Insulin-like growth factor-1 receptor expression in upper tract urothelial carcinoma.

Insulin-like growth factor-1 receptor (IGF1R) is a transmembrane tyrosine kinase receptor that plays a crucial role in cell proliferation, growth, differentiation, and apoptosis. IGF1R overexpression has been observed in several cancers, including invasive bladder carcinomas, as a potential prognostic factor. Given known biologic differences between upper and lower urinary tract urothelial carcinoma, we assessed the expression status and prognostic significance of IGF1R in upper tract urothelial carcinoma (UTUC). Two tissue microarrays (TMAs) were built from 99 Japanese patients with non-metastatic UTUC submitted to radical nephroureterectomy between 1997 and 2011. TMAs were constructed with triplicate tumor and paired benign urothelium. Membranous IGF1R staining was evaluated using immunohistochemistry. Two scoring methods were applied (Her2-score and H-score). The highest score was assigned to each tumor. IGF1R positivity was defined as Her2-score ≥ 1+. Association with clinicopathologic parameters and outcome was assessed using hazard ratios (HR) with 95% confidence intervals (CI) and adjusted P values. We found positive IGF1R expression in 70% of UTUC. Outcomes were as follows: tumor recurrence, 33%; tumor progression, 59%; overall mortality, 33%; and cancer-specific mortality, 30%. IGF1R was not associated with any clinicopathologic features. In addition, IGF1R expression was not associated with tumor recurrence (HR = 0.54, CI = 0.25-1.1, P = 0.11), tumor progression (HR = 1.6, CI = 0.8-3.1, P = 0.19), overall mortality (HR = 1.5, CI = 0.68-3.4, P = 0.31), or cancer-specific mortality (HR = 1.6, CI = 0.68-3.8, P = 0.27). Positive IGF1R expression was found in more than two thirds of UTUC. This finding provides a rationale to investigate IGF1R as a potential therapeutic target in UTUC. In contrast to bladder cancer, IGF1R expression in UTUC did not correlate with outcome, further pointing to biologic differences between UTUC and bladder cancer.

Expression of inflammatory and fibrogenetic markers in acne hypertrophic scar formation: focusing on role of TGF-ß and IGF-1R.

Acne vulgaris is a universal skin disease and it may leave a scar when the original skin lesion disappears. These scars can cause cosmetic problems and psychological burden, leading to poor quality of life of patients. Acne scars are classified into atrophic scars and hypertrophic scars. As most of the acne scars are atrophic, many studies have been conducted focusing on the treatment of atrophic lesions. This study was conducted to investigate the underlying pathogenesis of acne hypertrophic scars by identifying roles of fibrogenetic and inflammatory markers. Skin biopsy samples were obtained from hypertrophic scars of face and back and from adjacent normal tissues as control group. Some samples from back were immature hypertrophic scars and the other samples were in mature stages. Immunohistochemistry staining and quantitative PCR were performed for fibrogenetic and inflammatory markers. Both in mature and immature hypertrophic scars, vimentin and α-SMA were increased. Production of TGF-ß3 protein as well as transcription of TGF-ß3 was also significantly elevated. In contrast, expression of TGF-ß1 showed no increase. Instead, expression levels of SMAD2 and SMAD4 were increased. Elevations of CD45RO, TNF-α and IL-4 and reduction of IL-10 were observed. In immature hypertrophic scars, IGF-1R and insulin-degrading enzyme expression were increased. Increased apoptosis was observed in immature stages of hypertrophic scars but not in mature stages. Elevations of TGF-ß3, SMAD2 and SMAD4 in hypertrophic scars and increase of IGF-1R in immature stages may give some clues for acne hypertrophic scar formation.

The association of diabetes mellitus and insulin treatment with expression of insulin-related proteins in breast tumors.

