Insulin-like Growth Factor Binding Protein-related Protein 1 Research Articles

IGFBP‑rP1 affects the proliferation, apoptosis and macrophage polarization of endometrial cancer cells by regulating the PI3K/AKT pathway.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor gene in a variety of cancers including colorectal cancer and breast cancer. However, its role and the potential mechanism in endometrial carcinoma (EC) are still unclear. The purpose of this study was to explore the effect of IGFBP-rP1 on EC cell proliferation and apoptosis and its underlying mechanism. Western blot analysis and reverse transcription-quantitative PCR were used to assess protein and gene expression levels of IGFBP-rP1 in EC cells. Overexpression of IGFBP-rP1 and/or AKT serine/threonine kinase was used to analyze their effects on cell proliferation and apoptosis of the EC cells. Co-immunoprecipitation and glutathione S transferase pull-down assays were used to analyze the interaction between IGFBP-rP1 and AKT. The expression of IGFBP-rP1 in EC cells was downregulated. Overexpression of IGFBP-rP1 inhibited the proliferation and induced apoptosis of EC cells, which were abolished by the overexpression of AKT. In addition, IGFBP-rP1 directly interacted with AKT to inhibit PI3K/AKT signaling. Additionally, M0 macrophages were induced by EC cells to differentiate into M2 macrophages, which was reversed by IGFBP-rP1. Overexpression of AKT in EC cells abolished the inhibitory effect of IGFBP-rP1 on M2 polarization. IGFBP-rP1 as an oncogenic factor inhibits M2 polarization of TAMs through PI3K/AKT signaling pathway and may be a potentially valuable target for EC therapy.

IGFBP‑rP1‑silencing promotes hypoxia‑induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has been reported to have various functions in different cellular contexts. Our previous investigation discovered that IGFBP-rP1 inhibited retinal angiogenesis in vitro and in vivo by inhibiting the pro-angiogenic effect of VEGF and downregulating VEGF expression. Recently, IGFBP-rP1 was confirmed to be downregulated in the aqueous humor of patients with neovascular age-related macular degeneration compared with controls; however, its specific role remains unknown. The present study applied the technique of gene silencing, reverse transcription-quantitative PCR, western blotting, cell viability assays, cell motility assays and tube formation assays. Chemical hypoxic conditions and choroidal endothelial (RF/6A) cells were used to explore the effect of IGFBP-rP1-silencing on the phenotype activation of RF/6A cells under hypoxic conditions and to elucidate the underlying mechanisms. siRNA achieved IGFBP-rP1-silencing in RF/6A cells without cytotoxicity. IGFBP-rP1-silencing significantly restored the viability of RF/6A cells in hypoxia and enhanced hypoxia-induced migration and capillary-like tube formation of RF/6A cells. Furthermore, IGFBP-rP1-silencing significantly upregulated the expression of B-RAF, phosphorylated (p)-MEK, p-ERK and VEGF in RF/6A cells under hypoxic conditions; however, these upregulations were inhibited by exogenous IGFBP-rP1. These data indicated that silencing IGFBP-rP1 expression in RF/6A cells effectively promoted the hypoxia-induced angiogenic potential of choroidal endothelial cells by upregulating RAF/MEK/ERK signaling pathway activation and VEGF expression.

SIRT1/IGFBPrP1/TGF β1 axis involved in cucurbitacin B ameliorating concanavalin A-induced mice liver fibrosis.

