Insulin-like Growth Factor Binding Protein-3 mRNA Research Articles

Expression of insulin-like growth factor binding protein 5 in the vaginal wall tissues of older women with pelvic organ prolapse

This study aimed to investigate the expression and significance of insulin-like growth factor binding protein 5 (IGFBP5) and extracellular matrix (ECM) related proteins in anterior vaginal wall tissues among aged pelvic organ prolapse (POP) patients. Tissues from the anterior vaginal wall were collected from 28 patients with POP and 20 patients without POP. The expression of protein and mRNA levels of IGFBP5 and ECM related proteins were evaluated in the vaginal wall tissues using immunohistochemistry, western blotting, and RT-qPCR techniques. The expression levels were then compared with clinical parameters. The expression levels of protein and mRNA of IGFBP5, collagen I, and collagen III were significantly lower in the POP group. Protein and mRNA expression levels of MMP2 were significantly higher in the POP group. IGFBP5 protein and mRNA expression levels were negatively correlated with age and significantly lower in older POP patients (≥ 65 years old) compared to younger POP patients (< 65 years old). IGFBP5 protein and mRNA expression levels were also significantly lower in POP-Q stage IV patients compared to POP-Q stage III patients. IGFBP5 expression level is negatively correlated with the age and severity of prolapse. The significant decrease in IGFBP5 expression may play a crucial part in the aging process and the occurrence of POP.

Serum IGFBP-1 as a promising diagnostic and prognostic biomarker for colorectal cancer

Our previous study showed that levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) has potential diagnostic value for early-stage upper gastrointestinal cancers. This study aimed to assess whether serum IGFBP-1 is a potential diagnostic and prognostic biomarker for CRC patients. IGFBP-1 mRNA expression profile data of peripheral blood in colorectal cancer (CRC) patients were downloaded and analyzed from Gene Expression Omnibus database. We detected serum IGFBP-1 in 138 CRC patients and 190 normal controls using enzyme-linked immunosorbent assay. Blood IGFBP-1 mRNA levels were higher in CRC patients than those in normal controls (P = 0.027). In addition, serum IGFBP-1 protein levels in the CRC group were significantly higher than those in normal control group (P < 0.0001). Serum IGFBP-1 demonstrated better diagnostic accuracy for all CRC and early-stage CRC, respectively, when compared with carcinoembryonic antigen (CEA), carbohydrate antigen19-9 (CA 19-9) or the combination of CEA and CA19-9. Furthermore, Cox multivariate analysis revealed that serum IGFBP-1 was an independent prognostic factor for OS (HR = 2.043, P = 0.045). Our study demonstrated that serum IGFBP-1 might be a potential biomarker for the diagnosis and prognosis of CRC. In addition, the nomogram might be helpful to predict the prognosis of CRC.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy

Objective This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. Methods Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. Results Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). Conclusions The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity

To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. Laboratory based experimental study. Academic hospital and laboratory. Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

