Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2003) 5 P10

BES2003 Poster Presentations Bone (13 abstracts)

The Short Stature Homeobox containing Gene (SHOX) inhibits alkaline phosphatase (ALP) production by C3H10T1/2 cells treated with retinoic acid (RA)

JC Blair , D Sitara , TP Milligan , JM Burrin & AJL Clark

Department of Endocrinology, St Bartholomews and The Royal London School of Medicine and Dentistry, London, UK.

SHOX has been implicated in the regulation of bone growth and modelling. Haploinsufficiency and heterozygous mutations of SHOX are associated with Lerri Weil Dyschondrosteosis (LWD). The cardinal features of LWD are mesomelic limb shortening and Madelung deformity. Histological studies demonstrate premature fusion of the ulna border of the radial epiphysis and disordered osteoblast maturation and orientation. In view of these observations we investigated the role of SHOX in the osteoblastic differentiation of murine multi potential cells (C3H10T1/2) stimulated with RA.
SHOX was cloned from human foetal skeletal muscle RNA by RT-PCR and subcloned into pCR 2.1-TOPO. The product was subcloned into pCDNA 3.1. PCR and direct sequencing confirmed integrity and orientation of the subcloned SHOX gene. We performed calcium phosphate transfection of C3H10T1/2 cells with pCDNA3.1 SHOX or empty vector. Stable cell lines were established by selection with neomycin. 2 x 105 wild type C3H101/2 cells, sham transfected cells or cells transfected with SHOX were plated in 6 well plates and treated with 10-6M RA for 24 hrs. Cells were collected at baseline, 24, 48 and 72 hrs following RA stimulation and stored in 1ml cell lysis buffer at -20 degC. Following sonication, ALP activity was measured by spectrophotometric analysis of 4-nitrophenylphosphate hydrolysis by ALP. ALP activity and protein concentration of cell lysate from cells transfected with SHOX was compared to wild type and sham transfected cells using students T test.
ALP activity was stimulated by RA confirming osteoblastic differentiation in all 3 cell lines. At 72 hrs, ALP activity and protein concentration was significantly lower in cells transfected with SHOX than in the other 2 cell lines (p<0.05).
These data are supportive of the hypothesis that SHOX has a role in the regulation of osteoblast differentiation and are consistent with the histological features of premature epiphyseal fusion in LWD.

Volume 5

22nd Joint Meeting of the British Endocrine Societies

British Endocrine Societies 

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