The present study investigated the improving effect of cucurbitacin B on liver fibrosis induced by concanavalin A in mice and explored its possible mechanism. AST, ALT and TB were detected by kits. ELISA was performed to detect the levels of IL 5, IL 6, IL 13 and TNF-α in serum. Haematoxylin-eosin (HE) staining and Masson's trichrome staining were used to evaluate pathological changes. Western blotting was performed to observe expression levels of sirtuin (SIRT) 1, insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) and TGF β1. The activity of SIRT 1 also was detected. Results showed that cucurbitacin B could effectively improve the abnormal liver function, inhibit liver fibrosis and suppress releases of inflammatory factors in mice induced by concanavalin A. Furthermore, cucurbitacin B could down-regulate the expressions of TGF β1 and IGFBPrP1, increase the expression and activity of SIRT 1. Interestingly, when SIRT1 activity was inhibited by EX 527, a selective inhibitor of SIRT 1, the preventive effect of cucurbitacin B was significantly attenuated. Taken together, the above results showed that cucurbitacin B could significantly suppress releases of inflammatory cytokines and improve liver fibrosis induced by concanavalin A in mice, and those may be achieved through SIRT1/IGFBPrP1/TGF β1 axis.

Insulin-Like Growth Factor Binding Protein-Related Protein 1 Activates Primary Hepatic Stellate Cells via Autophagy Regulated by the PI3K/Akt/mTOR Signaling Pathway

BackgroundAutophagy is a self-degrading process. Previously, we showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel transforming growth factor β1 (TGFβ1)-interacting factor in liver fibrosis; the role of TGFβ1-mediated autophagy in hepatic stellate cells (HSCs) activation has been investigated. However, whether autophagy is regulated by IGFBPrP1 remains unknown.AimsWe investigated the interactions among IGFBPrP1, autophagy, and activation of primary rat HSCs.MethodsPrimary HSCs were separated from Sprague Dawley rats by two-step enzymatic digestion, and then, we overexpressed or inhibited IGFBPrP1 expression in HSCs under serum-starved condition. Autophagy inducer rapamycin or inhibitor 3-methyladenine (3MA) was used to assess the relationship between autophagy and HSCs activation.ResultsWe observed the expression of activation marker α-SMA and autophagy markers such as LC3B and Beclin1, which were significantly increased in HSCs treated with adenovirus vector harboring the IGFBPrP1 gene (AdIGFBPrP1) compared to cells cultured under serum-starved. In comparison, HSCs treated with shIGFBPrP1 showed opposite results. Furthermore, HSCs activation and autophagy increased when cells were treated with rapamycin, whereas opposite results were obtained when cells were treated with 3MA. AdIGFBPrP1 treatment downregulated the phosphorylation of Akt and mTOR.ConclusionAutophagy was induced in IGFBPrP1-treated primary HSCs, and IGFBPrP1-induced autophagy promoted the activation of HSCs and extracellular matrix expression, the underlying mechanism of which may involve the phosphatidylinositide 3-kinase/Akt/mTOR signaling pathway.

Expression and significance of insulin-like growth factor binding protein-related protein 1 in pancreatic neoplasms and its relationship with tumor microenvironment

