Absence Of Insulin-like Growth Factor-1 Research Articles

Increased Insulin-like Growth Factor 1 Activity Can Rescue KLE Endometrial-like Cells from Apoptosis

The peritoneal fluid (PF) of women with endometriosis contains protease(s) activity able to hydrolyze insulin-like growth factor-binding protein 3 (IGFBP-3), increasing the bioavailability of insulin-like growth factor-1 (IGF-1) locally. Therefore, we characterized the effects of IGF-1 on KLE endometrial-like cells in vitro. The mitogenic effect of IGF-1 was assessed by the analysis of the DNA content and cell count. Apoptosis was triggered experimentally by the 48 hr exposure of KLE cells to 100 nM of adriamycin in the presence and absence of IGF-1 (50 ng/ml). Adriamycin apoptosis of KLE cells was determined by the number of dead KLE cells using trypan blue exclusion and by the DNA fragmentation on simple agarose gel and flow cytometry of propidium iodide and HOECHST 33342-stained KLE cells using an EPICS 753 pulse cytometer. IGF-1 stimulated the growth of KLE cells in a dose-dependent manner (optimal dose of 50 ng/ml) and protected KLE cells from adriamycin (100 nM)-induced apoptosis. These data suggest that IGF-1 is a survival factor for KLE cells. Conceivably, increased IGF-1 activity in the PF can optimize both the survival and ectopic growth of endometrial cells in the peritoneal cavity.

Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells.

Wild-type (F/B), constitutively active (F/B*), and three kinase-inactive (F/Ba-, F/Bb-, F/Bc-) forms of Akt/protein kinase B (PKB) were permanently overexpressed in FDCP2 cells. In the absence of insulin-like growth factor-1 (IGF-1), activities of PKB, cyclic nucleotide phosphodiesterase 3B (PDE3B), and PDE4 were similar in nontransfected FDCP2 cells, mock-transfected (F/V) cells, and F/B and F/B- cells. In F/V cells, IGF-1 increased PKB, PDE3B, and PDE4 activities approximately 2-fold. In F/B cells, IGF-1, in a wortmannin-sensitive manner, increased PKB activity approximately 10-fold and PDE3B phosphorylation and activity ( approximately 4-fold), but increased PDE4 to the same extent as in F/V cells. In F/B* cells, in the absence of IGF-1, PKB activity was markedly increased ( approximately 10-fold) and PDE3B was phosphorylated and activated (3- to 4-fold); wortmannin inhibited these effects. In F/B* cells, IGF-1 had little further effect on PKB and activation/phosphorylation of PDE3B. In F/B- cells, IGF-1 activated PDE4, not PDE3B, suggesting that kinase-inactive PKB behaved as a dominant negative with respect to PDE3B activation. Thymidine incorporation was greater in F/B* cells than in F/V cells and was inhibited to a greater extent by PDE3 inhibitors than by rolipram, a PDE4 inhibitor. In F/B cells, IGF-1-induced phosphorylation of the apoptotic protein BAD was inhibited by the PDE3 inhibitor cilostamide. Activated PKB phosphorylated and activated rPDE3B in vitro. These results suggest that PDE3B, not PDE4, is a target of PKB and that activated PDE3B may regulate cAMP pools that modulate effects of PKB on thymidine incorporation and BAD phosphorylation in FDCP2 cells.

Abnormal regulation of urokinase plasminogen activator by insulin-like growth factor 1 in human osteoarthritic subchondral osteoblasts.

