Leon A. Bach

IGF-binding proteins – the pieces are falling into place

The six insulin-like growth factor (IGF)-binding proteins (IGFBPs) are important regulators of IGF actions. IGF-independent actions of several IGFBPs have also been described. IGFBPs contain highly conserved N- and C-terminal domains, both of which are important for high-affinity IGF binding. The C-domain also binds a large number of other biomolecules, thereby modulating IGF binding and mediating IGF-independent effects. The 3D structures of the IGF-binding region of the N-domain of IGFBP-5 and the entire C-domain of IGFBP-6 have been solved recently, providing new insights into IGFBP modulation of IGF actions, and structural studies might be expected to do the same for IGF-independent actions. IGFBP-based therapies for diseases such as cancer are promising, and this recent progress will enhance their development.

Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration

Limited knowledge exists regarding the efficacy of insulin-like growth factor I (IGF-I) administration as a therapeutic intervention for muscular dystrophies, although findings from other muscle pathology models suggest clinical potential. The diaphragm muscles of mdx mice (a model for Duchenne muscular dystrophy) were examined after 8 weeks of IGF-I administration (1 mg/kg s.c.) to test the hypothesis that IGF-I would improve the functional properties of dystrophic skeletal muscles. Force per cross-sectional area was approximately 49% greater in the muscles of treated mdx mice (149.6 +/- 9.6 kN/m(2)) compared with untreated mice (100.1 +/- 4.6 kN/m(2), P < 0.05), and maintenance of force over repeated maximal contraction was enhanced approximately 30% in muscles of treated mice (P < 0.05). Diaphragm muscles from treated mice comprised fibers with approximately 36% elevated activity of the oxidative enzyme succinate dehydrogenase, and approximately 23% reduction in the proportion of fast IId/x muscle fibers with concomitant increase in the proportion of type IIa fibers compared with untreated mice (P < 0.05). The data demonstrate that IGF-I administration can enhance the fatigue resistance of respiratory muscles in an animal model of dystrophin deficiency, in conjunction with enhancing energenic enzyme activity. As respiratory function is a mortality predictor in Duchenne muscular dystrophy patients, further evaluation of IGF-I intervention is recommended.

The N-terminal Disulfide Linkages of Human Insulin-like Growth Factor-binding Protein-6 (hIGFBP-6) and hIGFBP-1 Are Different as Determined by Mass Spectrometry

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1–6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20–100-fold lower affinity. IGFBPs 1–5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.

Overexpression of insulin‐like growth factor binding protein‐6 inhibits rhabdomyosarcoma growth in vivo

Rhabdomyosarcoma is the most common soft‐tissue sarcoma of childhood. Rhabdomyosarcoma cell lines overexpress insulin‐like growth factor‐II (IGF‐II), an autocrine growth factor that is inhibited by insulin‐like growth factor binding protein‐6 (IGFBP‐6). IGFBP‐6 is associated with myoblast quiescence, and expression in rhabdomyosarcoma cells is low. The effect of IGFBP‐6 on 2 rhabdomyosarcoma cell lines, RD and Rh30, was studied. IGFBP‐6 inhibited anchorage‐dependent growth of RD and Rh30 cells in a dose‐dependent manner (p < 0.0001). IGFBP‐6 also inhibited anchorage‐independent growth of RD cells in soft agar in a dose‐dependent manner (p < 0.01). Anchorage‐independent growth of RD cells on polyhydroxyethylmethacrylate‐coated plates was decreased to a minimum of 48% of control after treatment with IGFBP‐6 (p < 0.001). In this system, IGFBP‐6 increased apoptosis 4‐fold (p < 0.001). IGF‐II partially reversed the IGFBP‐6‐induced decrease in growth and increase in apoptosis. Rh30 cells were stably transfected with an IGFBP‐6 cDNA and subcutaneous xenografts established in BALB/c nude mice. After 18 days, sizes of 2 independent clones of IGFBP‐6‐overexpressing Rh30 cells were reduced to 12% and 26% of vector control‐transfected tumors (p = 0.0006 and 0.002, respectively). IGFBP‐6 therefore inhibits proliferation and promotes apoptosis of rhabdomyosarcoma in vitro and dramatically inhibits xenograft growth in vivo, at least in part by inhibiting IGF‐II. Low expression of IGFBP‐6 may therefore contribute to rhabdomyosarcoma growth and metastasis.

Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6.

The present study examined the effects of all‐trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco‐2 human colon adenocarcinoma cells. tRA inhibited Caco‐2 cell proliferation in a dose‐dependent manner, with a 40 ± 2% decrease in cell number observed 48 h after the addition of 1 μM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco‐2 cells produced three IGFBPs of Mr: 34,000 (IGFBP‐2), 24,000 (IGFBP‐4), and 32,000 (IGFBP‐6). The concentrations of IGFBP‐2 and IGFBP‐4 decreased by 48 ± 6 and 70 ± 13%, respectively, whereas that of IGFBP‐6 increased by 698 ± 20% with 1 μM tRA. tRA decreased mRNA levels of IGFBP‐2 and IGFBP‐4 by 20 ± 3 and 50 ± 8%, respectively, whereas tRA increased IGFBP‐6 mRNA by 660 ± 20%. tRA did not alter levels of IGF‐II mRNA or peptide. To examine if endogenous IGFBP‐6 inhibits cell proliferation, Caco‐2 cells were transfected with an IGFBP‐6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP‐6 grew more slowly than vector controls and achieved final densities 30–55% lower than those of vector controls. Accumulation of IGFBP‐6 mRNA and concentrations of IGFBP‐6 peptide in conditioned media were increased by 200–250 and 220–250%, respectively, in the IGFBP‐6 clones compared with controls. Increased expression of IGFBP‐6, which has a high binding affinity for IGF‐II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP‐6‐mediated disruption of the IGF‐II autocrine loop in these colon cancer cells. J. Cell. Physiol. 190: 92–100, 2002.

Urokinase type plasminogen activator receptor is involved in insulin-like growth factor-induced migration of rhabdomyosarcoma cells in vitro.

Urokinase‐type plasminogen activator (uPA) binds to its receptor, uPAR, on the surface of cancer cells, leading to the formation of plasmin. Rhabdomyosarcoma (RMS) cell lines secrete high levels of insulin‐like growth factor II (IGF‐II), suggesting autocrine IGFs play a major role in the unregulated growth and metastasis of RMS. In vitro, IGF‐II and IGF‐I increased migration of RD cells to 124 ± 9% (P < 0.01) and 131 ± 8% (P < 0.05) of control, respectively. IGF‐II‐induced migration was abolished by insulin‐like growth factor binding protein‐6 (IGFBP‐6) (P < 0.01), a relatively specific inhibitor of IGF‐II, and by plasminogen activator inhibitor type 1 (PAI‐1) (P < 0.05). Aprotinin, a plasmin inhibitor, and mannosamine, which inhibits the synthesis of glycosylphosphatidylinositol (GPI), thereby preventing anchorage of GPI‐linked proteins such as uPAR to the cell membrane, also decreased IGF‐II‐ (P < 0.05 for both) but not IGF‐I‐induced migration. [Arg54,Arg55]IGF‐II and [Leu27]IGF‐II, which preferentially bind to the IGF‐I and IGF‐II/mannose‐6‐phosphate receptors (IGF‐II/M6PR), respectively, both induced RD cell migration to 146 ± 8% (P < 0.01) and 120 ± 7% (P < 0.05) of control, respectively. An anti‐uPAR anti‐serum reduced IGF‐II‐ and IGF‐I‐induced migration (P < 0.05 for both). An anti‐low density lipoprotein‐related protein (LRP) anti‐serum reduced IGF‐I‐induced migration (P < 0.05). IGF‐I and ‐II both increased specific 125I‐single chain uPA (scuPA) binding to RD cells in a dose‐dependent manner (P < 0.01). These results suggest involvement of the PA/plasmin system in IGF‐induced migration and indicate important roles these systems may have in RMS metastasis. J. Cell. Physiol. 197: 131–138, 2003© 2003 Wiley‐Liss, Inc.

Insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced but not basal proliferation and adhesion of LIM 1215 colon cancer cells.

