Insulin-like growth factor-I stimulates H4II rat hepatoma cell proliferation: dominant role of PI-3'K/Akt signaling

Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H4II rat hepatoma cell proliferation, as estimated by 3H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H4II cells, stimulation being maximal 3 h after the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H4II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H4II cells. Altogether, these data support the notion that the stimulation of H4II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signaling.     

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.     

Progression to metastatic stage in a cellular model of prostate cancer is associated with methylation of the androgen receptor gene and transcriptional suppression of the insulin-like growth factor-I receptor gene

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF1R) expression. DNA methylation of CpG islands is an epigenetic mechanism associated with gene silencing. Recent studies have demonstrated that methylation occurs early in prostate carcinogenesis and, furthermore, may contribute to androgen independence. The methylation status of the AR and IGF1R genes was evaluated in a series of prostate cancer cell lines corresponding to early (benign) and advanced (metastatic) stages of the disease. Results of 5-Aza-2′-deoxycytidine (5-Aza) experiments, methylation-specific PCR, and sodium bisulfite-direct DNA sequencing revealed that the AR promoter is hypermethylated in metastatic M12, but not in benign P69, cells. On the other hand, no methylation was seen in the IGF1R promoter at any stage of the disease. We show, however, that 5-Aza treatment, which caused demethylation of the AR promoter, led to a significant increase in IGF1R mRNA levels, whereas addition of the AR inhibitor flutamide decreased the IGF1R mRNA levels to basal values measured prior to the 5-Aza treatment. Given that the IGF1R gene has been identified as a downstream target for AR action, our data is consistent with a model in which the AR gene undergoes methylation during progression of the disease, leading to dysregulation of AR targets, including the IGF1R gene, at advanced metastatic stages.     

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

Growth factor regulation of human growth plate chondrocyte proliferation in vitro

Linear growth occurs as the result of growth plate chondrocytes undergoing proliferative and hypertrophic phases. Paracrine feedback loops that regulate the entry of chondrocytes into the hypertrophic phase have been shown and similar pathways likely exist for the proliferative phase. Human long-bone growth plate chondrocytes were cultured in vitro. The proliferative effects of a variety of factors were determined by [3H]thymidine uptake and the gene expression profile of these cells was determined by DNA microarray analysis. Serum, insulin-like growth factor (IGF)-I and -II, transforming growth factor-beta (TGF-beta, fibroblast growth factor (FGF)-1, -2, and -18, and platelet-derived growth factor (PDGF)-BB were potent stimulators of proliferation. FGF-10, testosterone, and bone morphogenetic proteins (BMP)-2, -4, and -6 inhibited proliferation. Microarray analysis showed that the genes for multiple members of the IGF-I, TGF-beta, FGF, and BMP pathways were expressed, suggesting the presence of autocrine/paracrine pathways that regulate the proliferative phase of growth plate-mediated growth.     

Plasma insulin-like growth factor-I,testosterone and morphological changes in the growth of captive agile gibbons ( Hylobates agilis) from birth to adolescence

We examined growth changes in concentrations of plasma insulin-like growth factor-1 (IGF-1) and testosterone, and somatometric parameters in two captive male agile gibbons from birth to about 4 years of age, to examine the evolution of growth patterns in primates. Plasma IGF-1 concentrations in agile gibbons generally increased with age with values ranging from 200 to 1,100 ng/ml. The growth profiles in plasma IGF-1 in the gibbons were similar to those reported for chimpanzees. The highest concentrations of plasma testosterone (230 and 296 ng/dl) were observed within the first 0.3 years from birth, then the concentrations rapidly decreased and fluctuated below 100 ng/dl. Continuously higher IGF-1 concentrations were observed after 2.6 and 3.5 years of age. The profiles of plasma testosterone in these gibbons also resembled those of other primates including humans. However, their plasma testosterone levels in both neonate and adult stages (60 ng/dl) were lower than those reported for macaques and chimpanzees of respective stages. The obtained growth profiles of plasma IGF-1 and testosterone suggest that the adolescent phase starts around 2.6 or 3.5 years of age in male agile gibbons. The growth trend in many morphological parameters including body weight showed a linear increase without a significant growth spurt at approximately the onset of puberty. Head length and first digit length had reached a plateau during the study period. Brachial index, which indicates the relative length of forearm to upper arm, significantly increased gradually through the growth period. This result indicates that forearm becomes relatively longer than the upper arm with growth, which may be an evolutionary adaptation for brachiation.     

Crosstalk between the extracellular domain of the ErbB2 receptor and IGF-1 receptor signaling

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in cell growth and malignant transformation. To investigate IGF-1R-dependent signaling events and its effects on apoptosis induction and cellular proliferation, we generated a constitutively active, ligand-independent IGF-1R variant. We fused the cytoplasmic domain of the IGF-1R to the extracellular and transmembrane domains of the oncogenic ErbB2 receptor (ErbB2^V→E/IGF-1). A fusion protein in which the wild-type sequence of the ErbB2 receptor was used, served as a control (ErbB2^V/IGF-1R). ErbB2^V/IGF-1R, ErbB2^V→E/IGF-1R and IGF-1R were stably transfected into interleukin 3 (IL-3)-dependent BaF/3 cells. ErbB2^V→E/IGF-1R expressing cells exhibited ligand-independent, constitutive tyrosine phosphorylation of the receptor fusion protein. Constitutively, activated ErbB2^V→E/IGF-1R conferred IL-3 independence for growth and survival to the transfected BaF/3 cells. Constitutive activation of the IGF-1R results in cellular growth and protection against apoptosis upon IL-3 withdrawal in BaF/3 cells.