The purpose of this study is to investigate stage- and hormone-dependent expression of chondrocyte-derived ezrin-like domain containing protein (CDEP), a putative guanine nucleotide exchange factor (GEF) for Rho in chondrocytes, and demonstrate the guanine nucleotide exchange activity of this protein in vitro, as well as the transforming activity in NIH3T3 cells.
The expression of CDEP mRNA in growth plate chondrocytes in vivo and in vitro was examined by RT-PCR Southern analysis. The guanine nucleotide exchange activity was determined using a recombinant CDEP peptide containing the DH and PH domains in Sf9 cell lysates. The transforming activity was examined using NIH3T3 cells transiently transfected with a truncated CDEP cDNA.
CDEP mRNA was expressed at the highest level in the hypertrophic (terminal) stage of chondrocytes in vivoand in vitro. Parathyroid hormone (PTH) elicited a biphasic increase of CDEP mRNA in chondrocytes. The CDEP mRNA level increased within 1 h, then decreased nearly to the control level at 3 h. Thereafter the mRNA level started to increase at 6 h, reaching a plateau at 24 h. Dibutyryl cyclic AMP had a similar effect on CDEP expression in chondrocytes. The dissociation of [3H]GDP from RhoA was stimulated dose-dependently by Sf9 cell lysates containing the CDEP peptide. Furthermore, transfection of a truncated CDEP cDNA induced focus formation in NIH3T3 cultures.
CDEP is a novel GEF for Rho family GTPases with the transforming activity. CDEP may play a role in mediating or modulating the action of cAMP-elevating hormones on maturing chondrocytes.