Alterations of the insulin-like growth factor system in patients with polycythemia vera

The molecular etiology of Polycythemia vera (PV) is still undetermined. Recently, enhanced tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) has been shown in PV bone marrow progenitors and peripheral blood mononuclear cells (PBMNC), and elevated levels of IGF binding protein-1 (IGFBP-1) in the serum of PV patients have been reported. To identify further alterations of circulating IGFBPs, the IGFBP profile in the serum of 12 PV patients was compared with age- and sex-matched controls by Western ligand blot (WLB), two-dimensional WLB, IGFBP-3 immunoblot and specific RIA for IGFBP-1, -2, -3 and IGFBP-4. To elucidate a role for the IGF-IR in the pathogenesis of PV, basal and IGF-I stimulated tyrosine phosphorylation of the IGF-IR beta-subunit in PBMNC of PV patients or controls was determined by WLB. Furthermore, exons 2, 3 and 15-21 of the IGF-IR were screened for mutations by PCR-single strand conformation polymorphism analysis (PCR-SSCP). We found alterations of the IGFBP profile in the serum of eight out of 12 examined patients including elevated levels of IGFBP-1, -2 and -4, decreased levels of IGFBP-3 and an increase in IGFBP-3 fragment. However, no differences in tyrosine phosphorylation of the IGF-IR in PV patients, neither basal nor IGF-I induced, were detected. Furthermore, no mutations within the screened exons of the IGF-IR could be identified by PCR-SSCP. We conclude that there is no direct impairment of IGF-IR structure or function, but an altered IGFBP profile in a significant portion of PV patients which might contribute to the pathogenesis of PV in these patients.     

Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase

Angiotensin II (AngII) activates phosphatidylinositol 3-kinase (PI3-kinase), a known effector of receptor tyrosine kinases. Treatment of smooth muscle cells with AngII has also been shown to promote phosphorylation of various tyrosine kinase receptors. We therefore investigated the relationship between AngII and IGF-I receptor activation in smooth muscle cells with a phosphorylation-specific antibody. Our experiments showed that IGF-I receptor phosphorylation was maximally stimulated within 10 min by AngII. Inclusion of an IGF-I-neutralizing antibody in the culture media did not prevent IGF-I receptor phosphorylation after AngII treatment, which argues that a paracrine/autocrine loop is not required. Furthermore, this process was blocked by losartan and 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP-1), indicating stimulation of IGF-I receptor phosphorylation occurs via AngII type 1 receptor-dependent activation of Src kinase. The functional significance of IGF-I receptor transactivation was examined with selective inhibitors of the IGF-I receptor kinase (AG1024, AG538). When AngII-treated cells were incubated with AG1024 or AG538, phosphorylation of the regulatory p85 subunit of PI3-kinase was blocked. Furthermore, phosphorylation of the downstream factor p70(S6K) did not occur. In contrast, AG1024 did not prevent MAPK or Src kinase activation by AngII. AG1024 also did not inhibit AngII-dependent cell migration, although this process was blocked by inhibitors of the epidermal growth factor and platelet-derived growth factor receptors. Transactivation of the IGF-I receptor is therefore a critical mediator of PI3-kinase activation by AngII but is not required for stimulation of the MAPK cascade.     

Specificity of insulin-like growth factor I and insulin on Shc phosphorylation and Grb2 recruitment in caveolae

Caveolae are lipid raft microdomains that regulate endocytosis and signal transduction. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates caveolin 1, supporting a role for these subcellular regions in the compartmentalization of IGF-I signaling. Src homology 2/alpha-collagen related protein (Shc) is the main mediator of IGF-I mitogenic action, coupling IGF-IR phosphorylation to Ras-MAPK activation. Here we show that IGF-I induces Shc tyrosine phosphorylation in the caveolae with a time course significantly different from that observed in the nonraft cellular fractions. In the same time, IGF-I recruits growth factor receptor bound protein 2 (Grb2) to caveolae and activates p42/p44 MAPKs in these microdomains. Src family kinases regulate IGF-I action through an Shc-dependent mechanism. In R-IGF-IRWT cells, IGF-I causes Fyn enrichment in the caveolae with a time course consistent with Shc phosphorylation and Grb2 recruitment in these regions. Finally, we have observed that after IGF-I stimulation, IGF-IR and Fyn colocalize in lipid raft caveolin 1-enriched microdomains. As insulin and IGF-I share common substrates, the effect of insulin on these cellular processes was measured. Here we show that insulin also induces Shc phosphorylation and Grb2 recruitment to caveolae, but with a significantly different time course compared with IGF-I. Our results suggest that 1) IGF-I causes the colocalization of signaling proteins in caveolae through a phosphorylation-regulated mechanism; and 2) the time course of phosphorylation and recruitment of substrates in caveolae by insulin receptor and IGF-IR could determine the specific actions of these receptors.     

