Adisak Suwanichkul

Insulin-like Growth Factor-binding Protein-3 Binds Fibrinogen and Fibrin

Following tissue injury, a fibrin network formed at the wound site serves as a scaffold supporting the early migration of stromal cells needed for wound healing. Growth factors such as insulin-like growth factor-I (IGF-I) concentrate in wounds to stimulate stromal cell function and proliferation. The ability of IGF-binding proteins (IGFBPs) such as IGFBP-3 to reduce the rate of IGF-I clearance from wounds suggests that IGFBP-3 might bind directly to fibrinogen/fibrin. Studies presented here show that IGFBP-3 does indeed bind to fibrinogen and fibrin immobilized on immunocapture plates, withK d values = 0.67 and 0.70 nm, respectively, and competitive binding studies suggest that the IGFBP-3 heparin binding domain may participate in this binding. IGF-I does not compete for IGFBP-3 binding; instead, IGF-I binds immobilized IGFBP-3·fibrinogen and IGFBP-3·fibrin complexes with affinity similar to that of IGF-I for the type I IGF receptor. In the presence of plasminogen, most IGFBP-3 binds directly to fibrinogen, although 35–40% of the IGFBP-3 binds to fibrinogen-bound plasminogen. IGFBP-3 also binds specifically to native fibrin clots, and addition of exogenous IGFBP-3 increases IGF-I binding. These studies suggest that IGF-I can concentrate at wound sites by binding to fibrin-immobilized IGFBP-3, and that the lower IGF affinity of fibrin-bound IGFBP-3 allows IGF-I release to type I IGF receptors of stromal cells migrating into the fibrin clot.

Plasminogen binds the heparin-binding domain of insulin-like growth factor-binding protein-3

Limited proteolysis lowers affinity of insulin-like growth factor (IGF)-binding protein (IGFBP)-3 for bound IGFs, resulting in greater IGF bioavailability. Plasmin is one of many proteases that cleave IGFBP-3, and the plasmin system may regulate IGFBP-3 proteolysis and IGF bioavailability in cultured cells in vitro. A role for the plasmin system in IGFBP-3 proteolysis in vivo is suggested by data presented here showing that IGFBP-3 binds plasminogen (Pg; Glu-Pg) with a dissociation constant (Kd) ranging from 1.43 to 3.12 nM. IGF-I and Glu-Pg do not compete for IGFBP-3 binding; instead, the binary IGFBP-3/Glu-Pg complex binds IGF-I with high affinity (Kd = 0. 47 nM) to form a ternary complex. Competitive binding studies suggest that the kringle 1, 4, and 5 domains of Glu-Pg and the heparin-binding domain of IGFBP-3 participate in forming the IGFBP-3/Glu-Pg complex, and other studies show that Glu-Pg in this complex is activated at a normal rate by tissue Pg activator. Importantly, IGFBP-3/Glu-Pg complexes were detected in both human citrate plasma and serum, indicating that these complexes exist in vivo. Binding of IGFBP-3 to Glu-Pg in vivo suggests how Glu-Pg activation can specifically lead to IGFBP-3 proteolysis with subsequent release of IGFs to local target tissues.Limited proteolysis lowers affinity of insulin-like growth factor (IGF)-binding protein (IGFBP)-3 for bound IGFs, resulting in greater IGF bioavailability. Plasmin is one of many proteases that cleave IGFBP-3, and the plasmin system may regulate IGFBP-3 proteolysis and IGF bioavailability in cultured cells in vitro. A role for the plasmin system in IGFBP-3 proteolysis in vivo is suggested by data presented here showing that IGFBP-3 binds plasminogen (Pg; Glu-Pg) with a dissociation constant ( K d) ranging from 1.43 to 3.12 nM. IGF-I and Glu-Pg do not compete for IGFBP-3 binding; instead, the binary IGFBP-3/Glu-Pg complex binds IGF-I with high affinity ( K d= 0.47 nM) to form a ternary complex. Competitive binding studies suggest that the kringle 1, 4, and 5 domains of Glu-Pg and the heparin-binding domain of IGFBP-3 participate in forming the IGFBP-3/Glu-Pg complex, and other studies show that Glu-Pg in this complex is activated at a normal rate by tissue Pg activator. Importantly, IGFBP-3/Glu-Pg complexes were detected in both human citrate plasma and serum, indicating that these complexes exist in vivo. Binding of IGFBP-3 to Glu-Pg in vivo suggests how Glu-Pg activation can specifically lead to IGFBP-3 proteolysis with subsequent release of IGFs to local target tissues.

Effect of chronic renal failure and growth hormone therapy on the insulin-like growth factors and their binding proteins.

