Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.

An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

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Objective: Late diagnosis hinders proper management of small intestine neuroendocrine carcinoma (SI-NEC) patients. The olfactory receptor, family 51, subfamily E, member 1 (OR51E1) has been reported as a potential novel SI-NEC marker, without protein expression recognition. Thus, we further studied whether the encoded protein may be a novel SI-NEC clinical biomarker. Design: OR51E1 coding sequence was cloned using total RNA from SI-NEC patient specimens. Quantitative real-time PCR analysis explored OR51E1 expression in laser capture microdissected SI-NEC cells and adjacent microenvironment cells. Moreover, immunohistochemistry investigated OR51E1 protein expression on operation and biopsy material from primary SI-NECs, mesentery, and liver metastases from 70 patients. Furthermore, double immunofluorescence studies explored the potential co-localization of the vesicular monoamine transporter 1 (SLC18A1, generally referred to as VMAT1) and OR51E1 in the neoplastic cells and in the intestinal mucosa adjacent to the tumor. Results: OR51E1 coding sequence analysis showed absence of mutation in SI-NEC patients at different stages of disease. OR51E1 expression was higher in microdissected SI-NEC cells than in the adjacent microenvironment cells. Furthermore, both membranous and cytoplasmic OR51E1 immunostaining patterns were detected in both primary SI-NECs and metastases. Briefly, 18/43 primary tumors, 7/28 mesentery metastases, and 6/18 liver metastases were 'positive' for OR51E1 in more than 50% of the tumor cells. In addition, co-localization studies showed that OR51E1 was expressed in O50% of the VMAT1 immunoreactive tumor cells and of the enterochromaffin cells in the intestinal mucosa adjacent to the tumor. Conclusion: OR51E1 protein is a potential novel clinical tissue biomarker for SI-NECs. Moreover, we suggest its potential therapeutic molecular target development using solid tumor radioimmunotherapy.