BACKGROUND: The insulin receptor (INSR) and the insulin growth factor 1 receptor (IGF1R) play important roles in the etiology of both diabetes mellitus and breast cancer. We aimed to evaluate the expression of hormone and insulin-related proteins within or related to the PI3K and MAPK pathway in breast tumors of women with or without diabetes mellitus, treated with or without insulin (analogues). METHODS: Immunohistochemistry was performed on tumor tissue of 312 women with invasive breast cancer, with or without pre-existing diabetes mellitus, diagnosed in 2000-2010, who were randomly selected from a Danish breast cancer cohort. Women with diabetes were 2:1 frequency matched by year of birth and age at breast cancer diagnosis to those without diabetes. Tumor Microarrays were successfully stained for p-ER, EGFR, p-ERK1/2, p-mTOR, and IGF1R, and scored by a breast pathologist. Associations of expression of these proteins with diabetes, insulin treatment (human insulin and insulin analogues) and other diabetes medication were evaluated by multivariable logistic regression adjusting for menopause and BMI; effect modification by menopausal status, BMI, and ER status was assessed using interactions terms. RESULTS: We found no significant differences in expression of any of the proteins in breast tumors of women with (n = 211) and without diabetes (n = 101). Among women with diabetes, insulin use (n = 53) was significantly associated with higher tumor protein expression of IGF1R (OR = 2.36; 95%CI:1.02-5.52; p = 0.04) and p-mTOR (OR = 2.35; 95%CI:1.13-4.88; p = 0.02), especially among women treated with insulin analogues. Menopause seemed to modified the association between insulin and IGF1R expression (p = 0.07); the difference in IGF1R expression was only observed in tumors of premenopausal women (OR = 5.10; 95%CI:1.36-19.14; p = 0.02). We found no associations between other types of diabetes medication, such as metformin, and protein expression of the five proteins evaluated. CONCLUSIONS: In our study, breast tumors of women with pre-existing diabetes did not show an altered expression of selected PI3K/MAPK pathway-related proteins. We observed an association between insulin treatment and increased p-mTOR and IGF1R expression of breast tumors, especially in premenopausal women. This observation, if confirmed, might be clinically relevant since the use of IGF1R and mTOR inhibitors are currently investigated in clinical trials.

Analysis of the IGF-system in milk from farm animals - Occurrence, regulation, and biomarker potential.

IGFs and IGF-binding proteins (IGFBPs) are abundantly present in milk and in dairy products. Compared to the IGFs, the IGFBP have received less attention in milk, although truncated IGFBPs and IGFBP-glycosylation have been described in milk. Thereby, complex control of local IGF-effects can be assumed on the levels of IGFBPs, proteases, and protease inhibitors. The present review collects the current knowledge both on presence and regulation of IGFs and IGFBPs in milk particularly from dairy animal species. As a rule higher levels of IGF-I, IGF-II, and IGFBPs are measured around parturition if compared to later time-points of lactation. In all farm animal species included in this review, it is found that the relative abundancies of IGFBPs in milk and serum are similar, with IGFBP-3 and -2 characterized by higher concentrations if compared to IGFBP-4 or -5. The concentrations of IGFs and IGFBPs in milk or dairy products can be altered by hormones, dairy processing, or fermentation. Because milk can be used for non-invasive biomarker research, quality management, and health monitoring, we discuss novel directions of IGF-analysis and potential on-site biomarker research in milk.

Insulin-like growth factor 1 receptor affects the survival of primary prostate cancer patients depending on TMPRSS2-ERG status.

BACKGROUND: Prostate cancer (PCa) is characterized by clinical and biological heterogeneity and has differential outcomes and mortality rates. Therefore, it is necessary to identify molecular alterations to define new therapeutic strategies based on the risk of progression. In this study, the prognostic relevance of the insulin-like growth factor (IGF) system was examined in molecular subtypes defined by TMPRSS2-ERG (T2E) gene fusion within a series of patients with primary localized PCa. METHODS: A cohort of 270 formalin-fixed and paraffin-embedded (FFPE) primary PCa samples from patients with more than 5 years' follow-up was collected. IGF-1R, IGF-1, IGFBP-3 and INSR expression was analyzed using quantitative RT-PCR. The T2E status and immunohistochemical ERG findings were considered in the analyses. The association with both biochemical and clinical progression-free survival (BPFS and PFS, respectively) was evaluated for the different molecular subtypes using the Kaplan-Meier proportional risk log-rank test and the Cox proportional hazards model. RESULTS: An association between IGF-1R overexpression and better BPFS was found in T2E-negative patients (35.3% BPFS, p-value = 0.016). Multivariate analysis demonstrated that IGF-1R expression constitutes an independent variable in T2E-negative patients [HR: 0.41. CI 95% (0.2-0.82), p = 0.013]. These data were confirmed using immunohistochemistry of ERG as subrogate of T2E. High IGF-1 expression correlated with prolonged BPFS and PFS independent of the T2E status. CONCLUSIONS: IGF-1R, a reported target of T2E, constitutes an independent factor for good prognosis in T2E-negative PCa. Quantitative evaluation of IGF-1/IGF-1R expression combined with molecular assessment of T2E status or ERG protein expression represents a useful marker for tumor progression in localized PCa.

