Insulin-like Growth Factor Receptor Activation Research Articles

Role of Insulin Like Growth Factor (IGF) in Lung Cancer with Underlying Diabetes Melitus

Lung cancer is one of the diseases which the highest incidence and mortality in the world. Lung cancer consists of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) with various subtypes. Diabetes mellitus is one of the comorbidities that is often found in lung cancer patients, which is around 6% - 18% in lung cancer patients. Diabetes is a comorbidity factor that has a serious effect in increasing cancer cell progressivity through the activity of insulin growth factor (IGF) which consists of receptors (IGF-R) and binding proteins (IGFP). The role of IGF produces oncogenic mutations at the DNA level with the process of lung cancer immunity stimulates mitogenesis initiated by insulin and initiates neoplasm cells. IGF receptor mechanism has antineoplastic properties, to fulfill glycemia control that will reduce lung cancer progressivity and prevent clinical worsening so that it can help reduce lung cancer mortality with oral hypoglycemia drugs along with lung cancer therapy modalities such as cytotoxic agents, radiotherapy, immunotherapy, and surgery. The role of IGF is to amplify insulin levels in glucose, lipid, and protein metabolism. IGF can induce cell differentiation and initiation of neoplasm. Diabetes mellitus as a comorbidity can poor clinical outcomes and promote progression through IGF receptor activation in the tumorigenesis process.

Abstract P3-06-10: The impact of the Chinese medicine ShenLingLan on triple negative breast cancer, the metabolic and signalling pathways and clinical implications

Abstract Background. Triple negative breast cancer (TNBC) is a challenging both in the choice of therapies and clinical outcomes. In the present study, we investigated the potential prospect of a Chinese medicine formation, ShenLingLan, shown to have benefit to patients with cancer and able to influence the biological behaviour of cancer cells, on breast cancer cells in particular the differential response from TNBC and non-TNBC cells and, on the discovery that TNBC cells were particularly sensitive to the medicine, we went on to determine the signalling and mechanism of action. Methods. A panel of three TNBC (MDA MB-231, BT20 and BT549) and a panel of three non-TNBC (MCF-7, ZR 75-1 and T47D) cells were used. A soluble extract from ShenLingLan, designated as SLDM, was utilised during this study. The proliferation, cellular migration and adhesiveness were tested using conventional and biophysical methods. Signalling profiling was conducted using a protein kinase array platform (Kinexustm). Metabolic profiling was conducted using the Seahorse platform. Expression of insulin receptor (IR) and insulin-like growth factor receptor (IGFR) gene transcripts (quantitative transcript analysis) and proteins (IHC) were conducted using a fresh breast cancer cohort and tissue array, respectively. Results. SLDM had little effects on the growth of breast cancer cells. However, it had profound inhibitory effects on the migration of both TNBC and non-TNBC cells in a concentration dependent manner. Interestingly, TNBC cells were 5-20 times more sensitive than the non-TNBC cells in their migration and cell adhesion responses to SLDM. The protein array platform further revealed that, of the wide range of protein kinases, IR and IGRR1 were the most affected in that SLDM resulted in 25-50% reduction in the phosphorylation of IR and IR substrate in TNBC cells. SLDM also caused a contrasting response in IGFR1 phosphorylation in TNBC and non-TNBC cells. Metabolically, TNBC and non-TNBC cells responded to SLDM in very different fashions. For example, in TNBC cells SLDM resulted in a significant decrease in glycolytic activities, in particular that driven by insulin (30.2±12.2 pmol/min in control, 47.7±10 pmol/min with insulin and 35.4±4.2 pmol/min with insulin/SLDM, p=0.01). There is evidence that the mitochondria oxygen consumption (OCR) was also affected by SLDM in TNBC cells (p=0.01). These changes induced by SLDM were in clear contrast to non-TNBC cells which did not respond with significant reduction. Both TNBC and non-TNBC breast cancer tissues have higher IR staining than normal mammary tissues (p<0.001). TNBC tumours also demonstrated significantly more positive IR staining than the non-TNBC tumours (p=0.04). Conclusion. ShenLingLan has a profound inhibitory effect on the migration and cell-matrix adhesion of TNBC cells, with marked effect on the metabolics of these cells. This effect connects with reduction of the IR and IGFR activation, mainly through the reduction in glycolysis. Together with the clinical implication of IR and IGFR in breast cancer, ShenLingLan has an important role in the treatment of breast cancer with an emphasis in TNBCs. Citation Format: Jiang WG, Owen S, Ruge F, Gao Y, Wang H, Wei C, Wu Y, Davies E. The impact of the Chinese medicine ShenLingLan on triple negative breast cancer, the metabolic and signalling pathways and clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-10.

