MDCK cells secrete neutral proteases cleaving insulin-like growth factor-binding protein-2 to -6

Abstract

Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may be an important mechanism to regulate IGF availability and IGF-independent functions of IGFBPs. We analyzed the secretion of IGFBP proteases in Madin-Darby canine kidney (MDCK) cells. The results showed that several specific proteases were secreted, cleaving IGFBP-2 to -6 at neutral pH. The proteolytic activity against IGFBP-6 differed at least from IGFBP-5 protease activity in its sensitivity both to IGF-II and to the hydroxamic acid-based disintegrin metalloprotease inhibitor, as well as serine protease inhibitors. During partial purification steps, the serine protease inhibitor-sensitive fraction with IGFBP-6 protease activity was separated from fractions characterized by the presence of a 30-kDa disintegrin immunoreactive band. Whereas the IGFBP-4 and -6 proteases are predominantly secreted across the basolateral membrane, the majority of IGFBPs are sorted to the apical medium from filter-grown cells. These studies indicate that the side-specific secretion of several distinct IGFBP proteases with partially overlapping IGFBP specificities may be another level in the regulation of IGF-dependent epithelial functions.

Multiple post-translational modifications of mouse insulin-like growth factor binding protein-6 expressed in epithelial Madin–Darby canine kidney cells

“…The strips were rehydrated with 0.2 ml of rehydration buffer containing 8 M urea, 2% (w/v) Chaps and 0.5% (v/v) IPG buffer at room temperature for 16 hours, as recommended by the manufacturer. Proteins precipitated with acetone from conditioned media (Shalamanova et al, 2001) were resuspended in 0.1 ml of rehydration buffer and applied on IPG gels. Isoelectric focusing was carried out for 18,100 Volt-hours (200 V x 30 min, 3000 V x 6 h) at 20°C.…”

“…The endogenous IGFBPs in MDCK cells are preferentially secreted to the apical side of the cells, whereas proteases are delivered to the basolateral medium (Shalamanova et al, 2001). We M a n u s c r i p t A c c e p t e d M a n u s c r i p t M a n u s c r i p t M a n u s c r i p t M a n u s c r i p t .…”

“…In contrast, N-and O-glycosylation appear to be non-essential for binding of IGFBP-3 or IGFBP-6 to IGFs, but have the potential to modulate cell surface binding and binding to extracellular glycosaminoglycans, respectively (Firth and Baxter, 1999;Marinaro et al, 2000b). At present, it is unclear whether post-translational modifications play a role in transport of IGFBPs in the secretory pathway or in differential sorting and M a n u s c r i p t Shalamanova et al 4 secretion of IGFBPs in polarized cells (Remacle-Bonnet et al, 1995;Shalamanova et al, 2001). …”

“…A c c e p t e d M a n u s c r i p t (Shalamanova et al, 2001). Serum-free medium conditioned for 24-72 hours was collected as described earlier (Shalamanova et al, 2001).…”

“…Serum-free medium conditioned for 24-72 hours was collected as described earlier (Shalamanova et al, 2001). Cells were transfected with 10 µg of pBEH plasmid and 1 µg of the pGK Hygro (hygromycin resistance) plasmid using the calcium phosphate technique.…”