P-760 Effect of relaxin on human endometrial stromal cell characteristics

Abstract Study question Does relaxin (RLX) regulate decidualization and pro- and antiangiogenic factors in primary human endometrial stromal cells (hESC), primary decidual cells (pD) and hTERT-immortalized hESC (T hESC)? Summary answer RLX treatment enhances decidualization and affects the mRNA expression levels of pro- and antiangiogenic factors. What is known already Patients undergoing frozen embryo transfer in an artificial cycle (AC-FET) are at a 2-fold increased risk for preeclampsia compared to embryo transfer in a natural cycle. This may be related to the absence of the corpus luteum in an artificial FET cycle, which produces important hormones such as estrogen and progesterone in early pregnancy. These hormones are supplied during AC-FET, whereas other products of the corpus luteum, such as RLX, are not. Low maternal RLX levels were found in women who developed preeclampsia, but the direct relationship between RLX and the occurrence of preeclampsia remains to be elucidated. Study design, size, duration Primary hESC were isolated from biopsies of women without endometrial pathology, pD cells from biopsies of the maternal side of the placenta of uncomplicated pregnancies and T hESC purchased from American Type Culture Collection. Cells were decidualized with either 0.5 mM cyclic adenosine monophosphate (cAMP) or a cocktail of 10 nM estradiol, 1 µM progesterone and 0.5 mM cAMP (EPC) and additionally treated with 0, 0.3 or 1 ng/ml RLX for twelve days. Participants/materials, setting, methods RNA was isolated on day zero and day twelve from all three cell types. mRNA expression level of decidualization markers (prolactin (PRL), insulin like growth factor binding protein 1 (IGFBP1)) as well as pro- and antiangiogenic factors (vascular endothelial growth factor (VEGF); placenta like growth factor (PlGF); soluble Fms-like tyrosine kinase-1 (sFlt-1); and endoglin (ENG)) were evaluated by quantitative real time polymerase chain reaction (qRT-PCR), N = 3 biological replicates. Main results and the role of chance Decidualization treatment of hESC with cAMP caused more noticeable changes to cell morphology than treatment with EPC. While there was no rise of decidualization markers PRL (p = 0.99) and IGFBP1 (p = 0.95) in the cAMP treatment group, hESC after EPC treatment showed a significant increase in both PRL (p = 0.0003) and IGFBP1 (p = 0.0001). RLX treatment in the EPC treated hESC and pD significantly increased the mRNA expression levels of PRL and IGFBP1 (both p &amp;lt; 0.05, respectively), as well as the expression of the proangiogenic marker VEGF (P = 0.05). A comparative analysis with T hESC showed a moderate effect on VEGF and PlGF. No effect of RLX was detected for IGFBP1 and PRL mRNA expression levels. The mRNA expression level of the antiangiogenic marker ENG increased in the EPC group in both, primary hESC (P = 0.04) and T hESC (P = 0.01) after treatment with 0.3 ng/ml RLX compared to control. ENG mRNA expression level increased in a dose dependent manner in pD but not in hESC and T hESC. RLX treatment with 1ng/ml of cAMP decidualized hESC significantly enhanced mRNA expression levels of VEGF (P = 0.002) and PlGF (P = 0.02) only in hESC but not in T hESC or pD. Limitations, reasons for caution Our pilot study indicates that RLX treatment improves decidualization and affects pro- and antiangiogenic factors in three different human endometrial stromal cell models. A wider approach is needed to investigate the effects of RLX in the decidualization process and pro- and antiangiogenic factors. Wider implications of the findings Our results provide the basis for further studies elucidating the relationship between low maternal RLX levels and the occurrence of preeclampsia. This could lead to the development of improved prediction models and new treatment options in assisted reproduction that reduce the risk of preeclampsia, e.g. in AC-FET. Trial registration number Not applicable

Abstract 3490: IGFBP1 mediates musculoskeletal defects in colorectal cancer

Abstract Colorectal cancer (CRC) is frequently accompanied by cachexia, an uncured multi-systemic wasting syndrome that affects the majority of patients, especially when the disease recurs with liver metastases (LMs). Muscle and bone loss are amongst the most detrimental symptoms of cachexia and directly cause increased morbidity and mortality. We and others have shown that CRC also promotes metabolic and genomic perturbations of the liver, and further, that formation of LMs exacerbates muscle and bone wasting. These observations, along with evidence that liver-derived factors (i.e., hepatokines) may poorly influence musculoskeletal health, suggest an endocrine role of the liver in mediating cancer-induced cachexia. In the present study, we identified the hepatokine insulin-like growth factor binding protein 1 (IGFBP1) as a novel mediator of musculoskeletal wasting in CRC. Plasma from CRC patients and preclinical mouse models of CRC (C26, MC38, HCT116, ApcMin/+) were assessed for circulating IGFBP1 levels. AML12 hepatocytes were cultured with CRC cells (C26, MC38, HCT116) to assess tumor induced hepatic IGFBP1 production, while C2C12 myotubes and osteoclast precursor cells were cultured to examine the in vitro effects of IGFBP1 on myotube atrophy and osteoclastogenesis. 8-week-old male wild-type (WT) C57BL/6J and IGFBP1-KO mice were intrasplenically injected with MC38 tumor cells (mMC38) to mimic hepatic dissemination of cancer cells, while sham-operated animals received saline. Animals were assessed for muscle force 24-hours prior to euthanasia, and skeletal muscles and bone were collected for molecular and morphological analyses. CRC patients and CRC tumor-bearing mice demonstrated markedly elevated circulating plasma IGFBP1, also supported by increased liver IGFBP1 mRNA expression in C26, MC38 and HCT116 hosts. Follow-up in vitro studies demonstrated that co-culturing CRC cells (lacking IGFBP1 expression) with AML12 hepatocytes promotes IGFBP1 production, thereby suggesting that IGFBP1 is purely host-derived. Further, treatment with recombinant IGFBP1 was sufficient to stimulate osteoclastogenesis, while also promoting atrophy of C2C12 myotubes. Conversely, use of anti-IGFBP1 neutralizing antibodies prevented osteoclastogenesis and preserved C2C12 myotube size when exposed to plasma from mice bearing CRCs. Notably, WT mMC38 bearers displayed reductions in muscle mass and strength, as well as in trabecular bone volume fraction (BV/TV) and trabecular number (Tb.N). Conversely, IGFBP1-KO tumor hosts exhibited preserved skeletal muscle and bone mass. Altogether, our data implicate IGFBP1, an exquisitely liver-derived factor, as a novel mediator of musculoskeletal deficits in CRC cachexia, and supports novel strategies to counteract host-derived factors in the treatment of cancer-associated multi-organ complications. Citation Format: Joshua R. Huot, Fabrizio Pin, Andrea Bonetto. IGFBP1 mediates musculoskeletal defects in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3490.