Smoking-associated lung cancer prevention by blockade of the beta-adrenergic receptor-mediated insulin-like growth factor receptor activation.

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via β-adrenergic receptor (β-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of β-AR and the downstream signaling including a Gβγ subunit of β-AR and phospholipase C (PLC). Consistently, β-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via β-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with β-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking β-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

A presurgical window-of-opportunity study of metformin in obesity-driven endometrial cancer

BackgroundMetformin use is associated with reduced cancer risk in several observational studies of patients with type 2 diabetes. Results from preclinical studies in endometrial cancer show that metformin reduces cellular proliferation by inhibition of the PI3K-AKT-mTOR pathway. We tested the hypothesis that metformin would reduce cellular proliferation in vivo in atypical endometrial hyperplasia and endometrial endometrioid adenocarcinoma. MethodsWe recruited women attending gynaecological oncology clinics in Manchester, UK, with atypical endometrial hyperplasia or endometrial endometrioid adenocarcinoma. Women received metformin (850 mg twice daily) or no drug (control) during the 1–4 week presurgical window between cancer diagnosis and hysterectomy according to patient preference. Paired blood and tumour samples were obtained at recruitment and hysterectomy. Cellular proliferation was assessed by Ki-67 proliferation index. Automated scoring on two separate occasions provided consistent replicate scores (SD <10%). This study is registered with the ISRCTN register, number ISRCTN81570194. FindingsSamples from 40 women have been analysed (28 metformin-treated [median age 64 years, IQR 58–69]; 12 control [70, 64–70]). 24 of the patients (60%) were obese. 22 patients (55%) had either undiagnosed diabetes (fasting glucose >7·0 mmol/L, n=4) or insulin resistance (homoeostatic model assessment of insulin resistance >2·8, n=18). Metformin was taken for a median of 20 days (IQR 17–24), and mild gastrointestinal side-effects were reported by 22 metformin-treated patients. In the metformin-treated group, Ki-67 was 12·9% lower at hysterectomy than at recruitment (95% CI 3·7–22·1, p=0·008) after adjustment for baseline Ki-67, Ki-67 change in controls, age, and body-mass index. No significant changes in phosphorylation of AKT or markers of insulin resistance after adjustment for treatment arm were seen. InterpretationUndiagnosed insulin resistance or diabetes were common in our study population. Short-term presurgical metformin was associated with a reduction in Ki-67 proliferation index. We are now exploring the hypothesis that metformin reduces Ki-67 expression by inducing phosphorylation of AMP-activated kinase and subsequent mTOR proproliferative inhibition, independent of insulin and insulin-like growth factor receptor activation. FundingWellbeing of Women, Wellcome Trust.

Colorectal Cancer: Histopathologic Differences in Tumor Characteristics Between Patients With and Without Diabetes

BackgroundCurrent literature suggests that diabetes is a possible predictor of risk and worse outcome in colorectal cancer (CRC). The objective of this study was to explore if there are histopathologic differences in CRC between populations with and without diabetes. Patients and MethodsRetrospective analysis was done on 534 patients with CRC. Patients were divided into diabetic and nondiabetic subgroups. Data were collected for lymphovascular invasion, tumor location, depth invasion, staging, level of differentiation, histologic type, and presence of tumor components (mucinous, signet ring, or neuroendocrine). ResultsUnivariately, patients with diabetes had deeper tumor invasion, greater lymphovascular invasion, and higher TNM staging (OR and 95% CI, 2.06 [1.37, 3.10], 2.52 [1.74, 3.63], and 2.45 [1.70, 3.52], respectively; P < .001). Covariate adjustment retained the significant effect of diabetes on tumor characteristics (P < .005). Multivariable adjustment significantly linked diabetes with signet ring cell carcinoma (log odds, 11.40 ± 5.28; P = .03) and tumor components (log odds, 0.58 ± 0.25; P = .02). Patients with diabetes with hyperlipidemia had more well-differentiated tumors (log odds, −0.96 ± 0.47; P = .04). Transverse tumors were more common in patients with diabetes (log odds, 1.74 ± 0.72; P = .02). ConclusionPatients with diabetes had worse histopathologic CRC features. Hyperinsulinemia, insulinlike growth factor receptor activation, and hyperglycemia in diabetes can activate mitogenic pathways stimulating proliferation, invasion, angiogenesis, and metastasis. Future research is needed to identify responsible pathways for targeted therapy and to examine the role of better glycemic control and treatment in patients with CRC and diabetes.

