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 Fshß 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).

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD.

BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Insulin-Like Growth Factor Binding Protein 5-A Probable Target of Kidney Renal Papillary Renal Cell Carcinoma.

Kidney renal papillary renal cell carcinoma (KIRP) accounts for 10-15% of renal cell carcinoma (RCC). The need to find more therapeutic targets for KIRP is urgent because most targeted drugs have limited effects on advanced KIRP. Insulin-like growth factor (IGF) binding protein 5 (IGFBP5) is a secreted protein related to cell proliferation, cell adhesion, cell migration, the inflammatory response and fibrosis; these functions are independent of IGF. In our study, we determined the expression and functions of IGFBP5 with data from the database of The Cancer Genome Atlas (TCGA). We found that IGFBP5 is down regulated in KIRP kidney tissues compared to its expression in control tissues and that the expression of IGFBP5 is negatively related to patient survival. Bioinformatic analysis showed the probable processes and pathways involved in altered IGFBP5 expression, including blood vessel development, the cellular response to growth factor stimulus, the response to transforming growth factor ß (TGF-ß), and extracellular matrix organization. We proposed that VEGFA and TGF-ß act as upstream regulatory factors of IGFBP5 and verified this in the Caki-2 cell line. Based on our results, we suggest that IGFBP5 might be a therapeutic target of KIRP.

Overexpressed IGFBP5 promotes cell proliferation and inhibits apoptosis of nucleus pulposus derived from rats with disc degeneration through inactivating the ERK/MAPK axis.

It is previously suggested that insulin-like growth factor binding proteins (IGFBPs) potentially share an association with disc degeneration (DD) that causes back pain. This study aimed at exploring the functional relevance of IGFBP5 in DD by establishing a rat model of DD. The nucleus pulposus (NP) cells were transduced with IGFBP5-shRNA or IGFBP5 overexpression to determine the cellular processes (proliferation, apoptosis, as well as colony formation). The protein levels of apoptosis-related proteins were evaluated. Furthermore, NP cells were treated with the extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK/MAPK) pathway inhibitor (PD98059) followed by measurement of ERK protein level and ERK phosphorylation content. The NP cells showed suppressed proliferation and colony formation ability, yet promoted apoptosis after transfection with IGFBP5-shRNA. It was found that silencing of IGFBP5 could lead to the ERK/MAPK axis activation, as indicated by an elevated ERK protein level and ERK phosphorylation content. However, overexpression of IGFBP5 could reverse all the reaction induced by silenced IGFBP5. These key findings demonstrate that overexpressed IGFBP5 inactivates the ERK/MAPK axis to stimulate the proliferation and inhibit apoptosis of NP cells in a rat model of DD.

Bald thigh syndrome in sighthounds-Revisiting the cause of a well-known disease.

Bald thigh syndrome is a common hair loss disorder in sighthounds. Numerous possible causes, including environmental conditions, trauma, stress, endocrinopathies and genetic components have been proposed, but only endocrinopathies have been ruled out scientifically. The overall goal of our study was to identify the cause of bald thigh syndrome and the pathological changes associated with it. We approached this aim by comparing skin biopsies and hair shafts of affected and control dogs microscopically as well as by applying high-throughput technologies such as genomics, transcriptomics and proteomics. While the histology is rather unspecific in most cases, trichogram analysis and scanning electron microscopy revealed severe structural abnormalities in hair shafts of affected dogs. This finding is supported by the results of the transcriptomic and proteomic profiling where genes and proteins important for differentiation of the inner root sheath and the assembly of a proper hair shaft were downregulated. Transcriptome profiling revealed a downregulation of genes encoding 23 hair shaft keratins and 51 keratin associated proteins, as well as desmosomal cadherins and several actors of the BMP signaling pathway which is important for hair shaft differentiation. The lower expression of keratin 71 and desmocollin 2 on the mRNA level in skin biopsies corresponded with a decreased protein expression in the hair shafts of affected dogs. The genetic analysis revealed a missense variant in the IGFBP5 gene homozygous in all available Greyhounds and other sighthounds. Further research is required to clarify whether the IGFBP5 variant represents a predisposing genetic risk factor. We conclude from our results that structural defects in the hair shafts are the cause for this well-known disease and these defects are associated with a downregulation of genes and proteins essential for hair shaft formation. Our data add important knowledge to further understand the molecular mechanisms of HF morphogenesis and alopecia in dogs.

Role of insulin like growth factor axis in the bleomycin induced lung injury in rats.

