Growth Factor Binding Protein Research Articles

Effect of the tumor promoter phenobarbital on the pattern of global gene expression in liver of connexin32-wild-type and connexin32-deficient mice

The antiepileptic drug phenobarbital (PB) is used frequently as a model tumor promoter in rodent liver. It is believed to increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. The molecular mechanism underlying this process is only partly understood but seems to require the function of connexin32 (Cx32), one of the 2 gap junction proteins expressed in hepatocytes. PB mediates transcriptional activation of various genes in liver but which of these are relevant for tumor promotion is unknown. We have used oligonucleotide microarrays to identify genes differentially modulated in expression by PB in liver of Cx32-wild-type and Cx32-null mice. Mice of both strains were kept on PB containing (0.05%) or control diet for 2 weeks. Total liver RNA was isolated from 3 mice per experimental group and reverse transcribed; cDNAs were hybridized to oligonucleotide microarrays and a gene-by-gene linear model was used for statistical analysis of data. Five genes were identified as induced or repressed in untreated Cx32-null as compared to untreated Cx32-wild-type mice. PB affected the expression of 53 genes, of which 13 code for members of Phase-I/II of drug metabolism, and 12 genes were differentially affected in expression by PB in Cx32-null as compared to Cx32-wild-type mice. Among the differentially affected genes that could be verified by quantitative RT-PCR or Western analysis were the insulin like growth factor binding protein-1, retinol dehydrogenase-6 and the Y-chromosomally located gene Dby, among which may be a candidate of relevance for PB-mediated tumor promotion.

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15–76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.

Insulin‐like Growth Factor Binding Protein 5: Contribution to Growth and Differentiation of Neuroblastoma Cells

Neuroblastoma (NB) is a childhood tumor that depends on insulin-like growth factors (IGFs) for its growth and metastatic spread. Some metastatic NBs acquire independence from the paracrine support of IGF by activating autocrine production of IGF-2. Insulin-like growth factor binding protein-5 (IGFBP-5), a member of the IGF binding protein family, is able to optimize binding between IGF itself and its receptor. NB cell lines retain the ability to differentiate in vitro toward neuronal, schwann-like or melanocytic phenotypes upon treatment with retinoic acid (RA). Retinoids are currently used in NB therapy to achieve a mature postmitotic phenotype. Here, we present evidence that the expression of IGFBP-5 is a common feature of neuroblastoma cell lines and that IGFBP-5 acts in concert with IGF-2 in inducing cell proliferation. RA-induced differentiation causes a sharp increase of IGFBP-5. Functional assays carried out in differentiating conditions demonstrate that IGFBP-5 transcription is sensitive to RA treatment. We show that the effect of RA on the IGFBP-5 promoter is exerted, at least in part, through a proximal 5'-CACCC-3' tandem repeat (-147 bp to -137 bp from the transcription start site) that has previously been described as a cis-acting element involved in the progesterone-mediated response in osteoblasts. Given the relevance of IGF-2 in determining the proliferative and metastatic behavior of NB, the role of IGFBP-5 as a modulator of the IGF signal transduction pathway should be studied further for potential therapeutic applications.

Structural Basis and Therapeutic Implication of the Interaction of CCN Proteins with Glycoconjugates

The CCN family of growth factors is composed of six structurally related proteins including the cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), nephroblastoma overexpressed (NOV), Wnt-1 induced secreted protein-1 (WISP-1), WISP-2 and WISP-3. Each family member consists of four conserved cysteine rich modular domains with sequence similarity to the insulin like growth factor binding proteins, von Willebrand factor, thrombospondin repeat and the growth factors cysteine knot. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. Both cancer promoting and inhibiting roles were proposed for several CCN proteins suggesting that contextual factors could regulate their activities. Consistent with this hypothesis, structural and experimental evidence indicate that the function of these proteins is modulated by their interaction with sulfated glycosaminoglycans. Because the CCN proteins are implicated in the tumorigenic process, they are potential targets for the development of cancer therapeutics. Modulation of their glycosaminoglycan interaction by exogenous, highly sulfated polysaccharides, oligosaccharides or glycosaminoglycan mimetics could prevent their participation in cancer progression. Understanding the structural requirements for their polysaccharide interaction should provide important information to generate glycosaminoglycan-based cancer therapeutics targeting the CCN family of proteins.