Potentiating the efficacy of molecular targeted therapy for hepatocellular carcinoma by inhibiting the insulin-like growth factor pathway.

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.

Pregnancy-associated Plasma Protein A (PAPP-A) Modulates the Early Developmental Rate in Zebrafish Independently of Its Proteolytic Activity

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin‐like growth factor receptor pathway

The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans. SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin-like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis. Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor alpha/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3-kinase, phosphoinosite-dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1-mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down-regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased. For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down-regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage.

1432 INSULIN-LIKE GROWTH FACTORS MEDIATE INFLAMMATION-DRIVEN PROLIFERATION OF THE MOUSE PROSTATE

You have accessJournal of UrologyProstate Cancer: Basic Research VIII1 Apr 20101432 INSULIN-LIKE GROWTH FACTORS MEDIATE INFLAMMATION-DRIVEN PROLIFERATION OF THE MOUSE PROSTATE Travis Jerde, Eliza McFarland, Sanghee Lee, and Wade Bushman Travis JerdeTravis Jerde Madison, WI More articles by this author , Eliza McFarlandEliza McFarland Medford, MA More articles by this author , Sanghee LeeSanghee Lee Madison, WI More articles by this author , and Wade BushmanWade Bushman Madison, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1125AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inflammation in the prostate is of considerable importance to urological research because of its association with prostate cancer. Yet, mechanisms by which inflammation promotes cancerous or benign prostatic growth remain obscure. Insulin-like growth factor (IGF) signaling plays a role in prostate development and is postulated to drive prostate tumor and benign prostatic growth in men. Since inflammation is associated with growing prostate cancer, we sought to determine if IGF signaling is involved in inflammation-induced epithelial proliferation. METHODS We initiated inflammatory reactive hyperplasia in mice using our bacterially-induced acute inflammation model. Prostates were harvested 1, 2, 3, 5, 7, and 14 days post-instillation. IGF-1 and IGF-2 expression was determined by RT-PCR, and peptide concentration and release were determined by ELISA. IGF receptor activation was quantified by immunoblotting and spatially detected by immunohistochemistry using phospho-specific antibodies to activated IGF receptor. Normal and cancerous human prostate specimens were also stained for IGF receptor activation. Additional inflamed and control animals were treated with an IGF receptor antagonist (picropodophyllin [PPP]) or vehicle i.p. twice daily for 3 days. Inflammatory index, proliferation, IGF receptor activation, and IGF target pathway activity (phosphorylated Akt) were determined. RESULTS Inflammation caused a significant increase in IGF-1 expression and regulated release within two days of inducing inflammation. This correlated with substantially increased proliferative index and IGF receptor activation. The human prostatic epithelium showed substantial induction of IGF signaling in areas juxtaposed to inflammation. IGF inhibition substantially reduced proliferation rates and hyperplastic response of the mouse prostate. PPP treatment predictably reduced IGF receptor activation and Akt pathway activation, but had minimal effect on inflammation itself indicating that IGF inhibition targets proliferation of the epithelium and stroma but not inflammatory infiltrate. CONCLUSIONS Our data indicate that IGF plays a critical role in the hyperplastic response of the prostate to inflammation. While using IGF inhibition therapeutically has been previously proposed based on an associative increase in gene expression, our work provides clear evidence for a causative role for IGF in inflammation-induced growth and establishes IGF signaling as a logical therapeutic target in reducing epithelial expansion in the prostate. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e552 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Travis Jerde Madison, WI More articles by this author Eliza McFarland Medford, MA More articles by this author Sanghee Lee Madison, WI More articles by this author Wade Bushman Madison, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Insulin‐like growth factor‐induced signals activate mitochondrial respiration