BACKGROUND: Alveolar epithelial cell injury has been proposed as a causative factor for the onset and progression of pulmonary fibrosis. However, the role of type II alveolar epithelial cells (AECs) in the epithelial mesenchymal transition (EMT) is controversial. AIMS: The present study performed in rats instilled with bleomycin investigated a) the expressions of the insulin growth factor (IGF-1) and insulin growth factor binding protein 5 (IGFBP-5) and transforming growth factor (TGF-ß1) in the type II AECs, b) the role of type II AECs in EMT and extracellular matrix (ECM) formation and, c) the effect of pioglitazone on all the above parameters. METHODS: Male Wistar rats were divided into three Groups: Group I (saline control), Group II (Bleomycin, given as a single intratracheal instillation, 7U/kg) and Group III (Bleomycin+Pioglitazone (40mg/kg/day orally, starting 7days post bleomycin instilled as in Group II). From lung tissues, the protein expressions of IGF-1, IGFBP-5, TGF-ß1, surfactant protein C (SP-C, as a marker for type II AECs) and α-smooth muscle actin (α-SMA, as a marker for EMT), were determined on day 7 in Groups I and II and on days 14, 21 and 35 in all the three groups. RESULTS: IGFBP-5 and IGF-1 expressions were reduced significantly and TGF-ß1 expression increased significantly in type II AECs in Group II from day 7 till day 35 as compared to Group I. An increase in SP-C and α-SMA expression and their co-localization were seen in the type II AECs undergoing EMT from day 7 till day 35. A concomitant remodeling and laying down of ECM was observed also. In Group III, with pioglitazone, there was a reversal with significant up-regulation in IGFBP-5 and IGF-1 expressions and down-regulation of TGF-ß1 in the type II AECs along with a significant decrease in the solid area fraction, EMT and ECM in the lung tissue. CONCLUSIONS: IGFBP-5, IGF-1 and TGF-ß1 in the type II AECs play a key role in lung injury caused by bleomycin and pioglitazone attenuates the lung injury/fibrosis by restoring IGFBP-5 and IGF-1 and decreasing TGF-ß1 expressions in the type II AECs.

Dysregulated DNA Methyltransferase 3A Upregulates IGFBP5 to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia.

Preeclampsia is a unique multiple system disorder during human pregnancy, which affects ≈5% to 8% of pregnancies. Its risks and complications have become the major causes of maternal and fetal morbidity and mortality. Although abnormal placentation to which DNA methylation dysregulation is always linked is speculated to be one of the reasons causing preeclampsia, the underlying mechanisms still remain elusive to date. Here we revealed that aberrant DNA methyltransferase 3A (DNMT3A) plays a critical role in preeclampsia. Our results show that the expression and localization of DNMT3A are dysregulated in preeclamptic placenta. Moreover, knockdown of DNMT3A obviously inhibits trophoblast cell migration and invasion. Mechanistically, IGFBP5 (insulin-like growth factor-binding protein 5), known as a suppressor, is upregulated by decreased DNMT3A because of promoter hypomethylation. Importantly, IGFBP5 downregulation can rescue the defects caused by DNMT3A knockdown, thereby, consolidating the significance of IGFBP5 in the downstream of DNMT3A in trophoblast. Furthermore, we detected low promoter methylation and high protein expression of IGFBP5 in the clinical samples of preeclamptic placenta. Collectively, our study suggests that dysregulation of DNMT3A and IGFBP5 is relevant to preeclampsia. Thus, we propose that DNMT3A and IGFBP5 can serve as potential markers and targets for the clinical diagnosis and therapy of preeclampsia.

Orthodontic forces modulate insulin-like growth factor binding protein-5 changes in gingival crevicular fluid.

Orthodontic tooth movement results from the response of the periodontal tissue to orthodontic force, which leads to modeling and remodeling of the surrounding alveolar bone. The response is considered to occur through the activation of specific signaling pathways, many of which are known, all acting to ultimately result in tooth movement. Much is known about the actions of these two cells, and the signaling pathways that affect them, both in bone and orthodontic literature, however, to date, little work has been carried out to examine the effect of the insulin-like growth factor binding proteins (IGFBP) in orthodontics. Therefore, we investigated the presence of IGFBP-5 in the gingival crevicular fluid (GCF) of 6 healthy subjects, and assessed the effects of orthodontic treatment on the levels and molecular state of this protein.

IGFBP-4 and -5 are expressed in first-trimester villi and differentially regulate the migration of HTR-8/SVneo cells.

