Tumour necrosis factor alpha-stimulated gene-6 inhibits osteoblastic differentiation of human mesenchymal stem cells induced by osteogenic differentiation medium and BMP-2.

To better understand the molecular pathogenesis of OPLL (ossification of the posterior longitudinal ligament) of the spine, an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients. We found that TSG-6 (tumour necrosis factor alpha-stimulated gene-6) is down-regulated during osteoblastic differentiation. Adenovirus vector-mediated overexpression of TSG-6 inhibited osteoblastic differentiation of human mesenchymal stem cells induced by BMP (bone morphogenetic protein)-2 or OS (osteogenic differentiation medium). TSG-6 suppressed phosphorylation and nuclear accumulation of Smad 1/5 induced by BMP-2, probably by inhibiting binding of the ligand to the receptor, since interaction between TSG-6 and BMP-2 was observed in vitro. TSG-6 has two functional domains, a Link domain (a hyaluronan binding domain) and a CUB domain implicated in protein interaction. The inhibitory effect on osteoblastic differentiation was completely lost with exogenously added Link domain-truncated TSG-6, while partial inhibition was retained by the CUB domain-truncated protein. In addition, the inhibitory action of TSG-6 and the in vitro interaction of TSG-6 with BMP-2 were abolished by the addition of hyaluronan. Thus, TSG-6, identified as a down-regulated gene during osteoblastic differentiation, suppresses osteoblastic differentiation induced by both BMP-2 and OS and is a plausible target for therapeutic intervention in OPLL.

Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49-99 of beta-amyloid precursor protein.

A novel cleavage of beta-amyloid precursor protein (APP), referred to as epsilon-cleavage, occurs downstream of the gamma-cleavage and generates predominantly a C-terminal fragment (CTFgamma) that begins at Val-50, according to amyloid beta-protein (Abeta) numbering. Whether this cleavage occurs independently of, or is coordinated with, gamma-cleavage is unknown. Using a cell-free system, we show here that, although Abeta40 and CTFgamma 50-99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFgamma 49-99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Abeta42. Furthermore, a gamma-secretase inhibitor, which suppresses Abeta40 production and paradoxically enhances Abeta42 production at low concentrations, caused the proportion of CTFgamma 50-99 to decrease and that of CTFgamma 49-99 to increase significantly. These results strongly suggest a link between the production of Abeta42 and CTFgamma 49-99 and provide an important insight into the mechanisms of altered gamma-cleavage caused by mtAPP and mtPS1/2.

Distinct mechanisms by mutant presenilin 1 and 2 leading to increased intracellular levels of amyloid beta-protein 42 in Chinese hamster ovary cells.

To characterize the properties of presenilin (PS) 1- and PS2-associated gamma-secretases, we established stable transfectants overexpressing amyloid precursor protein and wild-type (wt) or a number of mutant (mt) PS1 or PS2. Quantification of the intracellular amyloid beta-protein (Abeta) levels in mtPS1 and mtPS2 cell lines revealed the presence of two subtypes. One group consists of N141I, M239V, and T122P mutations of the PS2 gene and homologous mutations of PS1, N135D and M233T. These mutations led to an increase in the intracellular Abeta42 levels and a concomitant decrease in the intracellular Abeta40 levels. A cell-free assay for Abeta production using the membranes prepared from these transfectants exhibited predominant cleavage at position Abeta42 with marginal production of Abeta40. The other group consists of M146L, H163R, and G384A mutations of PS1, leading only to an increase in the intracellular Abeta42 levels. While the intracellular Abeta levels in M146L cells were consistent with the results from cell-free Abeta production, H163R and G384A cells showed significant discrepancies between the intracellular Abeta levels and cell-free Abeta production. Thus, all the mtPS1/2 examined here result in increases in the intracellular Abeta42 levels. This suggests that the underlying mechanisms for this shared phenotype may be diverse.