In the present study we demonstrate that the promoter SNPs rs585017 and rs2602160 cooperate in increasing the transcriptional activity of RHOB in both, a chondrosarcoma line and in HeLa cells. The VNTR sequence located 820 bp upstream of the transcriptional start site also influences RHOB expression in that an elevated number of TR increases the transcriptional activity. However, this latter regulation is restricted to the chondrosarcoma line.
The synergistic effect of the promoter SNPs is likely to be provoked by conformational changes in the secondary structure of the DNA which in turn may modify the binding of transcription factors. Indeed, the presence of the rs2602160 SNP-haplotype generates a consensus recognition site for the nuclear factor 1 (NF-1) which is absent in the presence of the wildtype-haplotype. We did not find obvious transcription factor binding sites at rs585017 however, there may be cooperative binding of transcription factors which is also dependent on a conformational change at −165.
Genotype analyses revealed no association of the VNTR polymorphism with OA. However, we here show that a VNTR sequence carrying 13 repeats increases the transcriptional activity of RHOB compared to a VNTR with only 9 repeats. Our findings may seem contradictory to what has been published before (Tovar et al. 2003): Tovar et al. analyzed the effect of the VNTR sequence in combination with the SV40 promoter and did not find an impact of the number of repeats on the transcriptional activity. We here investigated the VNTR in combination with its native RHOB promoter and we used the chondrocyte-like SW1353 and HeLa cells while it was human MCF7 cells in the Tovar study. As both HeLa and MCF7 cells are of epithelial origin, a cell type restricted regulation of the VNTR polymorphism on RHOB gene expression is likely.
RHOB encodes a small GTPase which is involved in a broad range of cellular processes like vesicular transport, actin organization and apoptosis (Prendergast 2001; Ridley 2001) and indeed, we recently described a significant increase of apoptosis in RHOB overexpressing chondrocyte-like SW1353 cells (Mahr et al., submitted manuscript). Our data in combination with published reports describing significant apoptosis of the articular chondrocytes during OA (Blanco et al. 1998; Thomas et al. 2007) point at a functional role of RHOB overexpression in OA pathogenesis. Because OA is a disease of the elderly, we speculate that even a slightly enhanced RHOB expression over decades would influence the physiology of the articular chondrocytes leading to cell death and a loss of articular cartilage.
Taken together our data indicate that both the SNPs and the VNTR within the promoter region of the RHOB gene are true regulatory polymorphisms which via differential expression contribute to OA pathogenesis.