Characterization of lethal alleles of D-elg, an ets proto-oncogene related gene with multiple functions in Drosophila development.

We have used genetic complementation rescue to identify two lethal alleles of D-elg, an ets proto-oncogene related gene of Drosophila. Animals that are hemizygous or trans-heterozygous for the alleles die as pharate adults, demonstrating normal gene function is required to complete Drosophila development. Females trans-heterozygous for the 1(3)902 lethal allele and the previously characterized tne female sterile allele produce embryos with abdominal segmentation defects. This finding implicates a role for the D-elg gene in anterior-posterior patterning. The cloning and sequencing of the lethal alleles identified molecular mutations that may result in ELG protein truncation, altered ELG protein interactions, or defective D-elg mRNA splicing.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Binding protein-3-selective insulin-like growth factor I variants: engineering, biodistributions, and clearance.

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.

The mouse osteocalcin gene cluster contains three genes with two separate spatial and temporal patterns of expression.

Osteocalcin is the most abundant noncollagenous protein of bone. Here we report that the mouse genome contains an osteocalcin cluster composed of three genes arranged within a 23-kilobase span of genomic DNA. We named them osteocalcin gene 1 (OG1), osteocalcin gene 2 (OG2), and osteocalcin-related gene (ORG) in order from the 5' end to the 3' end of the cluster. Hybridization of polymerase chain reaction-amplified cDNAs with specific oligonucleotides and RNase protection assays showed that OG1 and OG2 are expressed only in bone, whereas ORG is transcribed in kidney but not in bone. Furthermore, during embryogenesis, OG1 and OG2 begin to be expressed at day 15.5, while ORG is transcribed as early as day 10.5. The protein encoded by ORG has a similar pattern of expression and identical structural features to nephrocalcin, a calcium-binding protein partially purified from kidney that plays a role in calcium reabsorption and in prevention of nephrolithiasis. The nephrocalcin gene has not been cloned in any species; we propose that ORG is the mouse nephrocalcin gene. The existence of several osteocalcin or osteocalcin-related sequences is not restricted to mouse but is present in every species we examined.

Ets oncogene-related gene Elg functions in Drosophila oogenesis.

Members of the ets gene family encode transcription factors that regulate the expression of a variety of cellular and viral genes including several protooncogenes. We have utilized Drosophila to elucidate the in vivo function of one family member. We show by complementation rescue and sequence analysis that the female sterile mutant tiny eggs (tne) is an allele of the Drosophila Ets-related gene Elg (also called D-elg). The mutation of a highly conserved tyrosine residue in the ETS DNA-binding domain of the Elg gene product demonstrates that normal gene function is required for proper follicle cell migration, chorion formation, and nurse cell-chromosome decondensation during Drosophila oogenesis.

Pathways by which interleukin 17 induces articular cartilage breakdown in vitro and in vivo.

Overexpression of interleukin (IL-)17 has recently been shown to be associated with a number of pathological conditions. Because IL-17 is found at high levels in the synovial fluid surrounding cartilage in patients with inflammatory arthritis, the present study determined the direct effect of IL-17 on articular cartilage. As shown herein, IL-17 was a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants. These effects were mediated in part by leukemia inhibitory factor (LIF), but did not depend on interleukin-1 activity. The mechanism whereby IL-17 induced matrix breakdown in cartilage tissue appeared to be due to stimulation of activity of aggrecanase(s), not matrix metalloproteinase(s). However, IL-17 upregulated expression of matrix metalloproteinase(s) in chondrocytes cultured in monolayer. In vivo, IL-17 induced a phenotype similar to inflammatory arthritis when injected into the intra-articular space of mouse knee joints. Furthermore, a related protein, IL-17E, was found to have catabolic activity on human articular cartilage. This study characterizes the mechanism whereby IL-17 acts directly on cartilage matrix turnover. Such findings have important implications for the treatment of degenerative joint diseases such as arthritis.

Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes.

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation.