Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.     

Fertilization and early embryology: Comparison of mouse embryo development in open and microdrop co-culture systems

Co-culture with numerous cell lines has been shown to improvein-vitro embryo development. It is usually performed in openculture without an oil overlay, or in relatively large volumesof medium (e.g. 0.5 ml) under oil. We compared the efficacyof open and microdrop co-culture systems using human endometrialand tuba] cell lines and mouse zygotes; Although the mean pHvalues of the media from the tubal cell cultures (both openand oil-covered) decreased significantly over 5 days of culture,this did not appear to impair embryo development Both co-cultureand microdrop culture significantly improved blas-tocyst andhatching blastocyst formation rates. The combination of thetwo techniques (microdrop and co-culture) demonstrated the highestblastocyst formation and hatching blastocyst formation rates,as well as the highest mean cell numbers in hatching blastocysts.Co-culture in a microdrop is a superior system for mouse embryoculture.     

Insulin-like growth factor (IGF)-1 and IGF binding protein-1 and -3 in the follicular fluid of infertile patients with endometriosis

BACKGROUND: Endometriosis is associated with pituitary-ovarian axis dysfunction. The study of the follicular fluid in patients with endometriosis is important to elucidate the pathophysiological mechanism of this disease. The objective of this present paper was to analyse the dosages of insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 and 3 (IGFBP-1 and IGFBP-3) in the follicular fluid environment of infertile patients with endometriosis. METHODS: A total of 41 infertile patients undergoing IVF between January 1999 and January 2000 participated in the cross-sectional prospective study. Patients were divided into three groups: group I, minimal/mild endometriosis (n = 12); group II, moderate/severe endometriosis (n = 10); and group III, tubal obstruction (n = 19). The ultra-short protocol was used in association with recombinant FSH for ovulation induction. Follicular fluid analysis was performed using radioimmunoassay with specific kits. RESULTS: Follicular fluid IGF-1 and IGFBP-3 levels were not significantly different among the groups; however, follicular fluid IGFBP-1 levels were lower in those patients with moderate/severe endometriosis (P < 0.05). Comparison of ovulation induction time, number of recombinant FSH units, number of follicles, oocytes and embryos, and fertilization and gestation/cycle rates showed non-significant differences. CONCLUSION: Infertile patients with moderate/severe endometriosis, which is associated with ovulatory dysfunction, presented lower levels of IGFBP-1 in the follicular fluid when undergoing IVF.     

Low levels of low molecular weight insulin-like growth factor-binding protein in patients with polycystic ovarian disease

Insulin-like growth factors IGF-I and IGF-II are bound to specificbinding proteins in serum. The lower mol. wt binding protein(IGF-BP) has been detected in various tissues, including secretoryendometrium and preovulatory follicles of the ovary. This groupstudied the circulating levels of IGF-BP in the serum of 23patients with polycystic ovarian disease (PCOD) and found thatone-third of them have a subnormal level. In comparison withPCOD patients with a normal level, those with a subnormal levelhad a higher degree of obesity and a tendency to be more hirsute.They also had a higher serum insulin concentration and testosterone/sexhormone-binding globulin (SHBG) ratio, but lower serum SHBGconcentration than those with a normal IGF-BP level. PCOD isthe second abnormal clinical condition, after insulinoma, inwhich subnormal serum IGF-BP concentrations have been reported.The significance of low serum IGF-BP levels to pathophysiologyof PCOD remains to be elucidated by studies on local interactionbetween IGF-BP and insulin in the polycystic ovary     

Insulin-like growth factors and their binding proteins in human follicular fluid.

Increasing evidence suggests that insulin-like growth factors I and II (IGF-I, IGF-II) have a regulatory role in animal granulosa cells. This study was undertaken to investigate the presence of IGF-I and IGF-II, as well as that of their binding proteins (BP), IGFBP-1 and IGFBP-3 in human serum and follicular fluid (FF). Preovulatory FF was obtained from 51 patients undergoing in-vitro fertilization. The IGFBP-1 level was found to be significantly higher (P less than 0.01) in FF than in serum, whereas IGF-I and IGFBP-3 values remained markedly lower (P less than 0.01) in FF. Serum IGF-II levels were slightly but not significantly elevated compared to values obtained in the FF of patients. A positive correlation (P less than 0.001) between individual serum and FF levels was observed only for IGF-I. When a group of poor responders was compared to patients with normal stimulation characteristics, no significant difference was found in either IGF or IGFBP levels in the FF. It is concluded that IGFBP-1 is produced locally, whereas the serum may possibly be the major source of IGF-I. No clear conclusions can be drawn regarding the source of FF IGF-II and IGFBP-3. Neither the absolute level nor the relationship of IGFs to their transport proteins could explain the poor response to ovarian stimulation.     