Clinical Characterization of the Expression of Insulin-Like Growth Factor Binding Protein 1 and Tumor Immunosuppression Caused by Ferroptosis of Neutrophils in Non-Small Cell Lung Cancer.

The efficacy of immunotherapy for non-small cell lung cancer (NSCLC) is limited owing to cold tumors and drug resistance. Therefore, it is important to identify the molecular mechanisms underlying immune evasion in NSCLC. Spontaneous ferroptosis of neutrophils has been suggested as a key mechanism of immunosuppression in cancer. Insulin-like growth factor binding protein 1 (IGFBP1) plays an important role in immune infiltration in several cancers. However, the role of IGFBP1 in NSCLC is unknown. Therefore, in this study, we aimed to investigate the association of IGFBP1 mRNA expression with infiltration of myeloid-derived suppressor cells and prognosis in NSCLC. Retrospective RNA-seq data from 990 patients in the Cancer Genome Atlas (TCGA) database were analyzed in relation to patient clinical characteristics. The Timer2 database was used to assess immune infiltration, and the FerrDb V2 database was used to obtain ferroptosis-related genes. Finally, the results were validated by the proteomic analysis of serum samples collected from six patients with NSCLC and six healthy individuals. IGFBP1 expression was enriched in lung adenocarcinoma samples and positively correlated with the pathological grade of NSCLC. IGFBP1 expression was an independent prognostic factor for the overall survival of patients with NSCLC. In addition, IGFBP1 expression correlated with myeloid-derived suppressor cell infiltration. Notably, Gene Ontology analysis of IGFBP1-related genes revealed that the major molecular functions of their protein products were related to NADP+ 1-oxidoreductase activity. Furthermore, expression levels of multiple ferroptosis suppressor genes positively correlated with IGFBP1 expression. High IGFBP1 expression indicates a poor prognosis in patients with NSCLC, which may be related to tumor immunosuppression caused by neutrophil ferroptosis.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshβ mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells.

Cystic Fibrosis (CF) patients are prone to contracting bacterial lung infections with opportunistic pathogens, especially Pseudomonas aeruginosa. Prolonged P. aeruginosa infections have been linked to chronic inflammation in the CF lung, whose hallmarks are increased levels of cytokines (i.e., TNF-α, IL-1β, IL-6) and neutrophil attraction by chemokines, like IL-8. Recently, insulin-like growth factor binding protein 6 (IGFBP-6) has been shown to play a putative role in the immune system and was found at higher levels in the sera and synovial tissue of rheumatoid arthritis patients. Moreover, it has been demonstrated that IGFBP-6 has chemoattractant properties towards cells of the innate (neutrophils, monocytes) and adaptive (T cells) immunity. However, it is not known whether IGFBP-6 expression is dysregulated in airway epithelial cells under infection/inflammatory conditions. Therefore, we first measured the basal IGFBP-6 mRNA and protein levels in bronchial epithelial cells lines (Wt and F508del-CFTR CFBE), finding they both are upregulated in F508del-CFTR CFBE cells. Interestingly, LPS and IL-1β+TNFα treatments increased the IGFBP-6 mRNA level, that was reduced after treatment with an anti-inflammatory (Dimethyl Fumarate) in CFBE cell line and in patient-derived nasal epithelial cultures. Lastly, we demonstrated that IGFBP-6 reduced the level of pro-inflammatory cytokines in both CFBE and primary nasal epithelial cells, without affecting rescued CFTR expression and function. The addition of a neutralizing antibody to IGFBP-6 increased pro-inflammatory cytokines expression under challenge with LPS. Together, these data suggest that IGFBP-6 may play a direct role in the CF-associated inflammation.