Estrogen signaling via a linear pathway involving insulin-like growth factor I receptor, matrix metalloproteinases, and epidermal growth factor receptor to activate mitogen-activated protein kinase in MCF-7 breast cancer cells

We present an integrated model of an extranuclear, estrogen receptor-alpha (ERalpha)-mediated, rapid MAPK activation pathway in breast cancer cells. In noncancer cells, IGF-I initiates a linear process involving activation of the IGF-I receptor (IGF-IR) and matrix metalloproteinases (MMP), release of heparin-binding epidermal growth factor (HB-EGF), and activation of EGF receptor (EGFR)-dependent MAPK. 17beta-Estradiol (E2) rapidly activates IGF-IR in breast cancer cells. We hypothesize that E2 induces a similar linear pathway involving IGF-IR, MMP, HB-EGF, EGFR, and MAPK. Using MCF-7 breast cancer cells, we for the first time demonstrated that a sequential activation of IGF-IR, MMP, and EGFR existed in E2 and IGF-I actions, which was supported by evidence that the selective inhibitors of IGF-IR and MMP or knockdown of IGF-IR all inhibited E2- or IGF-I-induced EGFR phosphorylation. Using the inhibitors and small inhibitory RNA strategies, we also demonstrated that the same sequential activation of the receptors occurred in E2-, IGF-I-, but not EGF-induced MAPK phosphorylation. Additionally, a HB-EGF neutralizing antibody significantly blocked E2-induced MAPK activation, further supporting our hypothesis. The biological effects of sequential activation of IGF-IR and EGFR on E2 stimulation of cell proliferation were also investigated. Knockdown or blockade of IGF-IR significantly inhibited E2- or IGF-I-stimulated but not EGF-induced cell growth. Knockdown or blockade of EGFR abrogated cell growth induced by E2, IGF-I, and EGF, indicating that EGFR is a downstream molecule of IGF-IR in E2 and IGF-I action. Together, our data support the novel view that E2 can activate a linear pathway involving the sequential activation of IGF-IR, MMP, HB-EGF, EGFR, and MAPK.     

Effect of epidermal growth factor on insulin-like growth factor-I (IGF-I) and IGF-binding protein synthesis by adult rat hepatocytes.

Growth hormone has been established as a primary regulator of IGF-I gene expression in adults, not only in liver but also in many extrahepatic tissues. We considered the possibility that IGF-I production by adult rat liver could also be stimulated by epidermal growth factor (EGF), a peptide known to be involved in liver regeneration. Chromatographic analysis performed after acid treatment of conditioned media revealed the presence of both immunoreactive (IR) IGF-I and IGF binding protein (IGFBP). Both IR IGF-I and IGFBP were present in the conditioned medium of adult rat hepatocytes in basal conditions. The stimulation of IGF-I and IGFBP secretion by EGF appears to be dose-dependent with a significant increment already evident at 5 nM. That EGF stimulates secretion is supported by the finding that IGF-I and IGFBP-1 mRNA levels are increased after EGF supplementation. We conclude that adult rat hepatocytes spontaneously produce IGF-I and IGFBP, and that EGF is able to increase their synthesis and secretion. This non-growth hormone-dependent regulation of IGF-I and IGFBP-1 production by adult rat hepatocytes in culture indicates an important autocrine/paracrine role for IGF-I, particularly during liver regeneration after extensive organ mass loss.     