BACKGROUND: Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -I and -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. The insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, including sequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and -5 in the placenta are unknown, despite consistent associations between pregnancy complications and the levels of two IGFBP-4 and/or -5 proteases, pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study was to elucidate the effects of IGFBP-4 and -5 on IGF-I and IGF-II in a model of EVT migration. A related objective was to determine the timing and location of IGFBP-4 and -5 expression in the placental villi. METHODS: We used wound healing assays to examine the effects of IGFBP-4 and -5 on the migration of HTR-8/SVneo cells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, -5 and PAPP-A2 was assessed by immunohistochemical staining of first trimester placental sections. RESULTS: 2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly more than IGF-II. IGFBP-4 and -5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blocked the effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to control levels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistry revealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placental villi as early as 5 weeks of gestational age. CONCLUSIONS: IGFBP-4 and -5 show different levels of inhibition on the migration-stimulating effects of IGF-I and IGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and their substrates within placental villi suggests undescribed roles of these molecules in early placental development.

Stable transfection and identification of a hair follicle-specific expression vector of IGFBP-5 in goat fetal fibroblasts.

The insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the 6 members of the IGFBP family and is involved in the regulation of cell growth, apoptosis, and other IGF-stimulated signaling pathways. To determine the significance of IGFBP-5 in the Inner Mongolia Cashmere goat (Capra hircus), a hair follicle-specific expression vector of IGFBP-5, pCDsRed2-K-IGFBP5 (6.7 kb), was constructed by cloning IGFBP-5 downstream of the keratin-association protein (KAP)6-1 promoter and inserting this fragment into pCDsRed2, which contains a red fluorescent protein (DsRed) expression unit. Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by using Lipofectamine(TM) 2000. Cell clones that stably expressed red fluorescence were obtained after selection with Geneticin (G418). The transgene in the cell clones was examined by polymerase chain reaction to verify that exogenous DNA (pKAP6-1 and IGFBP-5) had integrated stably into GFb cells. These data suggest that this method can be used for the construction of a hair follicle-specific expression vector for functional genetic analyses and for obtaining stable transfection donor cells for nuclear transfer.

Intramammary infusion of Panax ginseng extract in the bovine mammary gland at cessation of milking modifies components of the insulin-like growth factor system during involution.

The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng extract (GS) on insulin-like growth factors (IGF) in bovine mammary gland during early involution. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of ginseng extract solution (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Concentrations of IGF1 in mammary secretions were higher in GS-treated quarters than in placebo and uninoculated control quarters at 24, 48 and 72 h post-treatment (p<0.05). Treatment with GS did not affect mammary secretion of IGF2 (p=0.942). At 7 d of post-lactational involution, a decrease of immunostained area and mRNA expression for IGF1 was observed in mammary tissue of GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). The IGF2 immunostained area and mRNA expression for this growth factor were not affected by GS treatment (p=0.216 and p=0.785, respectively). An increase in protein levels and mRNA expression in mammary tissue of IGFBP3, IGFBP4 and IGFBP5 was observed in GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). These results provide evidence that intramammary inoculation of GS extract at cessation of milking may promote early mammary involution through the inhibition of IGF1 local production and bioavailability.

Gene expression in swine granulosa cells and ovarian tissue during the estrous cycle.

The components of the insulin-like growth factor (IGF) system appear to be involved in regulation of ovarian follicular growth and atresia in the pig. We investigated the expression pattern of mRNAs for IGF1 (IGF1), its binding proteins (IGFBP1, IGFBP2, IGFBP3, and IGFBP5), and epidermal growth factor in swine follicle cells and ovarian tissue throughout the estrous cycle using the real-time quantitative PCR technique. The results of gene expression were analyzed using linear regression with gene expression as a dependent variable and days of estrous cycle as an independent variable. Additionally, an analysis was made of the correlation of expression levels with plasma concentration of follicle-stimulating hormone, luteinizing hormone, estradiol-17ß, progesterone, and prolactin. Expression of mRNA of all of these genes was detected in granulosa cells and ovarian tissue. IGFBP3 mRNA showed a quadratic expression pattern (P ≤ 0.001) and was significantly and positively correlated with progesterone (r = 0.81; P ≤ 0.01) but negatively correlated with prolactin (r = -0.596; P ≤ 0.05). Expression of the other genes was unaffected by the stage of the estrous cycle. Real-time quantitative PCR effectively detected all transcripts, including the very low levels of IGFBP1 transcripts, and could be used for studies of follicle dynamics.

Fetal alcohol exposure increases mammary tumor susceptibility and alters tumor phenotype in rats.