A Mixture of Valine and Isoleucine Restores the Growth of Protein-Restricted Pigs Likely through Improved Gut Development, Hepatic IGF-1 Pathway, and Plasma Metabolomic Profile.

Valine (Val) alone or in combination with isoleucine (Ile) improves the growth under severe protein restriction; however, the underlying mechanisms remain unknown. In this study, we assessed whether Val/Ile-induced growth in protein-restricted pigs is associated with changes in gut development, hepatic insulin-like growth factor 1 (IGF-1) production, and blood metabolomics. Forty piglets were assigned to five dietary groups: positive control (PC) with standard protein content; low protein (LP) with very low protein content; and LP supplemented with Val (LPV), Ile (LPI), and Val and Ile (LPVI). LPVI reversed the negative effects of VLP diets on growth and gut morphology. Both LPV and LPVI restored the reduced transcript of IGF-1 while decreasing the transcript of insulin-like growth factor binding protein 1 (IGFBP1) in the liver. LPV and LPVI recovered the reduced plasma Val, glycine, and leucine concentrations, which were positively correlated with improved gut morphology and the hepatic IGF-1 gene expression and negatively correlated with hepatic IGFBP1 mRNA abundance. In conclusion, supplementation with a combination of Val and Ile into the VLP diets restored the decreased growth performance of pigs fed with these diets likely through improved gut development, hepatic IGF-1 expression and bioavailability, and plasma metabolomics profile.

LncRNA HOXA11-AS aggravates the keloid formation by targeting miR-148b-3p/IGFBP5 axis

Long non-coding RNA (lncRNA) homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism need to be further understood. HOXA11-AS, microRNA miR-148b-3p and Insulin like growth factor binding protein 5 (IGFBP5) expression were detected by RT-qPCR or western blot. CCK-8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p, HOXA11-AS and IGFBP5 was confirmed by luciferase reporter or RNA immunoprecipitation assay. The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis. These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

α7 Nicotinic Acetylcholine Receptor Agonists Regulate Inflammation and Growth Hormone Resistance in Sepsis.

During sepsis the normal induction of circulating insulin-like growth factor-I (IGF-I) by growth hormone (GH) action on liver is attenuated, a phenomenon called hepatic GH resistance. Hepatic GH resistance can be caused by cytokine-mediated activation of the NF-κB pathway which interferes with normal GH-signaling. The afferent and efferent fibers of the vagus nerve are integral to the cholinergic anti-inflammatory pathway (CAP) which attenuates hepatic TNFα production by activating the α7 nicotinic acetylcholine receptor (α7nAChR). We examined the effects of selective afferent vagotomy (SAV) and α7nAChR activation on sepsis-induced mortality, hepatic and systemic inflammation, the GH/IGF system and hepatic GH resistance using Sprague Dawley (SD) rats, C57BL/6 wild type (WT) mice, and α7nAChR knockout (KO) mice. Capsaicin was used to perform SAV and GTS-21 (α7nAChR agonist) was used to activate the α7nAChR. Sepsis-induced mortality, hepatic NF-κB activation, and plasma cytokine levels were increased in SAV rats and reduced in GTS-21-treated mice. The effects of sepsis on the GH/IGF-I system plasma IGF-I, IGF binding protein-1 (IGFBP-1), hepatic IGF-I, IGFBP-1, and GH receptor (GHR) mRNA and rhGH-responsiveness in mice were improved by GTS-21. Collectively these results confirm the protective effects of the anti-inflammatory CAP and α7nAChR activation in sepsis. They also provide evidence the CAP and α7nAChR activation could be used to attenuate hepatic GH resistance and anabolic failure in sepsis.