Inhibition of insulin-like growth factor-I signaling by ethanol in neuronal cells

BACKGROUND: Ethanol inhibits insulin-like growth factor-I receptor (IGF-IR) activation. However, the potency of ethanol for inhibition of the IGF-IR and other receptor tyrosine kinases varies considerably among different cell types. We investigated the effect of ethanol on IGF-I signaling in several neuronal cell types. METHODS: IGF-I signaling was examined in SH-SY5Y neuroblastoma cells, primary cultured rat cerebellar granule neurons, and rat NG-108 neuroblastoma x glioma hybrids. The tyrosine phosphorylation of IGF-IR, IRS-2, Shc, and p42/p44 MAP kinase (MAPK), and the association of Grb-2 with Shc, were examined by immunoprecipitations and Western blotting. RESULTS: IGF-I-mediated tyrosine phosphorylation of MAPK was inhibited by ethanol in all cell lines. IGF-IR autophosphorylation was markedly inhibited by ethanol in SH-SY5Y cells, was only mildly inhibited in cerebellar granule neurons, and was unaffected in rat NG-108 cells. In vitro tyrosine autophosphorylation of immunopurified IGF-IR obtained from all cell lines was inhibited by ethanol. There was also differential ethanol sensitivity of IRS-2 and Shc phosphorylation, and the association of Shc with IRS-2, among the different cell types. CONCLUSIONS: The findings demonstrate that IGF-I-mediated MAPK activation is a sensitive target of ethanol in diverse neuronal cell types. The data are consistent with ethanol-induced inhibition of IGF-IR activity, although the extent of IGF-IR tyrosine autophosphorylation per se is a poor marker of the inhibitory action of ethanol on this receptor. Furthermore, despite uniform inhibition of MAPK in the different neuronal cell types, tyrosine phosphorylation of proximal mediators of the IGF-IR are differentially inhibited by ethanol.     

C-terminal Src kinase (CSK) modulates insulin-like growth factor-I signaling through Src in 3T3-L1 differentiation

IGF-I stimulates both proliferation and differentiation of adipocyte-precursor cells, preadipocytes in vivo and in vitro. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of MAPK and MAPK activation by IGF-I is mediated through the Src family of nonreceptor tyrosine kinases. In addition, we have shown that when 3T3-L1 cells reach growth arrest and are stimulated to differentiate, IGF-I can no longer activate the MAPK pathway. We hypothesized that the loss of IGF-I signaling to MAPK in differentiating 3T3-L1 cells is due to loss of IGF-I activation of Src family kinases. We measured c-Src kinase activity in cell lysates from proliferating, growth-arrested and differentiating 3T3-L1 cells. Src activity increased 2- to 4-fold in IGF-I-stimulated proliferating cells; however, IGF-I had a marginal affect on Src activity in growth-arrested cells and inhibited Src activity localized at the membrane in differentiating cells. C-terminal Src kinase (CSK), a ubiquitously expressed nonreceptor tyrosine kinase, negatively regulates the Src family kinases by phosphorylation of the Src C-terminal tyrosine. IGF-I decreased phosphorylation of the Src C-terminal tyrosine in proliferating cells and increased phosphorylation of this site in differentiating cells. IGF-I stimulated CSK kinase activity 2-fold in differentiating 3T3-L1 cells. An association between CSK and c-Src was detected by immunoprecipitation following IGF-I stimulation of differentiating but not proliferating 3T3-L1 cells. These results suggest that the loss of IGF-I downstream mitogenic signaling in differentiating 3T3-L1 cells is due to a change in IGF-I activation of c-Src and CSK may mediate the inactivation of c-Src by IGF-I in 3T3-L1 adipogenesis.     

Effects of insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) on endometrial cancer (HHUA) cell apoptosis and EGF stimulated cell proliferation in vitro.

