Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors.

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.

Insulin-like growth factor binding protein (IGFBP) substrate zymography. A new tool to identify and characterize IGFBP-degrading proteinases.

Insulin-like growth factor binding protein (IGFBP) degrading proteinase activities have been described in biological fluids and conditioned media from numerous cell lines. To identify and characterize IGFBP-degrading proteinases, our laboratory has developed IGFBP substrate zymography. Herein, we illustrate how IGFBP substrate zymography can be used both to identify candidate IGFBP-degrading proteinases and characterize their degradative capabilities. For this purpose, human matrix metalloproteinase-3 (MMP-3), a proteinase that degrades IGFBP-3 in human fibroblast cultures, was first electrophoresed through a polyacrylamide gel containing IGFBP-3 as substrate and then analyzed for its ability to degrade the substrate into immunoreactive fragments that were absorbed onto a polyvinylidene difluoride membrane. IGFBP-3 substrate zymography was capable of detecting as little as 20 ng of human MMP-3, demonstrating a sensitivity similar to casein substrate zymography. Using the zymogram as a template, MMP-3 was identified in a standard SDS-polyacrylamide gel run in parallel with the zymogram, and the corresponding area of the gel was excised. Electroelution of the gel slice yielded active MMP-3 when examined by casein substrate zymography. Furthermore, digestion of IGFBP-3 in solution by the electroeluted MMP-3 revealed the same fragmentation pattern of the binding protein as that produced by MMP-3, which had not been electroeluted. Together, these studies demonstrate that IGFBP substrate zymography can be a useful tool for both the identification and the characterization of IGFBP-degrading proteinases.

Heparin-binding, highly basic regions within the thyroglobulin type-1 repeat of insulin-like growth factor (IGF)-binding proteins (IGFBPs) -3, -5, and -6 inhibit IGFBP-4 degradation.

MC3T3-E1 murine osteoblasts produce insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4)-degrading proteinase activity, which is inhibited by IGFBP-3 and a highly basic, C-terminal domain of IGFBP-3. Of all the other five IGFBPs, IGFBP-5 and -6 share the highest degree of homology with this domain of IGFBP-3; therefore, we investigated whether these two IGFBPs inhibit IGFBP-4 degradation. Both IGFBP-5 and IGFBP-6 inhibit the degradation of 125I-IGFBP-4 by MC3T3-E1-conditioned media, and their inhibitory effects are variably reversed by IGFs. Synthetic peptides containing highly basic, C-terminal regions of IGFBP-5 and IGFBP-6 inhibit 125I-IGFBP-4 degradation, as does an homologous IGFBP-3 peptide, yet each peptide displays a different IC50, with the IGFBP-5 peptide being the most potent and the IGFBP-6 peptide being the least potent. In contrast, a homologous, yet neutral, IGFBP-4 peptide does not inhibit 125I-IGFBP-4 proteolysis, confirming the role of basic residues in the inhibitory process. The IGFBP-3, -5, and -6 peptides, each of which contains the heparin-binding consensus sequence XBBBXXBX, bind heparin, yet the IGFBP-3 and -5 peptides bind heparin with the highest affinities, whereas the IGFBP-6 peptide binds heparin with approximately 10-fold less affinity. Consistent with these regions being involved in proteinase inhibition, heparin completely reverses their inhibitory effects on 125I-IGFBP-4 proteolysis. Together, these data demonstrate that IGFBP-3, -5, and -6 can function as IGF-reversible inhibitors of IGFBP-4 proteolysis, likely through homologous, highly basic, heparin-binding domains contained within the conserved thyroglobulin type-1 motif present in the C-termini of these IGFBPs.

Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3.

Matrix metalloproteinase 3 cleaves insulin-like growth factor-binding protein-3 (IGFBP-3) into six fragments, four of which bind heparin-Sepharose (Fowlkes, J. L., Enghild, J. J., Suzuki, K., and Nagase, H. (1994) J. Biol. Chem. 269, 25742-25746). Sequence analysis of IGFBP-3 heparin-binding fragments shows that all fragments contain at least one of two highly basic, putative heparin-binding consensus sequences present in IGFBP-3. Epitope-specific antibodies generated against synthetic peptides containing these domains recognized IGFBP-3, yet were significantly inhibited from binding in the presence of heparin, demonstrating that these regions of IGFBP-3 contain functional heparin-binding domains. IGFBP-3 peptides containing one of the two heparin-binding consensus sequences bound heparin in a solid phase binding assay in a dose-dependent and saturable manner. However, the IGFBP-3 peptide containing the heparin-binding consensus sequence 149KKGHA153 bound heparin with approximately 4-fold less affinity than the IGFBP-3 peptide containing the longer heparin-binding consensus sequence 219YKKKQCRP226. Examination of several well characterized glycosaminoglycans to inhibit the binding of heparin to both heparin-binding IGFBP-3 peptides revealed that the most potent inhibitors were heparin, heparan sulfate, and dermatan sulfate; chondroitin sulfate A and hyaluronic acid were intermediate in their inhibitory activities; and chondroitin sulfate C caused no inhibition. These studies identify and characterize the glycosaminoglycan-binding domains in IGFBP-3, providing a basis for the better understanding of IGFBP-3-glycosaminoglycan interactions at the cellular and extracellular interface.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) functions as an IGF-reversible inhibitor of IGFBP-4 proteolysis.

