Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions.

BACKGROUND: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. RESULTS: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. CONCLUSION: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.

Enhanced production and proteolytic degradation of insulin-like growth factor binding protein-2 in proliferating rat astrocytes.

Insulin-like growth factors (IGFs) protect neurons, are important for oligodendrocyte survival and myelin production, and stimulate the proliferation of astrocytes. The effects of IGFs are regulated by a family of IGF binding proteins (IGFBPs). Astrocytes express predominantly IGFBP-2. In the present study, primary neonatal rat astrocytes were cultivated in a chemically defined medium to initiate a differentiated cell status. After stimulation with fetal calf serum, astrocytes became hypertrophic and increased proliferation. Western blot analysis of cell lysate of proliferating astrocytes displayed an increased expression of IGFBP-2. This finding was supported by immunocytochemical images. Semiquantitative polymerase chain reaction analysis demonstrated equal mRNA levels in both differentiated and proliferating astrocytes, suggesting that the increase in IGFBP-2 production in proliferating astrocytes was exerted at the translational level. Concentrated medium of proliferating cells, however, displayed lower levels of IGFBP-2 than differentiated cells. When recombinant IGFBP-2 was incubated with culture media, we found degradation in the medium of proliferating cells, but not in medium of differentiated cells. This degradation could be inhibited with protease inhibitors, indicating that lower levels of IGFBP-2 in the medium of proliferating astrocytes are due to the presence of proteases. Our results suggest that, in proliferating astrocytes, IGFBP-2 may help target IGFs to IGF-1 receptors, and IGFBP-2 proteases may play a role in enhancing the availability of IGFs.

Insulin-like growth factor-binding protein 6 inhibits survival and differentiation of rat oligodendrocyte precursor cells.

Insulin-like growth factor 1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and promotes myelination. We demonstrate that IGF-binding protein 6 (IGFBP-6) is expressed and localized to the Golgi complex in rat oligodendrocyte precursor (O2A) cells. IGFBP-6 mRNA showed a developmentally regulated expression pattern, displaying a transient decrease during early development, and enhanced levels upon cell maturation. IGFBP-6 mRNA expression could be reduced by addition of basic fibroblast growth factor and progesterone while estrogen increased IGFBP-6 mRNA. IGF-1, platelet-derived growth factor, and insulin had no effect. When added exogenously, IGFBP-6 reduced O2A cell survival in the absence of IGF-1 and inhibited IGF-1-stimulated survival in a partially IGF-1-dependent and partially IGF-1-independent fashion. In addition, IGFBP-6 reduced the IGF-stimulated expression of two myelin proteins, CNPase and MAG. Taken together, the data show that IGFBP-6 is a new negative effector of oligodendrocyte survival and differentiation.

Insulin-like growth factor binding proteins-1 and -2 differentially inhibit rat oligodendrocyte precursor cell survival and differentiation in vitro.

Insulin-like growth factor-1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and induces myelination. Its actions are modulated by IGF binding proteins (IGFBPs) that are present in the extracellular fluids or on the cell surface. Additionally, IGFBPs are also known to exert actions that are independent of IGF-1. We studied whether IGF-binding proteins (IGFBPs)-1 and -2 modulate rat oligodendrocyte precursor (O2A) cell survival and differentiation in vitro both in the absence and presence of exogenously added IGF-1. The data reveal that IGFBP-1 and -2 reduced O2A cell survival in the absence and presence of exogenously added IGF-1. The effects of IGFBP-1 on cell survival in the presence of exogenously added IGF-1 were IGF-1-dependent, whereas IGFBP-2 displayed both IGF-1-dependent and IGF-1-independent effects. Furthermore, IGFBP-1 and -2 inhibited O2A cell differentiation in the presence of IGF-1 as reflected by decreased expression levels of two myelin proteins, CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase) and MAG (myelin associated glycoprotein). Analysis of medium samples revealed that O2A cells do not secrete proteases that degrade these IGFBPs. Taken together the data show that IGFBP-1 and -2 are negative effectors of oligodendrocyte survival and differentiation. Accordingly, the role of IGFBPs should be explicitly taken into account when investigating IGF-1 effects on oligodendrocytes, especially in the context of therapeutic purposes.