Objective To observe the expression of insulin-like growth factor binding protein-related protein 1(IGFBPrP1) in human pancreatic tumor tissues and investigate its significance and relationship with clinic pathological characteristics and tumor microenvironment of the pancreatic neoplasms. Methods A total of 236 patients with surgically resected pancreatic tissue from January 2007 to December 2017 were selected from the First Hospital of Shanxi Medical University. Totally 236 patients were divided into paracancer control group (normal pancreatic tissue adjacent to the tumor, n=111), benign group (benign or low-grade malignant tumor such as solid pseudopapillary tumor, n=37), and malignant group (malignant tumors such as pancreatic ductal adenocarcinoma, n=88). The histomorphology and collagen deposition were observed using hematoxylin-eosin (H-E) staining and Sirius red staining in the three groups. The expressions of IGFBPrP1, transforming growth factor β1 (TGFβ1), α-smooth muscle actin (α-SMA)and collagen type Ⅰ of pancreatic tissues in the three groups were detected by immunohistochemical staining. The relationship between IGFBPrP1 and TGFβ1, α-SMA or collagen type Ⅰ, and the relathionship between IGFBPrP1 and clinicopathological features of the pancreatic neoplasms were analyzed. T test and one-way analysis of variance were used for statistical analysis, and Spearman rank correlation test was used for correlation analysis. Results In benign group and malignant group, there were obvious cell atypia, and the cell atypia of malignant group was more significant than benign group. The contents of collagen fibers in benign group and malignant group were significantly higher than that in paracancer control group. IGFBPrP1, TGFβ1, α-SMA and collagen typeⅠwere highly expressed in the endochylema of the tumor cells and (or) the myofibroblast. The expression level of IGFBPrP1 in highly differentiated ductal adenocarcinoma was significantly higher than that in moderately and poorly differentiated ductal adenocarcinoma ((9.46±2.10)×104 vs. (6.48±1.38)×104 and (6.07±1.29)×104); t=7.430 and 6.767, both P<0.05). The expression of IGFBPrP1 in human pancreatic neoplasms was positively correlated with TGFβ1, α-SMA and collagen typeⅠ(r=0.530, 0.619, 0.625; all P<0.05). Conclusions IGFBPrP1 is highly expressed in pancreatic tumor tissue and its expression level may correlate with the histological grade of pancreatic neoplasms. The expression of IGFBPrP1 in human pancreatic tumor tissues may be accompanied by the activation of pancreatic stellate cells and the generation of cancer-related fibroblasts, and IGFBPrP1 may involve in the formation of tumor by changing the tumor microenvironment. Key words: Insulin-like growth factor binding protein-related protein 1; Pancreatic neoplasms; Tumor microenvironment; Transforming growth factor beta1; α-smooth muscle actin; Collagen type Ⅰ

Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic fibrosis via the regulation of MMPs/TIMPs in mice

BackgroundPrevious research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. MethodsHepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor β 1 (TGFβ1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis. ResultsWe found that hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. ConclusionsOur findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFβ1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

The role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) in endometrial carcinoma

Objective: Insulin resistance is a high risk factor of Endometrial Carcinoma (EC). Insulin like Growth Factor Binding Protein-Related Protein 1 (IGFBP-rP1) could lead to insulin resistance due to its high affinity with insulin. The role of IGFBP-rP1 in EC is still unclear. Method: The IGFBP-rP1 was overexpressed at the cellular level first, and then its effect on the growth of endometrial carcinoma cells was observed and its regulatory mechanism was investigated. Results: Our study showed that IGFBP-rP1 upregulation decreased the proliferation of cells and the proportion of S+G2/M phase, but increased the apoptosis of human endometrial cancer cells HEC-1A. The expression of p-ERK1/2 was inhibited, while the concentration of rhIGFBP-rP1 increased. Nevertheless, IGFBP-rP1 was highly expressed in EC tissues. Further examination revealed that the DNA hypomethylation in the promoter region of IGFBP-rP1 was correlated with the high mRNA and protein expression levels of IGFBP-rP1 in EC tissues. Conclusion: These results indicate that the DNA hypomethylation of IGFBP-rP1 can be used to predict high risk of EC. Further study may be required to confirm the predictor role of the methylation of IGFBP-rP1 in EC.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGFβ1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells (HSCs). We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin (α-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). We found that the adenovirus vector encoding the TGFβ1 gene (AdTGFβ1) induced IGFBPrP1 expression while that of α-SMA, collagen I, fibronectin, and TGFβ1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGFβ1, α-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA, collagen I, fibronectin, and p-Smad2/3. These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner, and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway.