OBJECTIVE: IGFBP-3 has been demonstrated to stimulate or inhibit cell proliferation independently of its ability to bind IGF and a specific IGFBP-3 receptor has been proposed. EGF has been implicated in the cancer development and carcinogenesis. Only limited data are available on the crosstalk between IGFBP-3 signaling and EGF induced cell survival and signal transduction. The current studies were undertaken to characterize IGFBP-3 binding to endometrial cancer cells (HHUA) and determine its biological effects, as well as whether IGFBP-3 exposure alters the cell proliferation stimulated by EGF. METHODS: Cell proliferation and apoptosis were analyzed by ELISA using specific antibodies. The interaction between HHUA cell and IGFBP-3 was analyzed using a biosensor. The phosphorylation abundance of specific proteins and their phosphorylation in response to EGF and IGFBP-3 was analyzed by immunoprecipitation followed by immunoblotting. RESULTS: Biosensor analysis showed that IGFBP-3 could bind to HHUA cell surface. IGFBP-3 inhibited BrdU uptake, potentiated ssDNA production and induced p53 in HHUA cells. Although EGF stimulated HHUA cell proliferation and Akt phosphorylation, IGFBP-3 inhibited cell proliferation and Akt phosphorylation that had been stimulated by EGF. However, EGF receptor phosphorylation and expression were not reduced by IGFBP-3. Since HHUA cells lack IGF receptors and do not show biological response to IGF these results suggest that IGFBP-3 can bind to HHUA cells, inhibit cell proliferation and induce apoptosis independently of its ability to bind to IGFs possibly by binding to an IGFBP-3 receptor. CONCLUSIONS: Taken together these findings demonstrate that IGFBP-3 binds to HHUA cell surface, and inhibits cell division induced by EGF, possibly by modulating the EGF-mediated signal transduction system.     

Insulin-like growth factor-I-dependent signal transduction pathways leading to the induction of cell growth and differentiation of human neuroblastoma cell line SH-SY5Y: the roles of MAP kinase pathway and PI 3-kinase pathway

IGF-I regulates cell growth, differentiation, and survival in many cultured nerve cell lines. The present study was undertaken in the human neuroblastoma cell line, SH-SY5Y, to elucidate whether there are differences in the IGF-dependent signal transduction pathways that stimulate proliferation compared to those that induce differentiation. Quiescent SH-SY5Y cells were treated with IGF-I in the presence or absence of PD98059 (an inhibitor of MEK, a MAP kinase kinase) or LY294002 (an inhibitor of PI 3-kinase). Cell growth was assessed by measuring [3H]thymidine incorporation into DNA and cell number. Cell differentiation was assessed by measuring mRNA levels of NPY and neurite outgrowth. IGF-I both induced cell proliferation and differentiation. It stimulated tyrosine phosphorylation of the type I IGF receptor (IGF-IR) beta-subunit, IRS-I, IRS-2, and Shc, and these changes were associated with activation of Erk and Akt. PD98059 inhibited activation of Erk and LY294002 repressed activation of Akt in response to IGF-I, but did not affect tyrosine phosphorylation of the IGF-IR, IRS-1, IRS-2, or Shc. Each PD98059 and LY294002 inhibited IGF-I-dependent cell proliferation in a concentration-dependent manner. In contrast, each of these inhibitors only partially depressed NPY gene expression induced by IGF-I and slightly inhibited IGF-I-mediated neurite outgrowth; however, when both PD98059 and LY294002 were present, IGF-I-dependent NPY gene expression and neurite outgrowth were abolished completely. These results suggest that in these nerve cells, 1) the IGF-I signals through the MAP kinase pathway and PI-3 kinase pathway are independently essential to induce IGF-I-dependent growth, and 2) alternate activation of the MAP kinase pathway and PI 3-kinase pathway is sufficient for the cells to undergo IGF-I-dependent differentiation.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.     

Increased hepatic expression of insulin-like growth factor-I receptor in chronic hepatitis C

AIM: Although increased insulin-like growth factor-I receptor (IGF-IR) gene expression has been reported in hepatocellular carcinoma, studies assessing IGF-IR in chronic hepatitis C (CHC) and cirrhosis are scarce. We therefore aimed to evaluate IGF-IR and IGF-I mRNA expression in liver from patient with CHC. METHODS: IGF-IR and IGF-I mRNA content were determined by semi-quantitative RT-PCR and IGF-IR protein expression was determined by immunohisto-chemistry in hepatic tissue obtained from patients with CHC before (34 patients) and after (10 patients) therapy with interferon-alpha and ribavirin. RESULTS: An increase of IGF-IR mRNA content was observed in hepatic tissue obtained from all CHC patients as well as from 6 cadaveric liver donors following orthopic transplantation (an attempt to evaluate normal livers) in comparison to normal liver, while no relevant modifications were detected in IGF-I mRNA content. The immunohistochemical results showed that the raise in IGF-IR mRNA content was related both to ductular reaction and to increased IGF-IR expression in hepatocytes. A decrease in IGF-IR mRNA content was observed in patients who achieved sustained virological response after therapy, suggesting an improvement in hepatic damage. CONCLUSION: The up-regulation of IGF-IR expression in hepatocytes of patients with CHC could constitute an attempt to stimulate hepatocyte regeneration. Considering that liver is the organ with the highest levels of IGF-I, our finding of increased IGF-IR expression after both acute and chronic hepatic damage highlights the need for additional studies to elucidate the role of IGF-I in liver regeneration.     