Anticancer mechanism of breviscapine in non-small cell lung cancer A549 cells acts via ROS-mediated upregulation of IGFBP4.

BackgroundThe overall 5-year survival rate of non-small cell lung cancer (NSCLC) is less than 15% because of multiple drug resistance to chemotherapy and the limitations of early diagnosis. Thus, safe and effective drugs to treat NSCLC are required. The present study aimed to investigate the effects of breviscapine (BVP) on NSCLC cell apoptosis and proliferation, and to study its possible mechanisms.MethodsUsing the NSCLC A549 cell line and BVP (0, 25, 50, and 100 µM), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect A549 cell proliferation, and flow cytometry was used to assess cell apoptosis. Insulin-like growth factor binding protein 4 (IGFBP4) levels was assessed using enzyme-linked immunosorbent assays and western blotting. Flow cytometry of hydrogen peroxide and superoxide was used to assess intracellular reactive oxygen species (ROS) generation. Western blotting was used to assess the levels of BCL2-associated X, apoptosis regulator (BAX) and B-cell CLL/lymphoma 2 (BCL2). Quantitative real-time reverse transcription PCR (qRT-PCR) was used to assess IGFBP4 mRNA expression.ResultsBVP induced apoptosis, inhibited cell proliferation, and increased ROS in A549 cells. Western blotting and qRT-PCR showed that BVP increased IGFBP4 protein and mRNA expressions in A549 cells. Compared with BVP treatment alone, IGFBP4 expression decreased in A549 cells treated with BVP and the ROS scavenger N-acetylcysteine. IGFBP4 overexpression increased BVP-induced proliferation inhibition, while increasing BAX expression and decreasing BCL2 expression. Silencing IGFBP4 had the opposite effects.ConclusionsBVP could inhibit the growth of NSCLC A549 cells by promoting apoptosis via ROS-mediated upregulation of IGFBP4.

BONE MORPHOGENETIC PROTEIN 2 PROMOTES HUMAN TROPHOBLAST CELL INVASION AND ENDOTHELIAL-LIKE TUBE FORMATION THROUGH ID1-MEDIATED UP-REGULATION OF IGF BINDING PROTEIN-3

Objective: Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Insufficient EVTs invasion and poor spiral arterial remodeling are associated with preeclampsia occurrence. Studies from us and others have demonstrated that the transforming growth factor-beta superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes endothelial precursor cell migration and angiogenesis. Moreover, Id1 shows regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Design and method: Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. RT-PCR and Western blot assay were used to study mRNA and protein levels. Small interfering RNA (siRNA) was used to check the roles of key molecules. Matrigel-coated transwell assay and endothelial-like tube formation assay were used to examine cell invasiveness and angiogenesis respectively. Results: BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 up-regulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Conclusions: Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 up-regulation.

Reduced Insulin-Like Growth Factor Family Member Expression Predicts Neurogenesis Marker Expression in the Subependymal Zone in Schizophrenia and Bipolar Disorder.

The generation of inhibitory interneurons from neural stem cells in the subependymal zone is regulated by trophic factors. Reduced levels of trophic factors are associated with inhibitory interneuron dysfunction in the prefrontal cortex and hippocampus in psychiatric disorders, yet the extent to which altered trophic support may underpin deficits in inhibitory interneuron generation in the neurogenic niche remains unexplored in schizophrenia and bipolar disorder. We determined whether the expression of ligands, bioavailability-regulating binding proteins, and cognate receptors of 4 major trophic factor families (insulin-like growth factor [IGF], epidermal growth factor [EGF], fibroblast growth factor [FGF], and brain-derived neurotrophic factor [BDNF]) are changed in schizophrenia and bipolar disorder compared to controls. We used robust linear regression analyses to determine whether altered expression of trophic factor family members predicts neurogenesis marker expression across diagnostic groups. We found that IGF1 mRNA was decreased in schizophrenia and bipolar disorder compared with controls (P ≤ .006), whereas both IGF1 receptor (IGF1R) and IGF binding protein 2 (IGFBP2) mRNAs were reduced in schizophrenia compared with controls (P ≤ .02). EGF, FGF, and BDNF family member expression were all unchanged in both psychiatric disorders compared with controls. IGF1 expression positively predicted neuronal progenitor and immature neuron marker mRNAs (P ≤ .01). IGFBP2 expression positively predicted neural stem cell and neuronal progenitor marker mRNAs (P ≤ .001). These findings provide the first molecular evidence of decreased IGF1, IGF1R, and IGFBP2 mRNA expression in the subependymal zone in psychiatric disorders, which may potentially impact neurogenesis in schizophrenia and bipolar disorder.