Insulin-Like Growth Factor Binding Protein-Related Protein 1 Inhibit Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

To explore the inhibitory effect of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) on retinal angiogenesis and its underlying molecular mechanisms in the mouse model of oxygen-induced retinopathy (OIR). C57BL/6J mice were classified into three groups as control group, OIR nonintervention group, and OIR intervention group. Postnatal day 12 (P12) mice in OIR intervention group were received recombinant mouse IGFBP-rP1 (50, 100, and 200 ng/mL) intravitreal injection. Five days later, the proliferative neovascular responses were estimated by quantifying the new vessel areas in flattening retinal tissues stained by high molecular fluorescein isothiocyanate-dextran and counting the numbers of neovascular cell nuclei breaking through the internal limiting membrane in cross sections. Expressions of phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, and vascular endothelial growth factor (VEGF) proteins in retinal tissues were assessed by western blot analysis. Irregular neovascularization, nonperfusion region, and fluorescence leakage were observed in OIR models. The expression of retinal p-ERK1/2 and VEGF proteins were significantly upregulated in OIR nonintervention group compared with control group. The area ratio of retinal new vessels and the number of neovascular cell nuclei in OIR intervention group both decreased significantly, following the downregulation of retinal p-ERK1/2 protein expression and VEGF protein expression in a dose-dependent manner. Moreover, there was no significant difference in retinal ERK1/2 protein expression. IGFBP-rP1 inhibits retinal angiogenesis by blocking ERK signaling pathway and downregulating VEGF expression in the mouse model of OIR. It highlights the potential importance of IGFBP-rP1 serving as a target of gene therapy for retinal neovascularization in the future.

Inhibitory effect of insulin-like growth factor binding protein-related protein 1 on retinal angiogenesis via ERK signaling pathway

Objective: To explore the inhibitory effect of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a novel anti-angiogenic factor, on retinal angiogenesis and its underlying molecular mechanisms. Methods: Experimental study. C57BL/6J mice were classified into three groups: control group (n=24), oxygen-induced retinopathy (OIR) non-intervention group (n=24) and OIR intervention group (n=72). The OIR mouse model was established using improved Smith's methods (n= 96). Twelve-day-old mice in the OIR intervention group were randomly assigned into three groups receiving intravitreal injection of recombinant mouse IGFBP-rP1 (50 μg/L, 100 μg/L and 200 μg/L, respectively). Five days later, the proliferative neovascular responses were estimated by quantifying the new vessel area relative to the total retinal area in flattening retinas stained by high molecular FITC-Dextran and counting the number of neovascular cell nuclei breaking through the internal limiting membrane (ILM) in cross-sections. Retinal phosphor-ERK1/2 (p-ERK1/2), ERK1/2 and vascular endothelial growth factor (VEGF) protein expression was assessed by Western blot. Results: In the fluorescence angiograms, irregular neovascularization and fluorescence leakage were observed in the OIR model. In the OIR non-intervention group, the expression of p-ERK1/2 and VEGF was significantly up-regulated in comparison with the control group (t=100.068, P=0.000. t=6.526, P=0.003). The area ratios of new retinal vessels and the number of neovascular cell nuclei in mice receiving intravitreal injection of recombinant mouse IGFBP-rP1 both decreased significantly (F=1920, P=0.000. F=852.387, P=0.000), following the down-regulation of retinal p-ERK1/2 protein expression (F=859.587, P=0.000) and VEGF protein expression (F=24.301, P= 0.000) in a dose-dependent manner (P<0.05). There was no significant difference in ERK1/2 protein expression (P>0.05). Conclusions: IGFBP-rP1 inhibits retinal angiogenesis by blocking ERK signaling pathway and down-regulating VEGF expression. This highlights the potential importance of IGFBP-rP1 serving as a target of gene therapy for retinal neovascularization. (Chin J Ophthalmol, 2017, 53: 207-211).