From experiments with lower eukaryotes it is known that the metabolic rate and also the rate of aging are tightly controlled by the insulin-like growth factor (IGF)/insulin signal transduction pathway. The mitochondrial theory of aging implies that an increased metabolic rate leads to increased mitochondrial activity; increased production of reactive oxygen species due to these alterations would speed up the aging process. To address the question if mitochondrial activity is influenced by insulin/IGF signaling, we have established an experimental system to determine the influence of IGF-I-dependent signaling on mitochondrial function. We used DU145 prostate cancer cells, known for the intact IGF signal transduction pathway, to address the influence of IGF receptor activation on mitochondrial function by high-resolution respirometry. These experiments revealed that indeed mitochondrial function is regulated by IGF signaling, and up-regulation of respiration seems to require phosphoinositide 3-kinase/AKT signaling, but is independent of IGF effects on cell cycle progression. Collectively these data establish a regulatory cross-talk between insulin/IGF signal transduction and mitochondrial function, two major pathways implicated in controlling the rate of aging.

Insulin receptor substrates mediate distinct biological responses to insulin-like growth factor receptor activation in breast cancer cells

Activation of the type I insulin-like growth factor receptor (IGF-IR) regulates several aspects of the malignant phenotype, including cancer cell proliferation and metastasis. Phosphorylation of adaptor proteins downstream of IGF-IR may couple IGF action to specific cancer phenotypes. In this study, we sought to determine if insulin receptor substrate-1 and -2 (IRS-1 and -2) mediate distinct biological effects in breast cancer cells. Insulin receptor substrate-1 and IRS-2 were expressed in T47D-YA breast cancer cells, which lack IRS-1 and -2 expression, yet retain functional IGF-IR. In the absence of IRS-1 and -2 expression, IGF-IR activation was unable to stimulate proliferation or motility in T47D-YA cells. Expression of IRS-1 resulted in IGF-I-stimulated proliferation, but did not affect motility. In contrast, expression of IRS-2 enhanced IGF-I-stimulated motility, but did not stimulate proliferation. The αIR-3, an inhibitor of the IGF-IR, was unable to affect these IGF-stimulated phenotypes unless IRS-1 or -2 was expressed. Thus, IGF-IR alone is unable to regulate important breast cancer cell phenotypes. In these cells, IRS proteins are required for and mediate distinct aspects of IGF-IR-stimulated behaviour. As multiple agents targeting the IGF-IR are currently in early clinical trials, IRS expression should be considered as a potential biomarker for IGF-IR responsiveness.

Role of N- and C-terminal Residues of Insulin-like Growth Factor (IGF)-binding Protein-3 in Regulating IGF Complex Formation and Receptor Activation

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu(77), Leu(80), and Leu(81) in the N terminus and Gly(217) and Gln(223) in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF.IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.

Insulin-Like Growth Factor (IGF) Binding Protein-3 Inhibits Type 1 IGF Receptor Activation Independently of Its IGF Binding Affinity

Insulin-Like Growth Factor (IGF) Binding Protein-3 Inhibits Type 1 IGF Receptor Activation Independently of Its IGF Binding Affinity

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.

In Vivo Regulation of CrkII and CrkL Proto-oncogenes in the Uterus by Insulin-like Growth Factor-I: DIFFERENTIAL EFFECTS ON TYROSINE PHOSPHORYLATION AND ASSOCIATION WITH PAXILLIN

Changes in CrkII and CrkL phosphorylation are associated with insulin-like growth factor receptor activation in cultured cells. We examined whether similar changes also occur following administration of recombinant human insulin-like growth factor-I to the intact animal. In female rats starved overnight, CrkL phosphorylation was significantly increased 12 min after insulin-like growth factor-I administration. Tyrosine phosphorylation of CrkII was not detectable in either control or treated animals. Paxillin, a 65-70-kDa phosphoprotein containing high affinity binding sites common for the Src homology 2 (SH2) domains of CrkII and CrkL, was observed in both CrkII and CrkL immunoprecipitates. Insulin-like growth factor-I treatment stimulated the association of CrkII with paxillin. In contrast, the same treatment resulted in the dissociation of the CrkL-paxillin complex. Similar effects of insulin-like growth factor-I treatment on the association of CrkL with tyrosine phosphorylated paxillin were observed in fibroblasts overexpressing CrkL. This study demonstrates that the activation of the insulin-like growth factor-I receptor induces changes in the tyrosine phosphorylation and protein-protein interactions of the Crk proteins in vivo. The different responses of CrkL and CrkII to insulin-like growth factor-I receptor activation suggest distinct roles for these two adapter proteins in signal transduction.

Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.