The molecular and cellular basis of exostosis formation in hereditary multiple exostoses.

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

Signal transducer and activator of transcription-1 (STAT1), but not STAT5b, regulates IGF-I receptor gene expression in an osteosarcoma cell line.

The IGF-I receptor (IGF-IR) exhibits potent mitogenic, antiapoptotic, and transforming activities. Previous studies have suggested that the expression of the IGF-IR gene is negatively regulated by certain cytokines, including interferon-gamma (IFN-gamma). The potential involvement of STAT proteins in transcriptional regulation of the IGF-IR gene by IFN-gamma was addressed by transient coexpression of vectors encoding STAT1 and STAT5b, together with an IGF-IR promoter luciferase reporter, in the osteosarcoma-derived cell line Saos-2. Physical interactions between IFN-gamma-induced transcription factors and the IGF-IR promoter region were examined by electrophoretic mobility shift assays (EMSA). The results obtained indicate that the mechanism of action of IFN-gamma involves stimulation of STAT1 which, in turn, binds IFN-gamma activation sites (GAS) in the IGF-IR regulatory region, thus suppressing promoter activity. Taken together, our results suggest that the IGF-IR gene is a novel target for STAT1 action and that at least part of the inhibitory effects of STAT1 may involve repression of the strongly antiapoptotic IGF-IR gene.

BRCA1-Sp1 interactions in transcriptional regulation of the IGF-IR gene.

The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in breast tumorigenesis and is overexpressed in most primary tumors. BRCA1 is a transcription factor involved in numerous cellular processes, including DNA damage repair, cell growth, and apoptosis. Consistent with its tumor suppressor role, we demonstrated that BRCA1 repressed the activity of co-transfected IGF-IR promoter reporter constructs in a number of breast cancer-derived cell lines. Results of electrophoretic mobility shift assay showed that BRCA1 did not exhibit any specific binding to the IGF-IR promoter, although it prevented binding of Sp1. Co-immunoprecipitation experiments demonstrated that BRCA1 action was associated with specific interaction with Sp1 protein. Furthermore, using a series of glutathione S-transferase-tagged BRCA1 fragments, we mapped the Sp1-binding domain to a segment located between aa 260 and 802. In summary, our data suggest that the IGF-IR gene is a novel downstream target for BRCA1 action.

The short-lived exostosis induced surgically versus the lasting genetic hereditary multiple exostoses.

Hereditary osteochondromas are often caused by mutation in the EXT1 gene. The lesions are typified by formation of a "pseudo" growth plate like lesion growing at 60 degrees to the normal growth direction of the bone. Such lesions can be mimicked surgically by reverting the position--the polarity of the zone of LaCroix. The current study attempts to compare the pathology between EXT1 gene expression in humans and surgically created osteochondroma pathology in a rat model. Tissues of human bunion, human embryonal tissue, and human adult cartilage as well as normal rat epiphyses served as controls. Rats were operated on and a 60 degree span of the ring of LaCroix was inverted as described by Delgado (Delgado, E., Rodriguez, J. I., Serada, A., Tellez, M., and Pariagoa, R.. Clin. Orthop. 201, 251-258 (1985)). The surgically created osteochondromas were assessed by histology, histochemistry, and immunohistochemistry. The findings show that the surgically created lesions contain only a small amount of FGF receptor 3 (FGFR3) expressed on mesenchymal stem cells located in the perichondrium, as compared to the cell population carrying FGFR3 in the contralateral limb. Indian hedgehog and Bcl2 are downregulated, while BMP-2 is overexpressed in the operated limb, compared to the LaCroix ring of the contralaetral limb. The shortage, as well as the disturbed migration routes of the residual mesenchymal stem cells in surgically created osteochondromas leads eventually to resorption of the pathological elements. In search of additional markers characterizing such pathological structures composed of mesenchymal stem cells and cartilaginous and bony cells, EXT1 gene was found to be expressed in the surgically created osteochondromas, like in normal growth plates. Nitric oxide synthase was also expressed like in adult cartilage, though tumor necrosis factor alpha typifying Bunion formation was absent. In summary, surgically created osteochondromas lack the massive and continuous population of mesenchymal stem cells with Bcl2 expression. However, the small residual mesenchymal cell population gives rise to short-lived EXT1-expressing cells that disappear eventually due to spontaneous resorption.