Strong association of insulin-like growth factor 1 receptor expression with histologic grade, subtype, and HPV status in penile squamous cell carcinomas: a tissue microarray study of 112 cases.

Insulin-like growth factor-1 receptor (IGF1R) plays a key role in cell growth and transformation. It is overexpressed in several solid tumors. This study evaluates IGF1R immunoexpression in penile squamous cell carcinoma (SCC). Four tissue microarrays were built from formalin-fixed, paraffin-embedded blocks of 112 penile SCC from Paraguay. Membranous IGF1R expression was evaluated by immunohistochemistry using two different approaches. An H-score was calculated in each spot (stain intensity by extent), and a median score per tumor was obtained. The second approach consisted of a score similar to the scoring system that was used for evaluating HER2 immunoexpression. For each case, the highest category obtained at any spot was used for statistical analyses. IGF1R expression was compared by histologic subtype, grade, and human papillomavirus (HPV) status. Median H-score was 22.5. The distribution of IGF1R expression by HER2 approach was as follows: 0 in 33.0% cases, 1+ in 46.4%, 2+ in 14.3%, and 3+ in 6.2%. IGF1R H-scores were associated with basaloid and warty/basaloid subtypes (p = 0.0026) and higher grade (p = 0.00052). Although weaker when using the HER2 approach, the association of IGF1R expression with subtype (p = 0.015) and grade (p = 0.015) remained significant. Furthermore, there was an association between IGF1R expression by HER2 approach and HPV status (p = 0.012). IGF1R was expressed in about two thirds of penile SCC cases, showing a strong positive association with histologic grade, subtype, and HPV status. Considering that grade is a predictor of outcome IGF1R expression may have prognostic relevance and could point to a potential role for IGF1R inhibitors in treating penile SCC.

Expression of GRK2 and IGF1R in hepatocellular carcinoma: clinicopathological and prognostic significance.

AIM: It has been shown that G-protein-coupled receptor kinase 2 (GRK2) negatively regulates the insulin-like growth factor 1 receptor (IGF1R) signalling pathway in hepatocellular carcinoma (HCC). The aim of this study was to evaluate the clinicopathological and prognostic significance of GRK2 and IGF1R in HCC. METHODS: Expression of GRK2 and IGF1R was first detected by tissue microarray-based immunohistochemistry in 156 patients with HCC. Staining results, termed the H-score, were then correlated with clinicopathological variables and patient survival. Finally, the prognostic value of GRK2 and IGF1R was validated in the publically available TCGA (The Cancer Genome Atlas) RNA-sequencing database. RESULTS: The H-score of GRK2 staining (which was significantly lower in tumour than non-tumour tissue) was negatively associated with that of IGF1R with a reverse trend. No clinicopathological significance of the proteins was found except for a relationship between tumoral IGF1R expression and tumour-node-metastasis stage. In univariate analysis, high IGF1R expression predicted poor overall and disease-free survival, whereas GRK2 was not prognostic. In multivariate analysis, IGF1R was significant for overall survival. Furthermore, IGF1R was also of prognostic value in the TCGA database. CONCLUSIONS: Our data indicate that GRK2 and IGF1R show a negative correlation in HCC. IGF1R could be a potential marker of poor prognosis for this malignancy.

Combined and individual tumor-specific expression of insulin-like growth factor-I receptor, insulin receptor and phospho-insulin-like growth factor-I receptor/insulin receptor in primary breast cancer: Implications for prognosis in different treatment groups.