BACKGROUND: Altered fetal programming because of a suboptimal in utero environment has been shown to increase susceptibility to many diseases later in life. This study examined the effect of alcohol exposure in utero on N-nitroso-N-methylurea (NMU)-induced mammary cancer risk during adulthood. METHODS: Study 1: Pregnant Sprague Dawley rats were fed a liquid diet containing 6.7% ethanol (alcohol-fed), an isocaloric liquid diet (pair-fed), or rat chow ad libitum (ad lib-fed) from day 11 to 21 of gestation. At birth, female pups were cross-fostered to ad lib-fed control dams. Adult offspring were given an I.P. injection of NMU at a dose of 50 mg/kg body weight. Mammary glands were palpated for tumors twice a week, and rats were euthanized at 23 weeks postinjection. Study 2: To investigate the role of estradiol (E2), animals were exposed to the same in utero treatments but were not given NMU. Serum was collected during the preovulatory phase of the estrous cycle. RESULTS: At 16 weeks postinjection, overall tumor multiplicity was greater in the offspring from the alcohol-fed group compared to the control groups, indicating a decrease in tumor latency. At study termination, 70% of all animals possessed tumors. Alcohol-exposed animals developed more malignant tumors and more estrogen receptor-α-negative tumors relative to the control groups. In addition, IGF-binding protein-5 (IGFBP-5) mRNA and protein were decreased in tumors of alcohol-exposed animals. Study 2 showed that alcohol-fed animals had significantly increased circulating E2 when compared to either control group. CONCLUSIONS: These data indicate that alcohol exposure in utero increases susceptibility to mammary tumorigenesis in adulthood and suggest that alterations in the IGF and E2 systems may play a role in the underlying mechanism.

Grade-specific expression of insulin-like growth factor-binding proteins-2, -3, and -5 in astrocytomas: IGFBP-3 emerges as a strong predictor of survival in patients with newly diagnosed glioblastoma.

BACKGROUND: Insulin-like growth factor (IGF)-binding protein (IGFBP) isoforms have been implicated in the pathogenesis of human neoplasms including glioma. In view of this, we evaluated the expression of IGFBP isoforms (IGFBP-2, -3, and -5) during malignant progression of astrocytoma and their prognostic significance in glioblastoma. METHODS: The expression of IGFBP isoforms was analyzed in diffusely infiltrating astrocytomas by real-time quantitative PCR (n = 203) and immunohistochemistry (n = 256). Statistical methods were used to assess their grade-specific expression pattern and mRNA-protein intercorrelation. Survival analyses were done on a uniformly treated, prospective cohort of adult patients with newly diagnosed glioblastoma (n = 136) by using Cox regression models. RESULTS: The mean transcript levels of IGFBP-2 and -3 were significantly higher in glioblastomas (GBM) relative to anaplastic astrocytoma (AA), diffuse astrocytoma (DA), and controls whereas IGFBP-5 mRNA was higher in GBM relative to AA and controls (P < 0.05). By immunohistochemistry, the mean labeling index of all isoforms was significantly higher in GBM compared with AA, DA, and control (P < 0.05). A strong positive correlation was observed between their respective mRNA and protein expressions (P < 0.01). Multivariate analysis revealed IGFBP-3 expression (hazard ratio, 1.021; P = 0.030) and patient age (hazard ratio, 1.027; P = 0.007) to be associated with shorter survival in glioblastoma. CONCLUSIONS: This study shows the associations of IGFBP-2, -3, and -5 expression with increasing grades of malignancy in astrocytomas. IGFBP-3 is identified as a novel prognostic glioblastoma biomarker. The strong correlation between their mRNA and protein expression patterns suggests their role in the pathogenesis of these tumors. IMPACT: IGFBP isoforms have emerged as biomarkers with diagnostic and prognostic utility in astrocytomas.

The effects of HIF-1alpha on gene expression profiles of NCI-H446 human small cell lung cancer cells.