Thrombopoietin regulates proliferation, apoptosis, secretory activity and intracellular messengers in porcine ovarian follicular cells: involvement of protein kinase A

Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.

Analysis of insulin like growth factor 1 and insulin like growth factor binding protein 3 levels in bronchoalveolar lavage fluid and serum of patients with lung cancer

Insulin like growth factor 1 (IGF-1) is recognized as a potent mitogen for many cancer cell lines and there is good evidence that lung cancer cells produce both IGF-1 and insulin like growth factor binding protein 3 (IGFBP-3). The aim of this study was to investigate the clinical significance of IGF-1 and IGFBP-3 levels in serum and in bronchoalveolar lavage (BAL) fluid by comparing lung cancer patients with healthy controls. BAL fluid and serum samples were obtained from 24 lung cancer patients and 12 healthy controls, and were analyzed for IGF-1 and IGFBP-3 levels by a two site immunoradiometric assay. The recovered BAL fluid was standardized by albumin to remove the variable of dilution and the data was expressed in epithelial lining fluid (ELF). Serum IGF-1 and IGFBP-3 levels were lower in lung cancer patients, but the difference between the groups did not reach a statistical significance. IGF-1/IGFBP-3 ratio in ELF was significantly lower in lung cancer patients (P=0.035). Mean IGF-1 level in ELF was determined to be significantly lower in patients with distant metastasis (P=0.04). Serum IGF-1/IGFBP-3 ratio was found to be significantly lower in patients with distant (P=0.04) and nodal metastasis (P=0.03). Tumor stage was negatively correlated with IGF-1 level in ELF (P=0.05, r=-0.4) and serum IGF-1/IGFBP-3 ratio (P=0.04, r=-0.4). IGF-1 and IGFBP-3 levels both in serum and ELF might serve a clinical significance in patients with lung cancer. However, further studies comprising more cases are needed to investigate the clinical significance of IGF-1 and IGFBP-3 in lung cancer.

Associations of insulin‐like growth factors, insulin‐like growth factor binding proteins and acid‐labile subunit with coronary heart disease

IGFs and their binding proteins (IGFBPs) are produced both systemically and locally by cells of the cardiovascular system. As growth promoters, they may play a role in atherosclerosis. Case-control, cross-sectional. A total of 95 nondiabetic male patients with coronary heart disease (CHD) and 92 probands from the Prospective Cardiovascular Munster (PROCAM) who were below the age of 60 years and matched by age, body mass index (BMI) and smoking habits. We analysed the strength and independence of associations of angiographically assessed presence of CHD with BMI, systolic and diastolic blood pressure, total, high-density lipoprotein (HDL) and LDL cholesterol, triglycerides, lipoprotein(a), apolipoproteins A-I and B, total and free IGF-I, IGF-II, IGFBP-1, IGFBP-3, IGFBP-5, acid-labile subunit (ALS), insulin, C-peptide, testosterone, DHEAS and sex hormone binding globulin. Using multivariate statistical analysis, the presence of CHD had significant positive associations with total IGF-I, IGFBP-5, ALS and IGFBP-3. These associations were independent of each other as well as of traditional risk factors, insulin and sex hormones. These observations may indicate a pathogenetic role of the GH/IGF axis in coronary atherosclerosis.

128 Increase of IGFBP1 following Hypoxia in The Piglet

Background:: Insulin Like Growth Factor Binding Proteins (IGFBPs) may be more directly related to ischaemic injury than factors such as pH and lactate, which relate to hypoxia and anaerobic metabolism. We sought to determine if IGFBP1 increases following a hypoxic insult in an animal model and to establish its possible role as an early predictor of outcome. Methods: 26 piglets were anaesthetised and ventilated. The oxygen intake was decreased to 3 to 7%, in order to create a hypoxic insult. Measurements of IGFBP1 were made before and in regular intervals after the insult. At 72 hours the piglets were sacrificed, the brains perfusion fixed and histologically examined. Results: There was no difference of birth weight or postnatal age of the animals between the groups. The histology score in the severe insult group was significantly higher than in the control or mild insult group (15.6 vs 0, p< 0.001). At 48 and 72 hours there was a significant difference of IGFBP1 between the groups with the short and prolonged hypoxia group having higher IGFBP1 levels (43.31 vs 77.9 μmol/L at 48 hours, 30.13 vs 79.68 μmol/L at 72 hours, p< 0.05) Conclusion: IGFBP1 is expressed after a hypoxic insult. The sustained increase may represent hepatic damage as is often seen in neonates following perinatal asphyxia. These findings need to be verified in neonates, however, timing of the insult is often not possible in babies. It may be that IGFBP1 may be used as a predictor of outcome following perinatal asphyxia in neonates.

Membrane type-matrix metalloproteinases and tumor progression

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that process growth factors, growth factor binding proteins, cell surface proteins, degrade extracellular matrix (ECM) components and thereby play a central role in tissue remodeling and tumor progression. Membrane-type matrix metalloproteinases (MT-MMPs) are a recently discovered subgroup of intrinsic plasma membrane proteins. Their functions have been extended from pericellular proteolysis and control of cell migration to cell signaling, control of cell proliferation and regulation of multiple stages of tumor progression including growth and angiogenesis. This review sheds light on the new functions of MT-MMPs and their inhibitors in tumor development and angiogenesis, and presents recent investigations that document their influence on various cell functions.

Insulin like growth factor-1 and insulin-like growth factor binding proteins: their possible roles in both maintaining normal retinal vascular function and in promoting retinal pathology.

Insulin like growth factor-1 and insulin-like growth factor binding proteins: their possible roles in both maintaining normal retinal vascular function and in promoting retinal pathology.

Binding site for the C-domain of insulin-like growth factor (IGF) binding protein-6 on IGF-II; implications for inhibition of IGF actions

Binding site for the C-domain of insulin-like growth factor (IGF) binding protein-6 on IGF-II; implications for inhibition of IGF actions

003 Gene Expression Profiles Following Electron Irradiation of Cultured Keloid and Normal Skin Fibroblasts by cDNA Microarray Analysis

Aim: When surgery with postoperative superficial electron irradiation is applied, the recurrence rate of keloid lesion has been found to be &lt;30%. In this study, we assessed the molecular changes underlying the effect of electron irradiation by differential global gene expression analyses of both cultured keloid and normal skin fibroblasts. Materials and Methods: Primary cultured fibroblasts from 4 active keloids and their adjacent normal dermal tissues were irradiated at a calibrated dosage of 15 Gy with 6 MeV electron beam generated by a linear accelerator. Corresponding paired non‐irradiated cells were used as control. RNA isolated from the collected cells was labeled with 33P, hybridized to the cDNA microarray gene filters and analyzed. Results: After irradiation, the gene expression profiles of keloid and normal skin fibroblasts were closely similar. Electron irradiated keloid fibroblasts showed suppressed levels of collagen typeI (alpha2), collagen typeVI (alpha1), matrix metalloproteinase 2, fibronectin 1, insulin‐like growth factor binding protein 3, alpha‐1‐antichymotorypsin and heparan glucosaminyl 1 as compared with their non–irradiated counterparts. Conclusions: Downregulation of matrix synthesis and upregulation of protease inhibitors and apoptosis promoting genes by electron irradiation may inhibit keloid development. This mode of therapy appears to exert a positive effect toward lowering the recurrence rate of keloid formation.

Molecular pathways executing the “trophic sentinel” response in HPV-16 E7-expressing normal human diploid fibroblasts upon growth factor deprivation

In response to oncogenic insults, normal human cells execute a defense response that culminates in cellular suicide, apoptosis. Normal human diploid fibroblasts expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein are predisposed to apoptosis when they are deprived of growth factors. Even though a dominant negative p53 mutant abrogates the cell death response, it is not accompanied by p53 phosphorylation, the DNA binding capacity of p53 remains unaltered, and no activation of common p53-dependent transcriptional targets is observed. Expression of two insulin-like growth factor-1 binding proteins, IGFBP-2 and -5, is increased presumably in response to enhanced NF-κB activity in HPV-16 E7-expressing serum-starved cells. Phosphorylation of AKT, an important modulator of IGF-1 survival signaling, is lower in serum-starved E7-expressing cells, and exogenously added IGF-1 can partially inhibit the cell death response. This suggests that IGFBP-2 and -5 may limit IGF-1 availability thus decreasing survival signaling. Caspase 3 but not caspase 8 is activated in serum-starved HPV-16 E7-expressing cells. Caspase inhibition affects nuclear DNA fragmentation, but cell death is not inhibited. Although mitochondria play important roles in caspase-dependent as well as -independent forms of cell death, there is no evidence for cytochrome c release and thus for mitochondrial permeabilization in growth factor deprived HPV-16 E7-expressing cells.

1alpha,25-dihydroxyvitamin D3 induced growth inhibition of PC-3 prostate cancer cells requires an active transforming growth factor beta signaling pathway.

Prostate cancer growth inhibition by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) is best characterized in the androgen dependent LNCaP cell line, where treatment with this hormone causes cell cycle arrest and apoptosis. 1,25(OH)2D3 also inhibits the growth of PC-3 prostate cancer cells, but not through the induction of G1 arrest or apoptosis. In this study, we have sought to elucidate the mechanism/s involved in PC-3 cell growth inhibition by 1,25(OH)2D3. We determined the effect of transforming growth factor beta (TGFbeta) blocking antibodies on 1,25(OH)2D3 mediated growth inhibition of PC-3 cells. In addition, we also studied the effects of 1,25(OH)2D3 on TGFbeta signaling and receptor expression. Finally, we assessed the role of TGFbeta signaling in the induction of the growth inhibitory protein, insulin like growth factor binding protein 3 (IGFBP-3), by 1,25(OH)2D3. We find that 1,25(OH)2D3 action in PC-3 cells is mediated through at least two distinct pathways, the TGFbeta pathway and the IGFBP-3 pathway. We show that 1,25(OH)2D3 treatment elevates TGFbeta production and signaling, as well as receptor levels, in PC-3 cells. Further, using a blocking antibody against TGFbeta substantially reduces 1,25(OH)2D3 mediated growth inhibition without affecting IGFBP-3 induction, suggesting that IGFBP-3, alone, is insufficient to inhibit the growth of PC-3 cells.

The ontogeny of the somatotropic axis in male and female Hereford calves from birth to one year of age.

Insulin-like growth factor-binding proteins-2 and -3 may play a role in age-dependent growth response to bovine ST (bST) treatment in cattle; however, samples have been collected at infrequent intervals and at limited time points. Therefore, the objective of this experiment was to examine the ontogeny of components of the somatotropic axis in Hereford calves from birth to 1 yr of age at weekly intervals to determine whether there is a certain age or time frame when the somatotropic axis may change and/or potentially become more responsive to exogenous bST administration. Blood samples and body weight measurements were collected from eight male and eight female Hereford calves once per week from birth to 1 yr of age. Serum concentrations of ST, IGF-I, IGFBP-2, and IGFBP-3 were determined. Males began to grow faster than females at approximately 16 wk of age (P < 0.05). Average concentrations of ST, IGF-I, and IGFBP-3 were greater in males than females (P < 0.01). Average concentrations of IGFBP-2 were greater in females than in males (P = 0.05). Concentrations of ST decrease with age (P < 0.01); however, the decrease occurred earlier in female calves. Concentrations of IGF-I and IGFBP-3 increased in males and females (P < 0.01), and concentrations of IGF-I began to plateau at approximately the same time as growth rate differences were observed (16 wk of age). Following an initial increase (birth to approximately 16 wk of age), concentrations of IGFBP-3 remained constant until approximately 43 wk of age. Concentrations of IGFBP-2 increased to approximately 10 wk of age (P < 0.05), followed by a decrease, and then, similar to IGFBP-3, remained constant until 43 wk of age. Correlations between average daily gain, ST, IGF-I, IGFBP-2, and IGFBP-3 were determined. Average daily gain was negatively (P < 0.01) correlated with ST and positively (P < 0.1) correlated with IGF-I. In females, ST was negatively (P < 0.01) correlated with IGF-I. Concentrations of ST were positively correlated (P < 0.01) with IGFBP-2 and IGFBP-3. Concentrations of IGFBP-2 were negatively correlated (P < 0.01) with IGF-I and positively correlated (P < 0.01) with IGFBP-3. In conclusion, serum concentrations of ST, IGF-I, IGFBP-2, and IGFBP-3 differed between male and fe-male calves. In addition, changes in components of the somatotropic axis occurred around the same time as males began to grow faster than females.

Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis.

The matrix metalloproteinases (MMPs) constitute a multigene family of over 23 secreted and cell-surface associated enzymes that cleave or degrade various pericellular substrates. In addition to virtually all extracellular matrix (ECM) compounds, their targets include other proteinases, chemotactic molecules, latent growth factors, growth factor-binding proteins and cell surface molecules. The MMP activity is controlled by the physiological tissue inhibitors of MMPs (TIMPs). There is much evidence that MMPs and their inhibitors play a key role during extracellular remodeling in physiological situations and in cancer progression. They have other functions that promoting tumor invasion. Indeed, they regulate early stages of tumor progression such as tumor growth and angiogenesis. Membrane type MMPs (MT-MMPs) constitute a new subset of cell surface-associated MMPs. The present review will focus on MT1-MMP which plays a major role at least, in the ECM remodeling, directly by degrading several of its components, and indirectly by activating pro-MMP2. As our knowledge on the field of MT1-MMP biology has grown, the unforeseen complexities of this enzyme and its interaction with its inhibitor TIMP-2 have emerged, often revealing unexpected mechanisms of action.

Membrane type-matrix metalloproteinases (MT-MMP)

Publisher Summary Matrix metalloproteinases (MMPs) belong to the family of zinc endopeptidases collectively referred to as metzincins. The metzincin superfamily is distinguished by a highly conserved motif containing three histidines that bind zinc at the catalytic site and a conserved methionine that sits beneath the active site. The metzincins are subdivided into four multigene families: serralysins, astacins, ADAMs/adamalysins, and MMPs. Once activated, MMPs degrade a variety of extracellular matrix components and assorted other proteins including growth factors, growth factor binding proteins, and protease inhibitors. The ability to degrade extracellular matrix proteins is essential for any cell to interact properly with its immediate surroundings and for multicellular organisms to develop and function normally. MMPs also generate matrix protein fragments, which have functional activity of their own. These various functions of MMPs modulate cell invasion and metastasis, cell migration, apoptosis, and angiogenesis. The belief that an MMP is involved in a given biologic/pathologic process is often based on the strength of its association with that process and the existence of plausible mechanisms that can be tested by experimental approaches. Because of extensive redundancy of MMP function in animals, it has been difficult to appreciate the role of individual genes in various experimental models. The multiplicity of MMPs with distinct but somewhat overlapping functions appears to act as a safeguard against loss of regulatory control. The known MMP genes have been divided into four subfamilies based on gene structure. Group I consists of the collagenase subfamily. Group 2 consists of the gelatinases/modular alteration (fimodular a-like domains) subfamily. Group 3 consists of the variant hemopexin exon subfamily. Group 4 consists of the variant catalytic exon and unique 3′-end exon (plasma membrane and cytoplasmic domains) subfamily, which consists of the membrane type-matrix metalloproteinases (MT-MMPs).

Stromal cells of the human postmenopausal ovary display a distinctive biochemical and molecular phenotype.

The stroma of the human postmenopausal ovary is postulated to produce androgens, but evidence for and against this idea exits in the literature. The purpose of this study was to determine whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma. Stromal cells were isolated from postmenopausal ovaries and expression for genes involved in steroidogenesis [steroidogenic acute regulatory protein (StAR), P450scc, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) P450c17, and P450c27] as well as for several growth factor binding proteins [gremlin, IGF binding protein-4, follistatin, and secreted frizzled-related protein (sFRP)-1 and -4], were compared with cultured human theca cells and dermal fibroblasts. Production of steroids (pregnenolone, progesterone, and hydroxysterol metabolites) and the metabolism of [3H] pregnenolone by ovarian stromal cells were also assessed. Isolated ovarian stromal cells from different subjects had a uniform morphology within and across cultures. Quantitative real time RT-PCR revealed that StAR, P450scc, and 3 beta-HSD transcripts were, respectively 30, 25, and 45 times more abundant in theca cells than in stromal cells. Mean levels of P450scc and 3 beta-HSD transcripts in stromal cells were similar to those found in dermal fibroblasts, whereas StAR transcripts in stromal cells were 285-fold more abundant than in fibroblasts. There was no significant expression of P450c17 in ovarian stromal cells or fibroblasts ( approximately 2000-fold less than in theca cells). Western analysis demonstrated the presence of the 30-kDa StAR mature protein in the cultured stromal cells, whereas P450c17 protein was not detectable. Ovarian stromal cells did not metabolize [3H] pregnenolone in the presence or absence of 8-Br-cAMP. Furthermore, pregnenolone and progesterone secretion by stromal cells was also undetectable, even in the presence of 22-hydroxycholesterol. P450c27 protein was detected in ovarian stromal cells and its metabolic products (i.e. 27-hydroxycholesterol and cholestenoic acid) were found in the culture media, reflecting functional cholesterol 27-hydroxylase activity. Follistatin, gremlin, IGF binding protein-4, and sFRP-1 and -4 transcripts were detected in the stromal cells in relative amounts significantly higher than theca cells, but not significantly different from fibroblasts, except for sFRP-1, which was significantly higher in stromal cells. Our observations demonstrate that stromal cells of the postmenopausal ovary have a signature biochemical and molecular phenotype that can be distinguished from fibroblasts. These cells do not appear to have significant steroidogenic potential in vitro, but they do metabolize cholesterol into hydroxysterols. We conclude that the predominant stromal cells of the postmenopausal ovary are not a significant site of androgen biosynthesis.

Risk of ovarian cancer in relation to prediagnostic levels of C-peptide, insulin-like growth factor binding proteins-1 and -2 (USA, Sweden, Italy).

To investigate the association of prediagnostic circulating levels of C-peptide, as a marker of pancreatic insulin secretion, and IGF binding proteins -1 and -2, as indicators of the biologically active IGF-I concentration, with risk of developing ovarian cancer. The study was nested within three prospective cohorts in New York (USA), Umeå (Sweden) and Milan (Italy). Case subjects were 132 women with primary invasive epithelial ovarian cancer diagnosed at least one year after blood donation. For each case, two control subjects were selected, matching the case subject on cohort, menopausal status, age and date of recruitment (n = 263). Only women who did not use exogenous hormones at blood donation were included in the study. Odds ratios and their 95% confidence intervals for risk of developing ovarian cancer over quartiles of peptides concentrations after adjustment for BMI and fasting were: 1.00, 0.66 (0.35-1.23), 0.96 (0.51-1.82) and 0.89 (0.44-1.81) for C-peptide; 1.00, 1.10 (0.58-2.09), 1.07 (0.55-2.04) and 0.79 (0.38-1.62) for IGFBP-1; and 1.00, 1.01 (0.54-1.89), 0.98 (0.51-1.88) and 0.87 (0.45-1.68) for IGFBP-2. In women who had ovarian cancer diagnosis before age 55 the ORs for the top tertiles of IGFBP-1 and IGFBP-2 were 0.51 (0.18-1.49) and 0.53 (0.18-1.54), respectively. This study does not support an independent direct etiological role of C-peptide in ovarian cancer pathogenesis, but suggests a possible protective effect of circulating IGFBP-1 and -2 in women who develop ovarian cancer before age 55.

Insulin-like growth factor binding protein 2 (IGFBP-2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF-I excess on adrenocortical cells in vivo.

GH and IGF-I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF-I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK-bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP-2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV-IGFBP-2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5-wk- and 4-month-old G mice vs. C and B mice. IGFBP-2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5-wk- and 4-month-old animals, respectively. GH-induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP-2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH-induced plasma corticosterone levels of 4-month-old G mice, but not of GB mice, were two- to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP-2 potently separates hypertrophic and hyperplastic effects of GH/IGF-I excess on adrenocortical cells.