The Regulatory Role of Apelin on the Appetite and Growth of Common Carp (Cyprinus Carpio L.).

Simple SummaryFood intake and growth are two interrelated physiological processes. Fish obtain energy from the outside to meet their own growth and development through food intake. Apelin, as a new endocrine factor, plays a wide range of biological roles. Among them, apelin’s role in regulating appetite and growth has attracted the attention of researchers. Based on our investigations, in vitro and in vivo experiments showed that Pyr-apelin-13 could induce significant changes in mRNA levels of appetite-related and growth-related genes, suggesting that Pyr-apelin-13 may regulate the appetite and growth of common carp by regulating the expression of these key genes.Apelin, a kind of active polypeptide, has many biological functions, such as promoting food intake, enhancing immunity, and regulating energy balance. In mammals, studies have indicated that apelin is involved in regulating food intake. However, there are relatively few studies about the regulatory effect of apelin on fish feeding, and the specific mechanism is not clear. Therefore, the purpose of this study was to preliminarily investigate the regulatory effects of apelin on key genes of feeding and growth in common carp (Cyprinus Carpio L.) through in vitro and in vivo experiments. In the present study, after incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in hypothalamic fragments, the expressions of Neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA were significantly up-regulated at 12 and 3 h, respectively, and the significant down-regulation of Cocaine and amphetamine-related transcript (CART) mRNA expression was observed at 1 and 3 h. In vivo, after Pyr-apelin-13 oral administration (0, 1, 10, and 100 pmol/g), the orexin mRNA level in the hypothalamus of common carp was significantly increased at 1, 6, and 12 h, while CART/(Proopiomelanocortin) POMC mRNA levels in the hypothalamus of common carp were significantly down-regulated. Following incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in primary hepatocytes, GHR (Growth hormone receptor), IGF2 (Insulin-like growth factor 2), IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 (Insulin like growth factor binding protein 3) mRNA levels were significantly increased at 3 h. In vivo, the levels of IGF1 (Insulin-like growth factor 1), IGF2, IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 mRNA were significantly increased after the oral administration of Pyr-apelin-13 in the hepatopancreas, in a time and dose-dependent manner. These results support the hypothesis that Pyr-apelin-13 might regulate the feeding and growth of common carp through mediating the expressions of appetite- and growth-related genes. Overall, apelin, which is an orexigenic peptide, improves food intake and is involved in the growth of common carp.

Insulin-like growth factor binding protein-2: a new circulating indicator of pulmonary arterial hypertension severity and survival

BackgroundPulmonary arterial hypertension (PAH) is a fatal disease that results from cardio-pulmonary dysfunction with the pathology largely unknown. Insulin-like growth factor binding protein 2 (IGFBP2) is an important member of the insulin-like growth factor family, with evidence suggesting elevation in PAH patients. We investigated the diagnostic and prognostic value of serum IGFBP2 in PAH to determine if it could discriminate PAH from healthy controls and if it was associated with disease severity and survival.MethodsSerum IGFBP2 levels, as well as IGF1/2 levels, were measured in two independent PAH cohorts, the Johns Hopkins Pulmonary Hypertension program (JHPH, N = 127), NHLBI PAHBiobank (PAHB, N = 203), and a healthy control cohort (N = 128). The protein levels in lung tissues were determined by western blot. The IGFBP2 mRNA expression levels in pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) were assessed by RNA-seq, secreted protein levels by ELISA. Association of biomarkers with clinical variables was evaluated using adjusted linear or logistic regression and Kaplan-Meier analysis.ResultsIn both PAH cohorts, serum IGFBP2 levels were significantly elevated (p < 0.0001) compared to controls and discriminated PAH from controls with an AUC of 0.76 (p < 0.0001). A higher IGFBP2 level was associated with a shorter 6-min walk distance (6MWD) in both cohorts after adjustment for age and sex (coefficient − 50.235 and − 57.336 respectively). Cox multivariable analysis demonstrated that higher serum IGFBP2 was a significant independent predictor of mortality in PAHB cohort only (HR, 3.92; 95% CI, 1.37–11.21). IGF1 levels were significantly increased only in the PAHB cohort; however, neither IGF1 nor IGF2 had equivalent levels of associations with clinical variables compared with IGFBP2. Western blotting shown that IGFBP2 protein was significantly increased in the PAH vs control lung tissues. Finally, IGFBP2 mRNA expression and secreted protein levels were significantly higher in PASMC than in PAEC.ConclusionsIGFBP2 protein expression was increased in the PAH lung, and secreted by PASMC. Elevated circulating IGFBP2 was associated with PAH severity and mortality and is a potentially valuable prognostic marker in PAH.