BACKGROUND: Gene targeted therapy refers to any therapy focused on one of the many biological features of the tumor. Such features are mediated by specific genes that are involved in tumor metastasis, recurrence, poor response to chemotherapy and others. Hypoxia is an important pathognomonic feature of many malignant tumors including SCLC (small cell lung cancer). HIF-1alpha, which is induced by hypoxia, is the most important regulatory factor of many specific genes that can influence the biological features of tumors. METHODS: In this study, we tried to elucidate the changes in gene expression profiles of SCLC NCI-H446 cells mediated by HIF-1alpha. According to different treatments of cells, three experimental pairwise comparisons were designed: hypoxia group vs. control group, Ad5-HIF-1alpha group vs. Ad5 group, and Ad5-siHIF-1 alpha group Vs Ad5 group. RESULTS: Results from the analysis of gene expression profiles indicated that there were 65 genes upregulated and 28 genes downregulated more than two-fold in all three experimental pairwise comparisons. These genes were involved in transport, signal-transduction, cell adhesion/motility, growth factor/cytokines, transcription, inflammatory response, metabolic process, in addition to others. SOCS1, IGFBP5, IL-6 and STAT3 were also upregulated at protein level. SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells but HIF-1alpha could induce growth and suppress apoptosis. CONCLUSIONS: Through this research, we are trying to find novel functional genes that are mediated by HIF-1alpha and provide the theoretical basis for new therapeutic targets. HIF-1 alpha maybe upregulate the expression of SOCS1 through mediation of STAT3 and IL-6. In addition, SOCS1 could significantly induce apoptosis and suppress growth of NCI-H446 cells. This was contrary to HIF-1alpha and it indicated that there might be an antagonism effect between HIF-1alpha and SOCS1 on regulating growth and apoptosis of NCI-H446 cells.

IGFBP2 and IGFBP5 overexpression correlates with the lymph node metastasis in T1 breast carcinomas.

The family of insulin-like growth factor binding proteins (IGFBPs) comprises six members which bind and regulate the functions of IGFs. Overexpression of IGFBP2 and IGFBP5 contributes to the invasiveness and progression of several human cancers, but their roles in the metastasis of breast cancer have not been investigated in detail. To determine their roles, we examined IGFBP2 and IGFBP5 expression levels in 164 T1 breast carcinomas using tissue microarrays and immunohistochemistry. The specimens were divided into those with (N1) or without (N0) axillary lymph node involvement. The results were associated with clinicopathologic parameters and prognostic molecular markers. No or very low expression of IGFBP2 and IGFBP5 was detected in normal breast epithelium or benign breast tissue with fibrocystic change. Moderate to strong cytoplasmic staining for IGFBP2 and IGFBP5 was detected in 49.1% and 50.3% of T1 invasive breast carcinomas, respectively. T1N1 carcinomas were more frequent to have moderate and strong-positive staining for IGFBP2 and IGFBP5 than in T1N0 carcinomas (p < 0.05). IGFBP2 and IGFBP5 expression correlated with the expression status of progesterone receptor and HER-2/neu in the overall T1 carcinoma group, but no association was found with tumor size or the expression status of estrogen receptor. Our data suggest that IGFBP2 and IGFBP5 play a role in the development of metastasis and may serve as useful markers to predict lymph node metastasis in patients with small (T1) invasive breast carcinomas.

The insulin-like growth factor pathway is altered in spinocerebellar ataxia type 1 and type 7.

Polyglutamine diseases are inherited neurodegenerative disorders caused by expansion of CAG repeats encoding a glutamine tract in the disease-causing proteins. There are nine disorders, each having distinct features but also clinical and pathological similarities. In particular, spinocerebellar ataxia type 1 and 7 (SCA1 and SCA7) patients manifest cerebellar ataxia with degeneration of Purkinje cells. To determine whether the disorders share molecular pathogenic events, we studied two mouse models of SCA1 and SCA7 that express the glutamine-expanded protein from the respective endogenous loci. We found common transcriptional changes, with down-regulation of insulin-like growth factor binding protein 5 (Igfbp5) representing one of the most robust changes. Igfbp5 down-regulation occurred in granule neurons through a non-cell-autonomous mechanism and was concomitant with activation of the insulin-like growth factor (IGF) pathway and the type I IGF receptor on Purkinje cells. These data define one common pathogenic response in SCA1 and SCA7 and reveal the importance of intercellular mechanisms in their pathogenesis.

Aging alters gene expression of growth and remodeling factors in human skeletal muscle both at rest and in response to acute resistance exercise.

The purpose of this investigation was to compare expression of genes that function in inflammation and stress, cell structure and signaling, or remodeling and growth in skeletal muscle of young (32 +/- 7 yr, n = 15) and elderly (72 +/- 5 yr, n = 16) healthy subjects before and after a bout of resistance leg exercises. A real-time RT-PCR method was used to screen 100 transcripts in v. lateralis biopsies obtained before and 72 h postexercise. The screen identified 15 candidates for differential expression due to aging and/or exercise that were measured quantitatively. The median levels of four mRNAs (insulin-like growth factor-1 and its binding protein IGFBP5, ciliary neurotrophic factor, and the metallopeptidase MMP2) were significantly affected by aging and were greater (1.6- to 2.3-fold, P

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells.

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.

Comparison of estrogen responsive genes in the mouse uterus, vagina and mammary gland.

Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.