Clinical trials examining insulin-like growth factor-I receptor (IGF1R)-targeting strategies have emphasized that better predictive biomarkers are required to improve patient selection.Immunohistochemical tumor-specific protein expression of IGF1R, insulin receptor (InsR), and phosphorylated IGF1R/InsR (pIGF1R/InsR) individually and combined in relation to breast cancer prognosis was evaluated in a population-based cohort of 1,026 primary invasive breast cancer patients without preoperative treatment diagnosed in Sweden. IGF1R (n = 923), InsR (n = 900), and pIGF1R/InsR (n = 904) combined cytoplasmic and membrane staining was dichotomized. IGF1Rstrong/InsRmod/strong/pIGF1R/InsRpos tumors were borderline associated with 2-fold risk for events, HRadj (2.00; 95%CI 0.96-4.18). Combined IGF1R and pIGF1R/InsR status only impacted prognosis in patients with InsRmod/strong expressing tumors (Pinteraction = 0.041). IGF1Rstrong expression impacted endocrine treatment response differently depending on patients' age and type of endocrine therapy. Phospho-IGF1R/InsRpos was associated with lower risk for events among non-endocrine-treated patients irrespective of ER status, HRadj (0.32; 95%CI 0.16-0.63), but not among endocrine-treated patients (Pinteraction = 0.024). In non-endocrine-treated patients, pIGF1R/InsRpos was associated with lower risk for events after radiotherapy, HRadj (0.31; 95%CI 0.12-0.80), and chemotherapy, HRadj (0.29; 95%CI 0.09-0.99). This study highlights the complexity of IGF hetero-and homodimer signaling network and its interplay with endocrine treatment, suggesting that combinations of involved factors may improve patient selection for IGF1R-targeted therapy.

Towards a multi protein and mRNA expression of biological predictive and distinguish model for post stroke depression.

Previous studies suggest that neurotrophic factors participate in the development of stroke and depression. So we investigated the utility of these biomarkers as predictive and distinguish model for post stroke depression (PSD). 159 individuals including PSD, stroke without depression (Non-PSD), major depressive disorder (MDD) and normal control groups were recruited and examined the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptors (VEGFR2), placental growth factor (PIGF), insulin-like growth factor (IGF-1) and insulin-like growth factor receptors (IGF-1R). The chi-square test was used to evaluate categorical variable, while nonparametric test and one-way analysis of variance were applied to continuous variables of general characteristics, clinical and biological changes. In order to explore the predictive and distinguish role of these factors in PSD, discriminant analysis and receiver operating characteristic curve were calculated. The four groups had statistical differences in these neurotrophic factors (all P < 0.05) except VEGF concentration and IGF-1R mRNA (P = 0.776, P = 0.102 respectively). We identified these mRNA expression and protein analytes with general predictive performance for PSD and Non-PSD groups [area under the curve (AUC): 0.805, 95% CI, 0.704-0.907, P < 0.001]. Importantly, there is an excellent predictive performance (AUC: 0.984, 95% CI, 0.964-1.000, P < 0.001) to differentiate PSD patients from MDD patients. This was the first study to explore the changes of neurotrophic factors family in PSD patients, the results intriguingly demonstrated that the combination of protein and mRNA expression of biological factors could use as a predictive and discriminant model for PSD.

Validation of a Preclinical Model of Diethylnitrosamine-Induced Hepatic Neoplasia in Yucatan Miniature Pigs.

OBJECTIVE: The purpose of this study was to reduce the time to tumor onset in a diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) swine model via partial liver embolization (PLE) and to characterize the model for use in translational research. METHODS: Eight Yucatan miniature pigs were injected intraperitoneally with either saline (n = 2) or DEN (n = 6) solution weekly for 12 weeks. Three of the DEN-treated pigs underwent PLE. The animals underwent periodic radiological evaluation, liver biopsy, and blood sampling, and full necropsy was performed at study termination (∼29 months). RESULTS: All DEN-treated pigs developed hepatic adenoma and HCC. PLE accelerated the time to adenoma development but not to HCC development. Biomarker analysis results showed that IGF1 levels decreased in all DEN-treated pigs as functional liver capacity decreased with progression of HCC. VEGF and IL-6 levels were positively correlated with disease progression. Immunohistochemical probing of HCC tissues demonstrated the expression of several important survival-promoting proteins. CONCLUSION: To our knowledge, we are the first to demonstrate an accelerated development of hepatic neoplasia in Yucatan miniature pigs. Our HCC swine model closely mimics the human condition (i.e., progressive disease stages and expression of relevant molecular markers) and is a viable translational model.

Expression and clinical significance of epidermal growth factor receptor and insulin-like growth factor receptor 1 in patients with ampullary adenocarcinoma.

Epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF-1R) play important roles in cell proliferation, antiapoptosis, angiogenesis, and metastasis and have been used for targeted therapies for patients with advanced colorectal and lung cancers. However, the expression and function of EGFR and IGF-1R in ampullary adenocarcinoma (AA) have not been examined in detail. We examined the expression of EGFR and IGF-1R in 106 AA patients at our institution using tissue microarrays and immunohistochemistry. The results were correlated with the clinicopathological parameters and survival. Overexpression of EGFR and IGF-1R was detected in 18 (17%) and 26 (25%) of AAs, respectively. Patients with EGFR-high tumors had shorter overall survival (mean, 109.8 ± 22.3 months) than those patients whose tumors were EGFR-low in overall study population (mean, 164.2 ± 10.6 months; P = .04). Overexpression of EGFR correlated with poor overall survival in patients with intestinal-type AA (P = .01) but not in those with pancreaticobiliary-type AAs (P = .47). In multivariate analysis, EGFR overexpression was an independent prognostic factor for overall survival (P = .02). In addition, we found that overexpression of IGF-1R correlated with AAs of pancreaticobiliary histology. No additional correlation between the expression of EGFR or IGF-1R and other clinicopathological factors was observed in our patient population. Our study demonstrates that EGFR and IGF-1R appear to be overexpressed in a subset of AAs and that strong membranous expression of EGFR is an independent predictor for overall survival in patients with AA. EGFR and IGF-1R represent potential therapeutic targets for treatment of patient with AAs.

Insulin-like growth factor receptor 1 (IGF1R) expression and survival in non-small cell lung cancer patients: a meta-analysis.

The insulin-like growth factor receptor-1 (IGF1R) plays an important role in cancer progression. Previous studies have been controversial with respect to the associations between IGF1R expression and non small cell lung cancer (NSCLC) prognosis. Thus, we performed a meta-analysis to investigate the prognostic value of IGF1R expression in NSCLC patients and the relationship between the expression of IGF1R and clinical characteristics. Two independent reviewers searched PubMed, Embase, Ovid Medline and CNKI to identify eligible studies. Overall survival (OS), disease free survival (DFS) and clinicopathological characteristics were collected from included studies. Pooled hazard ratios (HRs) or odds ratios (ORs) with 95% confidence interval (95% CI) were calculated to estimate the effect. 17 studies comprising 3,294 patients were included in this meta-analysis. The results showed IGF1R positive expression was associated with an unfavorable DFS in NSCLC patients on univariate analysis (HR = 1.26, 95% CI: 1.09-1.46, P = 0.002) and multivariate analysis (HR = 1.49, 95% CI: 1.01-2.20, p = 0.045), but the relationship between IGF1R expression and OS have no significant difference on univariate analysis (HR = 0.91, 95% CI: 0.82-1.01, P = 0.157) and multivariate analysis (HR = 0.79, 95% CI: 0.45-1.41, P = 0.427). Ever smoking and smaller tumor size (T1 or T2) were associated with IGF1R positive expression: pooled OR 1.45 (1.13-1.85) and pooled OR 0.61 (0.60-0.95). Our results suggested IGF1R positive expression as an unfavorable factor for DFS in NSCLC patients, and IGF1R expression was associated with smoking status and tumor size.

Differences in IGF axis-related proteins in amniotic fluid of trisomy 21 and trisomy 18 using a multiple reaction monitoring approach.

OBJECTIVE: Trisomy 21 and trisomy 18 are the two most common chromosomal anomalies in live births. To find new biomarkers for aneuploidies and pathogenesis of fetal malformations, we measured insulin-like growth factor (IGF) axis-related proteins in amniotic fluid (AF) of pregnant women carrying trisomies 21 or 18 affected fetuses using multiple reaction monitoring (MRM) approach. METHOD: Eighty-five AF samples from pregnant women carrying either trisomy 21, trisomy 18, or normal fetuses were collected. IGF axis-related proteins in AF after serial treatments were quantitated with MRM method. The differential protein levels were also confirmed by western blot in AF without any treatment. RESULTS: The IGF type I receptor and pregnancy-associated plasma protein-A in AF of trisomy 21 (1.35 ± 0.32 and 13.36 ± 3.64 µg/mg protein) and trisomy 18 (1.39 ± 0.40 and 12.80 ± 1.84 µg/mg protein) were decreased versus normal controls (2.16 ± 0.59 and 23.77 ± 6.18 µg/mg protein). IGF binding protein 5 was reduced in trisomy 18 (1.47 ± 0.33 vs 2.36 ± 0.77 µg/mg protein). These alterations were confirmed by western blot. The other proteins showed no significant difference between the three groups. CONCLUSION: Our data suggested that MRM can provide a powerful platform for the identification of biomarkers in AF that have crucial developmental effects in the aneuploid fetus.

