J774.1A responded in the same way as bone marrow-derived macrophages but required higher doses of LPS, whereas c-fos and TNF mRNA were induced in these cells at concentrations that did not inhibit growth. In PU5/1.8 cells, CSF-1 binding was already very low and was not further down-regulated, but c-fos and TNF mRNA was inducible by LPS. By contrast to primary macrophages, the cell lines did not respond to LPS with down-regulation of c-fms mRNA, which encodes the CSF-1 receptor. Hence, the resistance of macrophage-like tumour cells to LPS and pertussis toxin was specific to the pathways controlling growth, and was correlated with altered regulation of the CSF-1 receptor.