Previous studies have shown that insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is degraded only in the presence of exogenous IGFs; however, we found that cation-dependent proteinase activity present in conditioned medium of MC3T3-E1 osteoblasts degrades 125I-recombinant human (rh)IGFBP-4 in the absence of IGFs. Addition of IGF-I, IGF-II, or insulin to conditioned medium had little affect on 125I-rhIGFBP-4 proteolysis, while extraction of IGFs resulted in only a approximately 10% reduction in proteinase activity. Since factors other than IGFs appeared to be involved in regulating IGFBP-4 proteolysis, we hypothesized that IGFBP-3, an IGFBP produced by many cell lines, but not MC3T3-E1 cells, might function as an inhibitor of IGFBP-4 proteolysis. Addition of rhIGFBP-3 to conditioned media inhibited 125I-rhIGFBP-4 proteolysis by 90%, while IGF-I and IGF-II reversed the inhibitory effects of rhIGFBP-3 in a dose-dependent manner. 125I-rhIGFBP-4 proteolysis was not inhibited by N-terminal rhIGFBP-3 fragments that bind IGFs, but was inhibited by two synthetic peptides corresponding to sequences contained in the mid-region or C-terminal region of IGFBP-3. Both inhibitory peptides contain highly basic, putative heparin-binding domains and heparin partially reversed the inhibitory effects of rhIGFBP-3 on 125I-rhIGFBP-4 proteolysis. These data demonstrate that rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity and binding of IGFs or glycosaminoglycans to IGFBP-3 may induce conformational changes in the binding protein, causing disinhibition of the proteinase.

Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is uniquely regulated throughout MC3T3-E1 osteoblast differentiation: IGFBP-5 is first detectable in conditioned medium (CM) of replicating preosteoblasts (day 5); IGFBP-5 levels peak between culture days 8-12, then decline to almost undetectable levels in mature osteoblast cultures (> day 18) despite the persistence of IGFBP-5 messenger RNA. These observations suggest that IGFBP-5 concentrations may be regulated by posttranslational mechanisms. To determine whether proteolysis contributes to the disappearance of IGFBP-5 in CM of mature osteoblasts, serial samples of MC3T3-E1 cell CM obtained during a 30-day culture period were analyzed for IGFBP-5-degrading protease activity. Using [125I]recombinant human IGFBP-5 substrate zymography, we demonstrated that proteases with M(r) of 52-72 and 97 kilodaltons (kDa) were present in CM, and protease activity increased in concentration as cultures matured. The 52- to 72-kDa proteases were cation dependent and were inhibited by tissue inhibitor of metalloproteinase 1, a specific inhibitor of matrix metalloproteinases (MMPs), identifying them as MMPs. Furthermore, antisera to human MMP-1 and -2 immunoprecipitated IGFBP-5-degrading proteases with M(r) of 52 and 69/72 kDa, respectively, suggesting that homologous murine MMPs degrade IGFBP-5. Finally, MC3T3-E1 cell CM contained immunoreactive MMP-1 and -2, and MMP-2, in particular, increased significantly throughout differentiation. In contrast, the 97-kDa protease was neither inhibited by tissue inhibitor of metalloproteinase 1 nor immunoprecipitated by antisera to MMPs, suggesting that the 97-kDa protease is not a MMP. Together, these data suggest that MMPs along with an unidentified 97-kDa protease degrade IGFBP-5 in MC3T3-E1 cell cultures. Because truncated forms of IGFBP-5 have been shown to enhance the action of IGF in bone cells, IGFBP-5 proteases may be instrumental in IGF-mediated bone morphogenesis.

Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases.

Insulin-like growth factor (IGF) bioavailability is modulated by specific IGFBPs, six of which are known (IGFBPs 1-6). Since IGFBPs have equal or higher affinity for IGFs than do IGF receptors, it is believed that degradation of IGFBPs by IGFBP-degrading proteinases is an important step in regulating IGF bioactivity. Recent studies from our laboratory have demonstrated that at least two IGFBPs, i.e. IGFBP-3 and -5, are degraded under physiologic conditions by matrix metalloproteinases (MMPs). In vitro, we have demonstrated that IGFBP-3 is degraded in human dermal fibroblast cultures by MMPs using a variety of techniques, including proteinase inhibition with a specific inhibitor of MMPs, i.e. tissue inhibitor of metalloproteinases (TIMP-1), immunoabsorption with specific antisera to human MMPs and a unique method developed in our laboratory, IGFBP-3 substrate zymography. Using similar methods, we have also demonstrated that MMPs, along with an unidentified 97-kDa proteinase, degrade IGFBP-5 in murine osteoblast cultures. In rat pregnancy serum, we have shown that degradation of IGFBP-3 is associated with MMP activity present in the serum, which likely arises from the placental compartment. Analysis of the cleavage products of IGFBP-3 produced by MMPs 1, 2 and 3 reveals that MMPs cleave exclusively within the non-homologous, mid-region of the molecule. Together, these studies suggest that MMPs, beyond their previously described roles as extracellular matrix degrading enzymes, may also exert effects on cellular growth and differentiation via degradation of IGFBPs.