IGFBP-rP1 acts as a potential tumor suppressor via the suppression of ERK signaling pathway in endometrial cancer cells.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor‑suppressor gene in various cancers. However, its biological role and underlying mechanism has not been well investigated in endometrial cancer yet. The aim of the present study aimed to investigate the role and underlying molecular mechanisms of IGFBP‑rP1 in endometrial cancer cells invitro. The authors used transfection of IGFBP‑rP1 or small interfering (si)RNA in endometrial cancer HEC‑1A or Ishikawa cells, respectively. Biological functional alterations, such as cell growth and cell cycle were analyzed in endometrial cancer cells, combined with the use of PD98059. A panel of proteins including phospho‑retinoblastoma (p‑RB) and p‑extracellular signal‑regulated kinase (ERK)/ERK were detected by western blot analysis. It was observed that IGFBP‑rP1 transfection inhibited cell growth, and induced G1 phase arrest and cellular senescence in HEC‑1A cells while gene silencing presented the adverse functional changes. Moreover, p‑RB and p‑ERK were significantly downregulated or upregulated in HEC‑1A‑IGFBP‑rP1 cells or Ishikawa‑siRNA (IGFBP‑rP1) compared with control cells, respectively. These observations were reinforced in endometrial cancer cells by PD98059 treatment. The authors conclude that IGFBP‑rP1 acts as a potential tumor suppressor via the suppression of the ERK signaling pathway in endometrial cancer cells. These findings suggested that IGFBP‑rP1 may serve as a potential therapeutic target for cancer intervention in the future.

Screening for and validation of a hepatic fibrosis-related pathway induced by insulin-like growth factor-binding protein-related protein 1.

Our previous studies characterized insulin-like growth factor-binding protein-related protein 1 (IGFBPrP1) as a molecule that promotes hepatic fibrogenesis, but its mechanism has not been fully elucidated. Here, we have investigated the effect of IGFBPrP1 on gene expression in the hepatic fibrosis-related pathway. Sprague-Dawley rats received injections of an adenovirus carrying IGFBPrP1 or EGFP cDNA into their tail veins. In hepatic preparations, hepatic stellate cell activation was determined by α-smooth muscle actin expression and hepatic fibrosis by Sirius red staining and hydroxyproline content analysis. IGFBPrP1-inducible genes of the hepatic fibrosis-related pathway were assessed by PCR array. Expression of IGFBPrP1 and transforming growth factor β1 (TGFβ1) and array results were evaluated by quantitative real-time PCR and western blotting. IGFBPrP1-overexpressing rats showed an increase in α-smooth muscle actin expression and collagen and hydroxyproline content in the liver. The PCR array results indicated that some genes were upregulated and some were downregulated in Ad-IGFBPrP1-infected rats. Among these, Egr1, MAP2K2 (MEK2) and MAPK3 (ERK1) expression increased, whereas PTEN and Hhip mRNA expression decreased. Egr1 protein levels increased and peaked 2 weeks after infection and then decreased gradually. PTEN protein decreased gradually in Ad-IGFBPrP1-infected rats with a concurrent increase in the degree of hepatic fibrosis. TGFβ1 levels increased during hepatic fibrosis development in liver tissues. Egr1, PTEN, Hhip, MAP2K2 (MEK2) and MAPK3 (ERK1) were identified as candidate genes of the IGFBPrP1-induced hepatic fibrosis-related pathway. IGFBPrP1 promoted hepatic fibrosis mainly by enhancing the TGFβ1 expression that it triggered.

IGFBP-rP1 suppresses epithelial-mesenchymal transition and metastasis in colorectal cancer.

Epithelial–mesenchymal transition (EMT) was initially recognized during organogenesis and has recently been reported to be involved in promoting cancer invasion and metastasis. Cooperation of transforming growth factor-β (TGF-β) and other signaling pathways, such as Ras and Wnt, is essential to inducing EMT, but the molecular mechanisms remain to be fully determined. Here, we reported that insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a potential tumor suppressor, controls EMT in colorectal cancer progression. We revealed the inhibitory role of IGFBP-rP1 through analyses of clinical colorectal cancer samples and various EMT and metastasis models in vitro and in vivo. Moreover, we demonstrated that IGFBP-rP1 suppresses EMT and tumor metastasis by repressing TGF-β-mediated EMT through the Smad signaling cascade. These data establish that IGFBP-rP1 functions as a suppressor of EMT and metastasis in colorectal cancer.

Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) contributes to liver inflammation and fibrosis via activation of the ERK1/2 pathway.

