Dickkopf-1 directs periosteal bone formation in two murine models of inflammatory arthritis.

OBJECTIVE: The Wnt signalling antagonist Dickkopf-1 (DKK1) inhibits osteoblast differentiation and function and has been described to play a central role in promoting bone loss, while blockade of DKK1 increases bone formation. We investigated the effects of DKK1 on periosteal new bone formation in two murine models of inflammatory arthritis, the antigen-induced arthritis (AIA) and K/BxN serum transfer arthritis (STA) models. METHOD: The flare variant of AIA was induced in wild-type mice and a blocking antibody to DKK1, control rat immunoglobulin G (IgG), or phosphate-buffered saline (PBS) was administered starting on day 14, a time at which inflammation and erosions are known to be established. Knees were assessed for histological inflammation and periosteal new bone formation was quantitated. In addition, STA was generated in transgenic (Tg) mice with osteoblast-specific overexpression of Dkk1 and littermate controls. New bone formation around the wrists of these mice was quantified by micro-computed tomography. RESULTS: Blockade of DKK1 in arthritic mice resulted in significantly more periosteal new bone formation compared to mice treated with control rat IgG or PBS. Conversely, in the setting of increased Dkk1 expression, arthritic Dkk1 Tg mice developed significantly less periosteal new bone than arthritic controls. CONCLUSION: DKK1 is a regulator of periosteal bone formation in inflammatory arthritis. Thus, regulation of DKK1 may be considered as a therapeutic approach in inflammatory diseases in which patients suffer from excessive periosteal bone formation, such as spondyloarthritis.

Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/ß-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Production of a novel polyketide through the construction of a hybrid polyketide synthase.

The lactone rings of the polyketides platenolide and tylactone are synthesized by condensation of acetate-, proprionate-, and butyrate-derived precursors. A hybrid tylactone/platenolide synthase was constructed to determine if the choice of substrate is programmed by the polyketide synthase and to ascertain if a substrate different than that normally used in the first step of platenolide synthesis could be incorporated into the final polyketide. In this work, we report the successful incorporation of a propionate in place of the acetate normally used in the first step of platenolide synthesis. This result demonstrates that polyketide synthases choose a particular substrate at defined steps and provides strong evidence that substrate choice is programmed by the acyl transferase domain of a large, multifunctional polyketide synthase.

Analysis of the integration function of the streptomycete bacteriophage phi C31.

A 2.1 kb (1 kb = 10(3) base-pairs) segment of DNA from the streptomycete bacteriophage phi C31 was found to be sufficient to direct site-specific integration of plasmid vectors in Streptomyces ambofaciens and Streptomyces fradiae in the absence of any streptomycete origin of replication. Sequencing and analysis of phage, chromosomal and junction attachment sites of S. ambofaciens and S. fradiae revealed that recombination is conservative and that crossover takes place within three bases of homology between phage and host. Deletion analysis, sequencing and site-specific mutagenesis of the phi C31 DNA revealed a large open reading frame (ORF 613) whose expression was necessary for integration. This ORF begins near the point of crossover and reads away from the attachment site. A comparison of the predicted amino acid sequence of ORF 613 with known recombinases did not reveal any significant similarities. A genetic analysis of the amino-terminal region of ORF 613 suggested that translation could initiate at any one of three possible start codons. Primer extension experiments showed that transcriptional initiation occurred at a T and a C only four and five bases, respectively, from the site of crossover. This analysis suggested that ORF 613 would be separated from its promoter upon integration.

A thiostrepton-inducible expression vector for use in Streptomyces spp.

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp.

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).

Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis.

Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.

A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library.

A new shuttle cosmid vector, pKC505, was constructed for the cloning of Streptomyces DNA. This vector, which can be conjugally transferred between different streptomycetes, was used to construct a genomic library from a spiramycin-producing S. ambofaciens strain. By transformation of the spiramycin-sensitive S. griseofuscus with the library, three phenotypically different spiramycin-resistance genes were isolated. S. ambofaciens DNA in these clones was colinear with the chromosome, and the cloned DNA was stable in E. coli, S. griseofuscus and S. fradiae. These cosmids could be isolated easily from S. griseofuscus, an improvement over the previous shuttle cosmid vector, pKC462a [Stanzak et al., Bio/Technology 4 (1986) 229-232], which was somewhat difficult to isolate from S. lividans.

Expression in Streptomyces ambofaciens of an Escherichia coli K-12 gene which confers resistance to hygromycin B.

We have constructed two plasmid vectors (pKC293 and pKC305) that can replicate in Escherichia coli K-12 and Streptomyces ambofaciens. These shuttle vectors were used to demonstrate the expression of two E. coli genes, hygromycin B (Hm) resistance and Tn5 neomycin (Nm) resistance, in S. ambofaciens.