Subchondral bone sclerosis is a common feature of osteoarthritis (OA), but the mechanisms responsible for this condition remain unresolved. We investigated the role of insulin-like growth factor 1 (IGF-1) and urokinase plasminogen activator (uPA) in human osteoblasts from subchondral bone obtained from the tibial plateaus of OA patients and normal individuals. Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients at surgery and from normal individuals at autopsy. Levels of uPA and PA inhibitor 1 (PAI-1) levels were determined under basal conditions and after IGF-1 stimulation in conditioned media from osteoblasts by enzyme-linked immunosorbent assay. The activity of uPA was evaluated by specific substrate hydrolysis and zymography under basal conditions and after plasminogen stimulation, in the presence and absence of added IGF-1. Plasmin activity was also evaluated by specific substrate hydrolysis. Levels of uPA released by OA osteoblasts were significantly higher than normal. Addition of IGF-1 to osteoblasts significantly reduced uPA protein levels only in OA patients (P < 0.05). In contrast, the addition of uPA to osteoblasts did not modify IGF-1 levels in either normal or OA osteoblasts. Basal uPA activity was higher in OA than in normal osteoblasts. Interestingly, IGF-1 enhanced basal uPA activity in OA specimens in a dose-dependent manner. Addition of plasminogen promoted uPA activity in both normal and OA osteoblasts via a positive feedback loop due to plasmin generation, since this activity was inhibited by both PAI-1 and alpha2-antiplasmin. Unexpectedly, incubation with IGF-1 inhibited this positive feedback of plasminogen-dependent uPA activity in OA osteoblasts, but not in normal osteoblasts, in a dose-dependent manner. Hence, normal osteoblasts were relatively insensitive to IGF-1, whereas the same treatment reduced both uPA levels and plasminogen-dependent uPA activity in OA osteoblasts while it increased basal uPA activity in OA osteoblasts. This could not be explained by PAI-1 protein levels, which were similar in normal and OA osteoblasts in the presence and absence of IGF-1. IGF-1 also reduced plasmin activity in OA osteoblasts while it did not modify this activity in normal osteoblasts. These results suggest that in OA osteoblasts, the uPA/plasmin system functions normally, yet IGF-1 inhibits the positive feedback of plasmin on uPA activity. This inhibition may contribute to abnormal IGF-1- and uPA-dependent bone remodeling, ultimately leading to abnormal bone sclerosis in OA.

Insulin-like growth factor-1 modulation of intestinal epithelial cell restitution.

After superficial intestinal injury, the mucosal integrity is reestablished by rapid migration of epithelial cells from the adjacent area in a process called restitution. Our previous study suggested that growth hormone improves intestinal healing in an experimental small bowel ulceration, mediated by insulin-like growth factor-1 (IGF-1). The aim of the present study was to assess the role of IGF-1 in mucosal epithelial restitution using an in vitro epithelial wound model. Wounds were established in confluent monolayers of the intestinal cell line, IEC-6. Migration was quantitated in the presence or absence of IGF-1 as the number of cells migrating across the wound edge. Proliferation was assessed by thymidine incorporation. IGF-1-enhanced epithelial cell migration by 2- to 2.5-fold after 12- and 24-hour treatment, respectively, the first step involved in gastrointestinal wound healing. Cell proliferation was significantly stimulated by IGF-1 as well. In addition, expression of transforming growth factor-beta (TGF-beta) mRNA was significantly enhanced in the wounded monolayers treated with IGF-1. IGF-1 receptor mRNA was found to be detectable throughout the gastrointestinal mucosa and in the intestinal epithelial cells. In conclusion, these findings suggest that IGF-1 plays an important role in reconstitution of intestinal epithelial integrity after mucosal injury.

Up-Regulation of Integrin α5 by a C-Terminus Four-Amino-Acid Sequence of Substance P (Phenylalanine-Glycine-Leucine-Methionine-Amide) Synergistically with Insulin-like Growth Factor-1 in SV-40 Transformed Human Corneal Epithelial Cells

We previously reported that substance P and insulin-like growth factor-1 (IGF-1) synergistically facilitate corneal epithelial migrationin vivoandin vitro.We investigated whether the substance P-derived tetrapeptide (phenylalanine-glycine-leucine-methionine-amide, FGLM) can up-regulate the receptors for fibronectin and cellular adhesion to fibronectin matrix in the presence or absence of IGF-1 in cultured SV-40 transformed human corneal epithelial (HCE) cells. The combination of both FGLM and IGF-1 and substance P and IGF-1 significantly increased the number of cells adhered to the fibronectin matrix and the expression of integrin α5. The synergistic effect of FGLM and IGF-1 on the adhesion of HCE cells to the fibronectin matrix is partly carried through up-regulation of integrin α5 expression in HCE cells. These results showed that the four-amino-acid sequence at the C-terminus of substance P completely mimics the substance P molecule in terms of synergism with IGF-1 on adhesion of epithelial cells to the fibronectin matrix.