Insulin-like growth factor (IGF) binding protein-3 inhibits type 1 IGF receptor activation independently of its IGF binding affinity

Insulin-like growth factor binding proteins (IGFBPs) regulate the cellular actions of the IGFs owing to their strong affinities, which are equal to or stronger than the affinity of the type 1 IGF receptor (IGF-IR), the mediator of IGF signal transduction. We recently found that IGFBP-3 modulates IGF-I binding to its receptor via a different mechanism possibly involving conformational alteration of the receptor. We have now investigated the effects of IGFBP-3 on the initial steps in the IGF signaling pathway. MCF-7 breast carcinoma cells were preincubated with increasing concentrations of IGFBP-3 and then stimulated with IGF-I, des(1-3)IGF-I, or [Q(3)A(4)Y(15)L(16)]-IGF-I, the latter two being IGF-I analogs with intact affinity for the type 1 IGF receptor, but weak or virtually no affinity for IGFBPs. Stimulation of autophosphorylation of the receptor and its tyrosine kinase activity was dose-dependently depressed. At 2.5 nM, IGFBP-3 provoked more than 50% inhibition of the stimulation induced by 3 nM des(1-3)IGF-1 and, at 10 nM, more than 80% inhibition. Similar results were obtained with [Q(3)A(4)Y(15)L(16)]-IGF-I. Cross-linking experiments using iodinated or unlabeled IGFBP-3 and anti-IGF-IR antibodies indicated that the inhibitory effects do not involve direct interaction between IGFBP-3 and IGF-IR. The inhibition appeared to be specific to IGFBP-3, because IGFBP-1 and IGFBP-5 at 10 nM had no significant effect. Also, inhibition was restricted to the IGF receptor, because IGFBP-3 failed to inhibit the tyrosine kinase activity of the insulin receptor stimulated by physiological concentrations of insulin. Our results provide the first demonstration that IGFBP-3 can specifically modulate the IGF-I signaling pathway independently of its IGF-I-binding ability. They also reveal a regulatory mechanism specific to the type 1 IGF receptor, with no effect on insulin receptor activation.     

Caveolin-1 down-regulation inhibits insulin-like growth factor-I receptor signal transduction in H9C2 rat cardiomyoblasts

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.     

Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells

Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited ( approximately 80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca(2+) attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca(2+) mobilization or phosphorylation of Ca(2+)-dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II-induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II-stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease.     

Insulin-like growth factor-I receptor signaling in tamoxifen-resistant breast cancer: a supporting role to the epidermal growth factor receptor

There is considerable evidence that the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) cross-talk in breast cancer cells. In the present study, we have examined whether EGFR/IGF-IR cross-talk exists in EGFR-positive tamoxifen-resistant variants of MCF-7 (Tam-R) and T47D (T47D-R) breast cancer cell lines. Although Tam-R cells expressed reduced IGF-IR protein levels compared with their wild-type MCF-7 counterparts, phosphorylated IGF-IR protein levels were equivalent in the two cell lines under basal growth conditions, possibly as a consequence of increased IGF-II expression in Tam-R cells. IGF-II activated both IGF-IR and EGFR in Tam-R cells, whereas only activation of IGF-IR was observed in wild-type cells. In contrast, epidermal growth factor rapidly induced EGFR, but not IGF-IR, phosphorylation in Tam-R cells. IGF-II promoted direct association of c-SRC with IGF-IR, phosphorylated c-SRC, and increased EGFR phosphorylation at tyrosine 845, a c-SRC-dependent phosphorylation site. Pretreatment with either AG1024 (IGF-IR-specific inhibitor) or an IGF-II neutralizing antibody inhibited basal IGF-IR, c-SRC, and EGFR phosphorylation, and AG1024 significantly reduced Tam-R basal cell growth. The c-SRC inhibitor SU6656 also inhibited growth, reduced basal and IGF-II-induced c-SRC and EGFR phosphorylation, and blocked EGFR activation by TGFalpha. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGFalpha-induced EGFR activity. These results suggest the existence of a unidirectional IGF-IR/EGFR cross-talk mechanism whereby IGF-II, acting through the IGF-IR, regulates basal and ligand-activated EGFR signaling and cell proliferation in a c-SRC-dependent manner in Tam-R cells. This cross-talk between IGF-IR and EGFR is not unique to Tam-R cells because this mechanism is also active in a tamoxifen-resistant T47D-R cell line.     