Previously, we suggested that IGFBPrP1 played a major role in hepatic stellate cell (HSC) activation, yet the molecular mechanism of IGFBPrP1 in hepatic fibrosis is unclear. The ERK pathway is involved in activation of HSCs. This study investigated the involvement of the ERK1/2 pathway in IGFBPrP1-induced liver inflammation and fibrosis. An adenoviral vector encoding IGFBPrP1 (AdIGFBPrP1) was constructed. Rats received AdIGFBPrP1 or CAd (vector control) via their tail vein injection. One hour prior to adenoviral injections, rats were intraperitoneally administrated with 10 mg/kg U0126 (a specific MEK/ERK1/2 inhibitor) or DMSO (vehicle control). At weeks 2 or 4 post-gene transduction, serum samples were obtained and the levels of liver enzymes and hydroxyproline were determined. Liver tissue were histologically evaluated for inflammation and fibrosis. The expression of α-SMA and ECM were evaluated by qRT-PCR and western blotting. After transduction, IGFBPrP1 expression significantly increased in livers and transduced cells. MEK/ERK1/2 inhibition administration of AdIGFBPrP1-treated rats and cells significantly blocked AdIGFBPrP1-induced activation of ERK1/2. U0126 significantly down-regulated the number of F4/80-positive cells and CD3-positive cells (markers of liver inflammation), the expression of α-SMA and the concentration of ECM components in vivo. In addition, α-SMA and TGF-β1 levels in AdIGFBPrP1 HSCs were markedly inhibited by a MEK/ERK1/2 inhibitor, indicating that HSC activation was inhibited. These findings suggest that IGFBPrP1 acts as an initiator of liver fibrosis by inducing inflammation, HSC activation and ECM deposition through the ERK1/2 pathway.

Insulin‐like growth factor binding protein‐related protein 1 contributes to hepatic fibrogenesis

The aim of this study was to investigate the role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) in the development of hepatic fibrogenesis in experimental disease models and human liver samples. Cellular distribution patterns of IGFBP-rP1 were assessed by immunohistochemistry in fibrotic and cirrhotic human liver specimens. Gene silencing of IGFBP-rP1 was performed on cultured hepatic stellate cells (HSCs) by small interfering RNA (siRNA), and the silencing effect was determined by quantitative real-time polymerase chain reaction (PCR) and Western blot. We also determined the effects of siRNA-mediated gene silencing of IGFBP-rP1 on the production of extracellular matrix (ECM) components by Western blot. The expression of ECM components and transforming growth factor (TGF)-β1 was studied by immunohistochemistry and Western blot in C57BL/6 wild-type mice treated with recombinant IGFBP-rP1 (rIGFBP-rP1). Expression of IGFBP-rP1 was significantly elevated in fibrotic and cirrhotic human liver specimens, and this increase was positively correlated with the number of collagen fibers observed. siRNA-mediated gene silencing of IGFBP-rP1 resulted in significantly decreased levels of collagen I and fibronectin in HSCs. Moreover, IGFBP-rP1 overexpression significantly increased the production of collagen, fibronectin and TGF-β1 in rIGFBP-rP1-treated mice. IGFBP-rP1 contributes to the development of liver fibrosis and may be a novel molecule involved in the progression of hepatic fibrogenesis.

IGFBPrP1 induces liver fibrosis by inducing hepatic stellate cell activation and hepatocyte apoptosis via Smad2/3 signaling.