Assessment of the expression of IRß, IRS-1, IRS-2 and IGF-IRß in a rat model of intrauterine growth restriction.

OBJECTIVE: To investigate glomerular development and expression of insulin and insulin-like growth factor receptors in an experimental model of intrauterine growth restriction (IUGR). MATERIAL AND METHODS: We studied three groups of Sprague-Dawley fetuses: IUGR - restricted by ligation of the right uterine artery; C-IUGR - left horn controls, and EC - external controls (non-manipulated). Body and organs were weighed, and glomerular number and volume were analyzed. Expression of IRß, IRS-1, IRS-2 and IGF-IRß was analyzed in liver, intestine and kidneys by immunoblotting. RESULTS: Organ/body weight ratios were similar. In IUGR, glomerular number and volume were increased compared to C-IUGR and EC (p<0.001). In the IUGR liver, increases were found in IGF-IRß compared to C-IUGR and EC; IRß compared to EC, and IRS-2 compared to C-IUGR. However, decreases in IRß were noted in IUGR compared to C-IUGR; IRS-1 compared to C-IUGR and EC, and IRS-2 compared to EC. In IUGR intestine, increases were detected in IRß, IRS-1 and IGF-IRß compared to C-IUGR and EC. In IUGR kidneys, increases were observed in IRß and IGF-IRß compared to C-IUGR and EC, and IRS-1 compared to EC. Decreased IRS-2 in the intestine and kidney were noticed in IUGR compared to C-IUGR and EC. CONCLUSION: IUGR fetuses had less glomeruli and alterations in insulin receptors, which may be associated with an increased risk of disease occurrence in adulthood.

Effects of diet consistency on the expression of insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins during the development of rat masseter muscle soon after weaning.

A soft diet facilitates the development of faster-type fibres in rat masseter muscle in the 9 days after weaning compared with a hard diet. To determine whether insulin-like growth factors (IGFs), IGF receptors (IGFRs) and IGF binding proteins (IGFBPs) are involved in this fibre-type alteration, the expression of myosin heavy chain (MHC), IGF, IGFR and IGFBP mRNAs in the masseter muscle of rats fed a hard or soft diet for 9 days after weaning was analysed using competitive, reverse transcriptase-polymerase chain reaction. A soft diet decreased the expression of MHC IIa (slower type) by 70%, but increased the expression of MHC IIx (intermediate type) and IIb (faster type) by 80 and 582%, respectively, compared with a hard diet. These findings verified that a soft diet facilitates the development of faster-type fibres in rat masseter muscle compared with a hard diet. A soft diet induced reductions of 25-76% (P < 0.05-0.01) in the expression of IGF-I, IGF-II, IGFR2, IGFBP4 and IGFBP6 compared with a hard diet, but induced a 25% (P < 0.05) increase only in expression of IGFBP3. These findings suggest that the changes in expression of IGF-I, IGF-II, IGFR2, IGFBP3, IGFBP4 and IGFBP6 are associated with the fibre-type alteration of rat masseter muscle in response to diet consistency soon after weaning.

Analysis of chondrocyte functional markers and pericellular matrix components by flow cytometry.

Flow cytometry has been used as a procedure to characterize the phenotype and function of human articular cartilage cells cultured as monolayers or in gelled artificial matrices. Procedures allowing intact cells with their cell-associated matrix, to be obtained have been described. Appropriate monoclonal antibodies have allowed plasma membrane-associated proteins, e.g., growth factors and cytokine receptors, as well as the cell-associated extracellular matrix macromolecules, to be studied. Intracellular compounds have been traced in permeabilized cells after blocking of their intracellular transport and secretion mechanisms. We report the use of fluorescent dye-labeled monoclonal antibodies or specific binding proteins against extracellular matrix compounds such as hyaluronan, aggrecan, types I and II collagen, and fibronectin. The autocrine and paracrine growth factor and cytokine pathways considered include the insulin-like growth factor-1 (IGF-1)/IGF receptor I (IGFRI), and the transforming growth factor-beta1 (TGF-beta1)/TGF-beta receptor II (TGF-betaRII) cascades, as well as the interleukin-1alpha/beta (IL-1alpha/beta)/interleukin-1 receptors I and II (IL-1RI and II) systems. Catabolic enzymes that mediate extracellular matrix turnover, e.g., some matrix metalloproteinases and their natural inhibitors, were also studied. Finally, flow cytometry was used to assess the results of some pharmacological interventions on the aforementioned variables in cultured chondrocytes.