IGF-II is an autocrine growth factor for many colon cancer cells. This study aimed to determine the role of IGF-II in proliferation and adhesion of LIM 1215 colon cancer cells. RT-PCR demonstrated expression of IGF-I and IGF-II mRNA. Addition of IGF-I or -II increased monolayer proliferation in a dose-dependent manner. Although addition of IGFBP-6 had no effect on basal proliferation, coincubation of IGFBP-6 decreased IGF-II but not IGF-I-induced proliferation. Colony formation in agar was increased by IGF-II, an effect inhibited by coincubation with IGFBP-6. IGFBP-6 alone significantly decreased colony formation. Preincubation of cells with IGF-II increased adhesion to type IV collagen, fibronectin and laminin. IGFBP-6 had no effect on basal cell adhesion but completely inhibited the effects of IGF-II. LIM 1215 colon cancer cells are therefore IGF-responsive but IGF-II is not a major autocrine factor for these cells in monolayer, suggesting heterogeneity between colon carcinoma cell lines with respect to the role of the IGF system.

Localization of the Insulin-like Growth Factor System in a Rat Model of Heart Failure Induced by Myocardial Infarction

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (Ml) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after Ml. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to Ml and consequent failure.

Induction of insulin-like growth factor binding protein expression by ICI 182,780 in a tamoxifen-resistant human breast cancer cell line

Earlier studies in our laboratory demonstrated that the steroidal antiestrogen ICI 182,780 is very effective in abolishing the tamoxifen‐resistant proliferation of MCF 7/5‐23 cells [1]. In addition, preliminary binding studies showed that ICI 182,780 increased the binding of insulin‐like growth factor (IGF)‐I to the MCF 7/5‐23 cells, although this finding was not the result of an increase in the expression of the insulin‐like growth factor‐I receptor (IGF‐IR). Hence, we reasoned that the inhibition of tamoxifen‐resistant cell growth by ICI 182,780 might have been due to increased expression of insulin‐like growth factor binding proteins (IGFBPs).We observed the up‐regulation of non‐insulin‐suppressible IGF‐I binding in both the tamoxifen‐sensitive MCF 7/5‐21 cell line (1.5‐fold) and the tamoxifen‐resistant MCF 7/5‐23 cell line (2.5‐fold) after 5 days of treatment with ICI 182,780 (10−7 M) in serum‐free medium, suggesting a role for cell‐associated IGFBPs. Affinity cross‐linking experiments confirmed the presence of an IGF‐I:IGFBP complex of approximately 38‐kDa in tamoxifen or ICI 182,780‐treated cells. Western ligand blots showed higher levels of a soluble 30‐kDa IGFBP in media conditioned by either of the subclones that had been treated with ICI 182,780, an effect consistently opposed by estrogen (E2:10−9 M). RT‐PCR showed higher levels of IGFBP‐5 mRNA than any of the other known IGFBPs, suggesting that this was the major IGFBP subtype. The protein was subsequently identified by Western immunoblotting as IGFBP‐5. In conclusion, we postulate that this may be a mechanism contributing to the greater potency of ICI 182,780 in the growth inhibition of the MCF 7/5‐23, tamoxifen‐resistant cell line.

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells†

The insulin‐like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co‐modulated by other growth factors including fibroblast growth factor‐2 (FGF‐2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK‐N‐MC). In serum free conditions proliferation of the SK‐N‐MC cells was promoted by IGF‐I (25 ng/ml), but blunted by FGF‐2 (50 ng/ml). IGF‐I‐induced proliferation was abolished in the presence of FGF‐2 even when IGF‐I was used at 100 ng/ml. In addition to our previously described FGF‐2 induced proteolytic cleavage of IGFBP‐2, we found that FGF‐2 increased IGFBP‐6 levels in conditioned medium (CM) without affecting IGFBP‐6 mRNA abundance. Modulation of IGFBP‐2 and ‐6 levels were not significant mechanisms involved in the blockade of IGF‐I action since the potent IGF‐I analogues [QAYL]IGF‐I and des(1–3)IGF‐I (minimal IGFBP affinity) were unable to overcome FGF‐2 inhibition of cell proliferation. FGF‐2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF‐I alone showed no morphological change. When IGF‐I was combined with FGF‐2, however, cell morphology was indistinguishable from that seen with FGF‐2 alone. FGF‐2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF‐I alone was potently anti‐apoptotic (60% decreased), IGF‐I was unable to prevent apoptosis when administrated in combination with FGF‐2. Gene‐array analysis confirmed FGF‐2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti‐apoptotic signaling. FGF‐2, directly and indirectly, overcomes the proliferative and anti‐apoptotic activity of IGF‐I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF‐I induced anti‐apoptotic signalling. Modulation of IGF binding protein abundance by FGF‐2 does not play a significant role in inhibition of IGF‐I induced mitogenesis.