To investigate the role and mechanism of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in the development of liver fibrosis. An in vitro model using hepatic stellate cell (HSC)-T6 cells and an in vivo model of rat liver overexpressing IGFBPrP1 were established using an IGFBPrP1-expressing recombinant adenovirus. The expression of IGFBPrP1 was examined by immunofluorescence, and the expression of collagen I and fibronectin was measured by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The expression of Smad2/3 and p-Smad2/3 was examined by Western blot and immunohistochemistry. A shSmad3-expressing recombinant adenovirus (AdshSmad3) was designed and used to knockdown the Smad3 gene in HSC-T6 cells and rat liver fibrosis transfected with IGFBPrP1. The expression of collagen I, fibronectin, and α-smooth muscle actin (α-SMA) was determined by Western blot analysis and immunohistochemistry. Hepatocyte apoptosis was assessed using TUNEL assay. IGFBPrP1 overexpression induced collagen deposition and up-regulated the expression of α-SMA and p-Smad2/3, and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2/3 activation and the expression of α-SMA, collagen I and fibronectin in HSC-T6 cells. Similarly, increased hepatocyte apoptosis (38.56% ± 3.42% vs 0.24% ± 0.03%, P < 0.05), α-SMA positive stained cells (29.84% ± 1.36% vs 5.83% ± 1.47%, P < 0.05), and increased numbers of Smad3 (35.88% ± 2.15% vs 10.24% ± 1.31%, P < 0.05) and p-Smad2/3 positive cells (28.87% ± 2.73% vs 8.23% ± 0.98%, P < 0.05) were detected in the livers of IGFBPrP1-overexpressing rats compared with the control group. Moreover, AdshSmad3 reduced IGFBPrP1-stimulated Smad3 expression and attenuated α-SMA expression (29.84% ± 1.36% vs 8.23% ± 1.28%, P < 0.05), hepatocyte apoptosis (38.56% ± 3.42% vs 6.75% ± 0.52%, P < 0.05), and both collagen I and fibronectin deposition in the livers of AdIGFBPrP1-treated rats. IGFBPrP1 induces liver fibrosis by mediating the activation of hepatic stellate cells and hepatocyte apoptosis in a Smad3-dependent mechanism.

Serum insulin‐like, growth factor binding protein‐related protein 1 (IGFBP‐rP1) and endometrial cancer risk in Chinese women

Hyperinsulinemia and the metabolic syndrome confer increased risks of endometrial carcinoma. The roles of insulin, and, insulin-like growth factor-binding proteins (IGFBPs) in the etiology of endometrial carcinoma, remain unclear. We recruited 206 patients with endometrial carcinoma and 350 healthy women to a case-control study of fasting insulin and IGFBP-related protein 1 (IGFBP-rP1) in a Chinese tertiary centre. Patients with endometrial carcinoma had higher insulin concentrations (14.8 ± 16.7 vs. 8.1 ± 9.4 μU/mL; p < 0.001) and lower IGFBP-rP1 levels (17.5 ± 17.2 vs. 22.4 ± 22.8 μg/L; p = 0.018) than controls. High insulin and IGFBP-rP1 levels were both positively and negatively associated with endometrial cancer (odds ratio for the highest tertile versus the lowest tertile: insulin: 4.11; 95% CI = 2.61-6.47; IGFBP-rP1: 0.38; 95% CI = 0.24-0.60). Logistic regression analysis confirmed the associations between endometrial carcinoma and fasting insulin or IGFBP-rP1 after adjustments for age, BMI, serum glucose, cholesterol, triglycerides and high-density lipoprotein cholesterol (odds ratio for the highest tertile versus the lowest tertile: insulin: 2.13; 95% CI = 1.30-3.49; IGFBP-rP1: 0.57; 95% CI = 0.34-0.94). Hyperinsulinemia and high IGFBP-rP1 levels confer altered risks for endometrial carcinoma.

IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells

Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells. DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1. Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection. Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.