Involvement of the Src family kinase yes in bile salt-induced apoptosis

BACKGROUND AND AIMS: Hydrophobic bile acids induce CD95 (Fas, APO-1)-dependent hepatocyte apoptosis, which involves epidermal growth factor receptor (EGFR)-catalyzed CD95 tyrosine phosphorylation. The mechanisms underlying bile salt-induced EGFR activation remain unclear. METHODS: Bile acid-induced EGFR activation was studied in 24-hour cultured rat hepatocytes and perfused rat liver. RESULTS: The proapoptotic bile salts taurolithocholate-3-sulfate (TLCS), glycochenodesoxycholate (GCDC) and taurochenodeoxycholate (TCDC), but not taurocholate (TC), activate within 1 minute the Src kinase family member Yes, followed by an association of Yes with EGFR and subsequent EGFR activation. EGFR phosphorylation by TLCS involves tyrosines 845 and 1173 but not 1045. Yes/EGFR association and EGFR activation were sensitive to inhibition by SU6656 but not by PP-2. cAMP had no effect on TLCS and GCDC-induced Yes activation but induced Ser/Thr phosphorylation of Yes and prevented Yes/EGFR association and subsequent EGFR activation. Both SU6656 and cAMP had no effect on bile salt-induced c-Jun N-terminal kinase activation, but blocked bile salt-induced CD95 tyrosine phosphorylation, membrane trafficking of CD95, formation of the death-inducing signaling complex, and apoptosis. In 4-day cultured hepatocytes, knockdown of either Yes or EGFR strongly attenuated bile salt-induced CD95 activation and apoptosis. CONCLUSIONS: The data identify the Src kinase Yes as an upstream target of proapoptotic bile acids, which triggers EGFR activation, subsequent CD95 tyrosine phosphorylation, and apoptosis. The antiapoptotic effect of cAMP involves a protein kinase A-dependent inhibition of Yes/EGFR association, thereby preventing EGFR activation, which is required for CD95 activation.     

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.     

The endocytosis-linked protein dynamin associates with caveolin-1 and is tyrosine phosphorylated in response to the activation of a noninternalizing epidermal growth factor receptor mutant

Several studies have shown that an EGF receptor C-terminal truncation at residue 973 (CT973) attenuates ligand-induced receptor endocytosis and is associated with cell transformation. Previously, we have shown that EGF stimulation of murine B82L fibroblasts expressing CT973 EGF receptors can promote the tyrosine phosphorylation of caveolin-1, which is a major component of caveolae membranes. Because dynamin plays an essential role in receptor-mediated endocytosis via clathrin-coated pits and caveolae, and because dynamin has been localized to caveolae, we tested the hypothesis that dynamin associates with caveolin-1 and is differentially modified in response to the abnormal actions of internalization-defective EGF receptors. We found that dynamin coimmunoprecipitates with caveolin-1 in cells containing normal or CT973 EGF receptors, but EGF stimulated the tyrosine phosphorylation of dynamin only in cells expressing truncated/oncogenic EGF receptors. Maximum dynamin phosphorylation was observed within 15 min of EGF administration and decreased thereafter. Furthermore, phosphotyrosine-containing proteins in the dynamin immunocomplexes were observed to be reactive with anticaveolin-1 antibodies. The EGF receptor does not appear to directly phosphorylate dynamin because a Src antagonist, PP1, inhibited the EGF-induced tyrosine phosphorylation of dynamin at a concentration that does not block EGF receptor autophosphorylation. These results provide the first evidence that caveolin-1 and dynamin form a complex, and that the EGF-induced tyrosine phosphorylation of dynamin occurs via a Src inhibitor-sensitive signaling pathway that is associated with the aberrant actions induced by internalization-defective EGF receptors.