Expression of insulin-like growth factors (IGF)-1 and -2, IGF-binding proteins-2 and -3, and receptors for growth hormone, IGF type-1 and -2 and insulin in the gastrointestinal tract of neonatal calves.

Growth hormone (GH), insulin-like growth factors (IGFs) and insulin influence post-natal gastrointestinal development and function. We have measured by real-time PCR the mRNA levels of IGF-1 and -2, IGF-binding proteins (IGFBPs)-2 and -3, and receptors for GH, IGF type-1 and -2, and insulin in esophagus, rumen, fundus, pylorus, duodenum, jejunum, ileum and colon of calves on days 1 and 5 of life. Levels of mRNA of measured traits were different (P < 0.05) at different gastrointestinal sites. Furthermore, mRNA levels of IGFs, IGFBPs and of receptors for GH and IGF type-1 and -2 and insulin differed (P < 0.05) on days 1 and 5. Differences in mRNA abundance of IGFs, IGFBPs and of receptors for GH, IGFs, and insulin among gastrointestinal sites on days 1 and 5 of life suggest site-specific functional importance and demonstrate that changes are the consequence of ontogenetic development and/or due to feeding.

Involvement of insulin-like growth factor binding protein-2 in activated microglia as assessed in post mortem human brain.

In vitro studies suggest that insulin-like growth factor (IGF)-I is a mitogen for microglia/macrophages. The actions of IGF-I are mediated by IGF-I receptors and modulated by IGF binding proteins (IGFBPs). The aim of this study was to investigate IGF-I receptors and IGFBPs in human microglia in normal brain white matter and active lesions of multiple sclerosis, which contain activated microglia/macrophages. Methods used were immunohistochemistry and confocal laser microscopy. IGF-I receptors were demonstrated in both resting and activated microglia. In resting conditions, microglia displayed no immunoreactivity for any of the six IGFBPs, whereas activated microglia/macrophages were immunoreactive for IGFBP-2 only. Our data suggest an important function for IGFBP-2 in IGF-I actions in activated microglia/macrophages in human brain.

Gastric cancer: the role of insulin-like growth factor 2 (IGF 2) and its receptors (IGF 1R and M6-P/IGF 2R).

Insulin-like growth factor 2 (IGF 2) appears to be involved in the progression of many tumours. It binds to at least two different types of receptor: IGF type 1 (IGF 1R) and mannose 6-phosphate/IGF type 2 (M6-P/IGF 2R). Ligand binding to IGF 1R provokes mitogenic and anti-apoptotic effects. M6-P/IGF 2R has a tumour suppressor function--it mediates IGF 2 degradation. Mutation of M6-P/IGF 2R causes both diminished growth suppression and augmented growth stimulation. The aim of this study was to investigate the role of IGF 2 and its receptors (IGF 1R and IGF 2R) in human gastric cancer. The expression of IGF 2 and its receptors was measured in order to analyse the possible correlation between the activity of these genes and cell proliferation in two different gastric tumour types: diffuse and intestinal. The effect of IGF 1 receptor blockage on cell proliferation and anchorage-independent cell growth was also examined. Increased expression of IGF 2 and IGF 1R genes (at the mRNA and protein level) was found in gastric cancer when compared with non-tumour tissue. Furthermore, there was a significant difference between IGF 2 expression in the more aggressive diffuse type and that in the intestinal type of gastric cancer. Moreover, the IGF 2 peptide level in the culture media obtained from the diffuse type of cancer cells was significantly higher when compared with the intestinal type. The level of IGF 2 peptide in the conditioned media strongly correlated with [3H]thymidine incorporation and cell proliferation. On the contrary, IGF 2R mRNA expression was much higher in the intestinal type of cancer than in the diffuse type. In addition, IGF 2R protein expression was substantially lower with progression of the diffuse cancer type to a higher stage. The alphaIR3 monoclonal antibody strongly inhibited [3H]thymidine incorporation and decreased the number of colonies in soft agar of cells overexpressing IGF 2. These findings suggest that members of the IGF family are involved in the pathogenesis of gastric cancer, probably by autocrine/paracrine stimulation of cell growth. Such tumours might be excellent candidates for therapeutic strategies aimed at interference with this pathway.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.