WT1-p53 interactions in insulin-like growth factor-I receptor gene regulation.

The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in transformation. The expression of the IGF-IR gene is negatively regulated by a number of transcription factors, including the WT1 and p53 tumor suppressors. Previous studies have suggested both physical and functional interactions between the WT1 and p53 proteins. The potential functional interactions between WT1 and p53 in control of IGF-IR promoter activity were addressed by transient coexpression of vectors encoding different isoforms of WT1, together with IGF-IR promoter-luciferase reporter constructs, in p53-null osteosarcoma-derived Saos-2 cells, wild-type p53-expressing kidney tumor-derived G401 cells, and mutant p53-expressing, rhabdomyosarcoma-derived RD cells. Similar studies were also performed to compare p53-expressing Balb/c-3T3 and clonally derived p53-null, (10)1 fibroblasts and the colorectal cancer cell line HCT116 +/+, which expresses a wild-type p53 gene, and its HCT116 -/- derivative, in which the p53 gene has been disrupted by homologous recombination. WT1 splice variants lacking a KTS insert between zinc fingers 3 and 4 suppressed IGF-IR promoter activity in the absence of p53 or in the presence of wild-type p53. WT1 variants that contain the KTS insert are impaired in their ability to bind to the IGF-IR promoter and are unable to suppress IGF-IR promoter. In the presence of mutant p53, WT1 cannot repress the IGF-IR promoter. Coimmunoprecipitation experiments showed that p53 and WT1 physically interact, whereas electrophoretic mobility shift assay studies revealed that p53 modulates the ability of WT1 to bind to the IGF-IR promoter. In summary, the transcriptional activity of WT1 proteins and their ability to function as tumor suppressors or oncogenes depends on the cellular status of p53.

Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS-WT1 fusion protein.

The EWS family of genes is involved in numerous chromosomal translocations that are characteristic of a variety of sarcomas. A recently described member of this group is desmoplastic small round cell tumor (DSRCT), which is characterized by a recurrent t(11;22)(p13;q12) translocation that fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. The originally described chimera comprises exons 1-7 of EWS and exons 8-10 of WT1. We have previously reported that the WT1 protein represses the expression of the IGF-I receptor gene, whereas the EWS(1-7)-WT1(8-10) fusion protein activates IGF-I receptor gene expression. It has recently become apparent that EWS-WT1 chimeras produced in DSCRT are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We have recently characterized additional EWS-WT1 translocations that involve the juxtaposition of EWS exons 7 or 8 to WT1 exon 8, and an EWS-WT1 chimera that lacks EWS exon 6. The chimeric transcription factors encoded by these various translocations differ in their DNA-binding characteristics and their ability to transactivate the IGF-I receptor promoter. These data suggest that the molecular pathology of DSRCT is more complex than previously appreciated, and that this diversity may provide the foundation for predictive genotype-phenotype correlations in the future.

Use of confocal microscopy for the study of spermatogenesis.

Spermatogenesis consists of spermatogonial proliferation, meiosis and spermatid differentiation. Laser scanning confocal microscopy (LSCM) may be used as an advanced analytical tool to follow spermatogenesis inside the seminiferous tubules without performing histological sections. For this purpose, separated seminiferous tubules are fixed in 0.5% paraformaldehyde, stained for DNA with propidium iodide and analyzed by LSCM. By producing longitudinal optical sections in the layer of spermatogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followed within the tubules by LSCM. Longitudinal z-sections may be obtained to produce three-dimensional images of the seminiferous tubules. In addition, different proteins may be followed during spermatogenesis in a stage specific manner within the tubule by incubation of the fixed seminiferous tubules with appropriate antibodies. As an example of the spermatogenesis studies using described LSCM techniques, detailed examination of spermatogonia, spermatocytes and spermatids during golden hamster spermatogenesis is presented. LSCM analysis of c-kit and SC3 protein expression at different stages of hamster spermatogenesis is demonstrated.