Abstract Children with chronic renal failure (CRF) are often growth retarded, and abnormalities of the growth hormone (GH)/insulin-like growth factor (IGF) axis in CRF may contribute to this poor growth. Despite normal IGF levels in CRF serum, IGF bioactivity is low due to excess IGF-binding proteins (IGFBPs) in the 35-kDa serum fractions. Levels of IGFBP-1, -2, -4 and -6, and a 29-kDa IGFBP-3 fragment, are high in CRF serum, and levels of intact IGFBP-1 and -2 correlate negatively with height. IGFBP-1 levels may be high due to insulin resistance, suggesting that the FKHR family of transcription factors may play a role in the overexpression of IGFBP-1, and other growth inhibitors, in CRF. GH-treated CRF children show catch-up growth that correlates positively with a rise in each component of the 150-kDa serum ternary complex (IGF-I or -II/IGFBP-3 or -5/acid-labile subunit); IGFBP-1, -2 and -6 levels do not rise, but serum IGF bioactivity does. Thus, GH increases levels of IGFs and ternary complexes in CRF serum. It is likely that increased IGFs contribute to catch-up growth by overcoming the inhibitory effects of excess IGFBPs present in the CRF milieu.

Insulin-like growth factor (IGF) suppression of IGFBP-1 production: evidence for mediation by the type I IGF receptor

The regulation of insulin-like growth factor binding protein-1 (IGFBP-1) by its ligands, IGF-I and IGF-II, was studied in continuous cultures of HepG2 human hepatoma cells. Both IGF-I and IGF-II in concentrations as low as 1-10 nmol/l caused significant suppression of IGFBP-I protein levels. This suppression was accompanied by decreased IGFBP-1 mRNA levels occurring within 2-4 h of exposure to IGF-I or IGF-II, and by a significant decrease in IGFBP-1 promoter activity. IGF-I and IGF-II were equipotent in suppressing basal levels of IGFBP-1 protein, mRNA and promoter activity. IGF-I, IGF-II, and IGF-analogs with low IGFBP-1 affinity, (des 1-3)IGF-I and long R3IGF-I, all potently suppressed the previously characterized increase in IGFBP-1 protein levels and promoter activity induced by cAMP and theophylline. In contrast, [Leu-27]IGF-II, which interacts with the type II but not type I IGF receptor, had no effect on IGFBP-1 protein levels or promoter activity. Our data indicate that IGFBP-1 production is inhibited by its ligands, IGF-I and IGF-II, and that this effect is probably mediated at the transcriptional level. The effects of IGF-I and IGF-II apparently occur as a result of binding to the type I IGF receptor, and are similar to the previously characterized suppressive effects of insulin on IGFBP-1 transcription mediated through the insulin receptor. When considered with previous data regarding expression of IGFBP-1 and the type I IGF receptor, our results suggest that IGF regulation of IGFBP-1 may play an as yet undefined role in fetal development and postnatal hepatic regeneration.

Multihormonal regulation of IGFBP-1 promoter activity

Insulin-like growth factor binding protein-1 (IGFBP-1) is a 25 kiloDalton protein which can compete with IGF receptors for binding of IGF-I and IGF-II peptides. Such high affinity binding allows IGFBP-1 to influence IGF action; IGFBP-1 can inhibit or potentiate the effects of IGF peptides depending on experimental conditions and on post-translational modifications of this binding protein (1). IGFBP-1 is expressed in a tissue-specific manner, with significant expression essentially limited to liver and uterus in most individuals (2,3).

Differential expression of functional Fc-receptors and additional immune complex receptors on mouse kidney cells

The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Ig binding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury.

High titer anti-basement membrane antibodies in a subset of patients with pediatric systemic lupus erythematosus.

Background/Aims: There is a critical need for more noninvasive biomarkers to identify nephritis in patients with systemic lupus erythematosus (SLE). Recent studies in a model mouse and an adult SLE patient cohort suggest that anti-basement membrane antibody levels correlate well with lupus activity and kidney injury. The purpose of this study was to assess the anti-basement membrane reactivity in pediatric SLE (pSLE) patients with or without nephritis. Methods: Auto-antibodies to basement membrane antigens were assessed using an anti-matrigel ELISA. Endpoint titers were measured in pSLE patients and healthy children, as well as in autoimmune and non-immune mice, with good reproducing capabilities. Findings were also analyzed with respect to the presence or absence of nephritis, dsDNA antibodies, and other manifestations of pSLE. Results: MRL/lpr mice developed high-titer anti-matrigel antibodies, whereas C57BL/6 mice did not. In a cohort of 21 pSLE patients and 22 pediatric controls, high-titer anti-matrigel IgG, IgM and IgA antibody levels were specific for pSLE. High-titer anti-matrigel IgG3 levels could distinguish with good sensitivity the 13 pSLE patients with a history of nephritis from the 8 non-renal pSLE patients. High-titer anti-matrigel IgG, IgA, IgM or IgG3 did not correlate with positive anti-double stranded DNA, but defined an overlapping subset of patients. Conclusion: The addition of anti-basement membrane antibody testing to serologic testing in pSLE may help to monitor disease activity or to define important subsets of patients with risks for specific disease manifestations.