Insulin-Like Growth Factor Binding Protein-Related Protein 1 Mediates VEGF-Induced Proliferation, Migration and Tube Formation of Retinal Endothelial Cells

The potential role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) as an endogenous angiogenesis inhibitor in the prevention of vascular endothelial growth factor (VEGF)-induced retinal angiogenesis has not been explored. Expression of IGFBP-rP1 in rhesus macaque choroid-retinal endothelial cell line (RF/6A) cells was assessed by reverse transcription polymerase chain reaction analysis and Western blotting. RF/6A cells were treated with VEGF (10 ng/ml) alone or in the presence of IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml. The proliferation, migration and capillary-like tube formation of RF/6A cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric assay, the chemotactic motility assay and the Matrigel tube formation assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells, and oncogenic B-Raf expression was assessed to elucidate the pathway for IGFBP-rP1-mediated induction of apoptosis in the presence of VEGF. RF/6A cells expressed both IGFBP-rP1 transcripts and IGFBP-rP1 protein. VEGF markedly stimulated proliferation, migration and capillary-like tube formation of RF/6A cells (P<0.05), whereas those VEGF-induced parameters were significantly inhibited by IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml in a dose-dependent manner (P < 0.05). Following addition of IGFBP-rP1, expression of B-Raf was significantly decreased dose-dependently, and apoptosis occurred as evidenced by flow cytometry (P < 0.05). IGFBP-rP1 can inhibit the stimulatory effect of VEGF on retinal angiogenesis in vitro by inhibiting expression of B-Raf to induce apoptosis. It is a novel endogenous anti-angiogenic factor with potential therapeutic action in retinal neovascularization dependent disorders.

Inhibitory effect ofinsulin-like growth factor binding protein-related protein 1 on retinal angiogenesis invitro

：Background Antagonistsagainst vascular endothelial growth factor (VECF) play key roles in treating andpreventing neovascular ophthalmopathy. As a novel anti-angiogenic factor, insulin-likegrowth factor binding protein-related protein 1 (IGFBP-rP1) might be an antagonist againstVEGF in eye. Objective This study was to explore the inhibitory effect of IGFBP-rP1, anovel anti-angiogenic factor, on VEGF-induced retinal angiogenesis in vitro. Methods Theretina-choroid endothelial cell line ( RF/6A ) was cultured in DMEM containing 10% fetalbovine serum. Culture cells were divided into control group(free-serum culture group),10mg/L VEGF culture group and different concentrations of IGFBP-rP1 (50,100,200 mg/L) +10mg/L VEGF group. The expression of IGFBP-rP1 in the cells was detected byimmunofluorescence assay. The proliferation and migration of RF/6A cells were evaluatedusing MTS colorimetric assay and the chemotactic motility assay, respectively. Flowcytometry was used to detect the apoptosis of RF/6A cells. Results The immunofluorescenceassay RF/6A cells showed the green fluorescence in cytoplasm and red fluorescence innuclei after cells were exposed to any concentration of IGFBP-rP1 ,but only redfluorescence was seen in nuclei in control cells. After stimulation of 10 mg/L VEGF,theproliferation value (A490) was elevated and the numbers of cell migration were increasedin comparison with control group (t = -15. 191, P = 0. 000; t = -21. 274, P = 0. 000 ) ,but the cellular apoptosis rate was lower than the control group (t - 10. 228, P = 0. 000) . After treated with various concentrations of IGFBP-rP +10% VEGF, the proliferation andmigration of RF/6A cells were significantly decreased in comparison with only 10% VEGFgroup (F = 534. 158,P = 0. 000;F = 2742. 323,P = 0.000,respectively) ,and the inhibitoryeffects were gradually enhanced with the increase of IGFBP-rP1 levels (P<0. 05). Theapoptosis rate of RF/6A cells in 50,100 and 200 mg/L + 10 mg/L VEGF groups increased by (1. 26±0. 04)% ,( 1. 50±0. 07)% and ( 1. 93±0. 27)% respectively,showing significantdifferences among different groups ( F = 274. 273, P = 0. 000). Conclusion IGFBP-rP1inhibits the proliferation and activity of retina and choroid endothelial cells induced byVEGF at a concentration-independent manner. It appears to be as a novel endogenousinhibitory factor in retinal angiogenesis. Key words: Insulin-like growth factor binding protein-related protein 1; Vascular endothelial growth factor; Retina; Vascular endothelial cell; Angiogenesis