Growth Factor Binding Protein-related Protein Research Articles

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

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

Molecular identification and putative role of insulin growth factor binding protein-related protein (IGFBP-rp) in the swimming crab Portunus trituberculatus

The insulin-like growth factor/insulin-like polypeptide (IGF/ILP) signaling is vital for growth, physiological metabolism, development, and reproduction. Insulin-like growth factor-binding protein (IGFBP) is involved in the insulin signaling pathway in both vertebrates and invertebrates and is critical for various physiology functions. Herein, we cloned and characterized the full-length cDNA of IGFBP-rp in the swimming crab, Portunus trituberculatus (PtIGFBP-rp). The deduced amino acid sequence of PtIGFBP-rp was found to contain three key domains (insulin-like binding (IB) domain, the kazale-type serine protease inhibitor (KAZAL) domain, and the immunoglobulin-like C2 (IGc2) domain). Results showed that PtIGFBP-rp shared the same expression pattern as P. trituberculatus insulin androgenic gland hormone (PtIAG) transcripts during the embryonic larval, juvenile crab stage and the androgenic gland (AG) developmental cycle. Moreover, PtIGFBP-rp transcripts were also present in high abundance in hepatopancreas, muscle, and androgenic glands. The regulatory relationship between PtIGFBP-rp and PtIAG was investigated by RNA interference and co-localization assays, which showed a co-localization relationship and feedback regulation between them. Bilateral eye stalk ablation (ESA) increased the expression of PtIGFBP-rp in the AG at 7 d after surgery. These results demonstrate the involvement of PtIGFBP-rp in the signaling regulatory network of IAG in P. trituberculatus.

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.

The lncRNA NEAT1/miR-29b/Atg9a axis regulates IGFBPrP1-induced autophagy and activation of mouse hepatic stellate cells

AimsInsulin-like growth factor binding protein-related protein 1 (IGFBPrP1) promotes hepatic stellate cell (HSC) autophagy and activation. However, the underlying mechanism remains unknown. Noncoding RNAs (ncRNAs) including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), have received increasing attention. We aimed to investigate the roles of the lncRNA nuclear enriched abundant transcript 1 (NEAT1), miR-29b, and autophagy related protein 9a (Atg9a), and their relationships with each other during IGFBPrP1-induced HSC autophagy and activation. Main methodsLevels of NEAT1, miR-29b, Atg9a, and autophagy were detected in adenovirus-mediated IGFBPrP1 (AdIGFBPrP1)-treated mouse liver tissue and immortalized mouse hepatic stellate cell line JS1 transfected with either AdIGFBPrP1 or siIGFBPrP1. In AdIGFBPrP1-treated JS1 cells, autophagy and activation were detected after altering NEAT1, miR-29b, or Atg9a levels. In AdIGFBPrP1-treated JS1 cells, relationships among NEAT1, miR-29b, and Atg9a were explored using dual-luciferase reporter assays, Western blot, qRT-PCR, and immunofluorescence. Key findingsIGFBPrP1 increased levels of NEAT1, Atg9a, and autophagy while decreasing the level of miR-29b in mouse liver tissues and mouse HSCs. Moreover, NEAT1 increased HSC autophagy and activation while miR-29b decreased both processes. Atg9a also participated in IGFBPrP1-induced HSC autophagy and activation. Importantly, NEAT1, miR-29b, and Atg9a formed a NEAT1/miR-29b/Atg9a regulatory axis for IGFBPrP1-induced HSC autophagy and activation. SignificanceOur study unveiled the new NEAT1/miR-29b/Atg9a regulatory axis involved in IGFBPrP1-induced mouse HSC autophagy and activation. The study thus provides new insights in the pathogenesis and potential therapeutic strategies of liver fibrosis.

RETRACTED ARTICLE: 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

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.

Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development.

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.

Multiple Mechanisms for Anti-Fibrotic Functions of Statins on Radiotherapy Induced Fibrosis

Radiotherapy-induced fibrosis (RTIF) presents a challenge in radiotherapy for cancer patients. Although numerous studies have attempted to elucidate the mechanisms leading to RTIF, the pathogenesis of RTIF at the cellular and molecular level is still incompletely described. One key component involved in the post-radiation injury is the pleuripotent cytokine transforming growth factor (TGF)-β. TGF-β signaling pathway has been under intensive investigation about its critical role in radiation-induced fibroproliferative disease. Connective tissue growth factor (CTGF), also known as insulin-like growth factor binding protein-related protein 2 (IGFBP-rP2) is a potent regulator of fibroblast proliferation, cell adhesion, and stimulation of extracellular matrix production. CTGF is known as a major downstream mediator of the chronic fibrotic effects of TGF-β. Here we have demonstrated that irradiation and TGF-β induced CTGF, subsequently upregulates fibrotic factors such as fibronectin and type IV collagen. Furthermore, as HMG-CoA reductase inhibitors, statins inhibit expressions of CTGF and downstream fibrotic proteins in both normal human fetal fibroblasts (HFL-1) and human dermal fibroblasts (HDF) on TGF-β treatment or irradiation. Our study also demonstrates that simvastatin not only suppressed TGF-β-induced fibrosis through inhibition of CTGF production but also CTGF-induced fibrosis. We further show that simvastatin may act in a TGF-β-independent manner by inhibiting Rho kinase pathway. Taken together, these data suggest that radiotherapy may upregulate CTGF expression in a TGF-β-dependent and -independent manner, thereby enhancing expression of profibrotic factors and inducing lung fibrosis.

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.

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.