Characterization of Insulin-Like Growth Factor Binding Protein-5 (IGFBP-5) Gene and Its Potential Roles in Ontogenesis in the Pacific Abalone, Haliotis discus hannai.

Insulin-like growth factor binding protein family is known to be involved in regulating biological actions of insulin-like growth factors (IGFs). In the present study, a full-length cDNA encoding the IGFBP-5 gene was cloned and characterized from the cerebral ganglion of Haliotis discus hannai. The 921-bp full-length sequence of Hdh IGFBP-5 cDNA transcript had an open reading frame of 411 bp encoding a predicted polypeptide of 136 amino acids, sharing high sequence identities with IGFBP-5 of H. diversicolor. The deduced Hdh IGFBP-5 protein contained a putative transmembrane domain (13-35 aa) in the N-terminal region. It also possessed a signature domain of IGFBP protein family (IB domain, 45-120 aa). Six cysteine residues (Cys-47, Cys-55, Cys-73, Cys-85, Cys-98, and Cys-118) in this cloned sequence could potentially form an intrachain disulfide bond. Phylogenetic analysis indicated that the Hdh IGFBP-5 gene was robustly clustered with IGFBP-5 of H. diversicolor. Tissue distribution analysis based on qPCR assay showed that Hdh IGFBP-5 was widely expressed in all examined tissues, with significantly (p < 0.05) higher expression in the cerebral ganglion. In male and female gametogenetic cycles, Hdh IGFBP-5 mRNA was expressed at all stages, showing significantly higher level at ripening stage. The expression level of Hdh IGFBP-5 mRNA was significantly higher in the polar body stage than in other ontogenic stages. In situ hybridization revealed that Hdh IGFBP-5 mRNA was present in the neurosecretory cells of the cerebral ganglion. This is the first study describing IGFBP-5 in H. discus hannai that might be synthesized in the neural ganglia. Our results demonstrate Hdh IGFBP-5 is involved in regulating ontogenic development and reproductive regulation of H. discus hannai.

Decreased expression of IGFBP6 correlates with poor survival in colorectal cancer patients

BackgroundThe insulin-like growth factor binding protein 6 (IGFBP6), as a specific inhibitor of IGF-Ⅱ, is a candidate human anti-oncogene in multiple tumors. However, the expression of IGFBP6 in colorectal cancer (CRC) and prognostic significance are unclear. MethodsIn this study, we examined colorectal cancer tissues and adjacent normal tissues to determine the expression levels of IGFBP6 mRNA and protein by quantitative reverse-transcription polymerase chain reaction and tissue microarray immunohistochemistry analysis respectively. Moreover, we explored the effects of IGFBP6 on cell growth, migration and invasion by Cell Counting Kit-8(CCK8), colony formation and transwell migration assays. We also investigated whether IGFBP6 expression in tumor tissue correlated with various clinical parameters, including overall survival by univariate and multivariate analyses ResultsBoth IGFBP6 mRNA and protein levels were significantly lower in colorectal cancer tissues than in adjacent normal colon. Downregulating IGFBP6 using RNAi increased CRC cell proliferation, migration and invasion. Low IGFBP6 expression correlated with poor overall survival in both univariate and multivariate analyses. ConclusionOur data suggest that IGFBP6 may act as a tumor suppressor gene in the development of CRC, and that low IGFBP6 expression could be used as an independent prognostic biomarker in CRC.