The effect of Danazol on the circulating levels of 34K insulin-like growth factor-binding protein (PP12) and endometrial secretory protein PP14

Twenty-four women, 11 with endometriosis and 13 with fibrocysticmastopathy, were treated with a medium dose (300–400 mgdaily) of Danazol for 6 months. The circulating level of progesterone,the 34K insulin-like growth factor-binding protein (IGF-bp)and endometrial protein PP14 (placental protein 14) were measuredby specific radioimmunoassays before and during treatment. Theserum progesterone concentration decreased significantly duringDanazol treatment, as did the serum levels of 34K IGF-bp andPP14. By the third month of therapy amenorrhoea was observedin 22 out of 24 women and this was accompanied by a furtherdecline in the IGF-bp and PP14 levels. In light of the previousobservations on the IGF-bp and PP14 synthesis by secretory endometriumand the fact that Danazol causes endometrial atrophy, theseresults suggest that Danazol treatment has an effect on endometrialprotein secretion.     

Uterine endocrinology and paracrinology: insulin-like growth factor binding protein-1 and placental protein 14 revisited

A large number of proteins and peptides have been identifiedin the endometrium where they are likely to exert local biologicaleffects. These substances may be enzymes, their inhibitors,proteinases, proteinase inhibitors, hormones, or bioactive peptideswith diverse functions. Endometrial function and embryo—endometrialinteractions require synchronized actions between endocrineand local factors. As examples of local factors recent studieson the insulin-like growth factor (IGF) system and placentalprotein 14 (PP14) are reviewed. IGF binding protein-1 (IGFBP-1)and PP14 are products of the secretory phase endometrium: IGFBP-1is produced by decidualized stromal cells and PP14 by glandularepithelial cells. IGFBP-1 may inhibit the action of IGFs atthe endometrial-trophoblastic interphase, and it may also havea role in stromal—epithelial interaction. PP14 has immunosuppressiveproperties, and recent findings indicate that it may play apart in the fertilization process by inhibiting binding of spermatozoato the zona.     

Serum levels of insulin-like growth factor-1, IGF binding protein-1 and insulin and the response to human menopausal gonadotrophins in women with polycystic ovary syndrome

In order to determine which factors influence the large variationsin sensitivity to gonadotrophins witnessed in women with polycysticovary syndrome (PCOS), a prospective study was conducted ofthe correlation between basal clinical and endocrinologicalfeatures and gonadotrophin requirements of 20 women with clomiphene-resistantPCOS undergoing ovulation induction. Baseline evaluation ofserum concentrations of luteinizing hormone (LH), follicle stimulatinghormone (FSH), testosterone, fasting insulin, insulin-like growthfactor-1 (IGF-1), IGF binding protein-1 (IGFBP-1) and sex hormone-bindingglobulin (SHBG) were performed before administering gonadotrophin-releasinghormone agonist (GnRHa). Two weeks later, human menopausal gonadotrophin(HMG) was given in a standard individualized protocol accordingto ovarian response, until human chorionic gonadotrophin (HCG)was given. Serum concentrations of insulin, IGF-1, and IGFBP-1were unaffected by GnRHa. The BMI correlated positively withinsulin and inversely with IGFBP-1 serum concentrations andinsulin and IGFBP-1 were inversely correlated. The amount ofHMG required correlated positively with BMI and insulin concentrationsand inversely with IGFBP-1 in the whole group and these correlationswere maintained in the sub-group of lean women. No correlationwas observed between HMG requirements and IGF-1 or other hormones.Womenwith hyperinsulinaemia and low IGFBP-1 concentrations requiredsignificantly more HMG. Multiple regression analysis revealedthat insulin concentration is the most significant determinantof HMG requirement even when dissociated from BMI. We concludedthat requirement of HMG in PCOS is not merely determined byobesity but by a cardinal role of insulin concentrations which,when high, induce, hypothetically, a hyperandrogenic intrafollicularmilieu.     

The role of insulin-like growth factor binding protein-1 phosphoisoforms in pregnancies with impaired placental function identified by doppler ultrasound.

This study was performed to investigate the hypothesis that insulin-like growth factor binding protein-1 (IGFBP-1) is involved in the pathogenesis of trophoblast invasion and impaired placentation in human pregnancy. The role of total and non-phosphorylated IGFBP-1 in women with fetal growth restriction and in high risk pregnancies identified by uterine artery Doppler ultrasound screening was examined. This was a prospective study of women booked for antenatal care having second trimester anomaly scans and Doppler screening between 22-26 weeks gestation. Women were divided into three groups and compared: normal uterine artery Doppler and normal fetal growth (control group, n = 10); abnormal Doppler and normal fetal growth [bilateral uterine artery notches (BN; n = 16); abnormal Doppler and intrauterine growth restriction (IUGR; n = 8)]. Maternal serum was collected, stored and assayed simultaneously for total and non-phosphorylated IGFBP-1. There was elevated total and non-phosphorylated IGFBP-1 (mean 44.99 +/- 12.19 and 29.61 +/- 10.38 microg/l respectively) in the IUGR group compared with controls (mean 17.96 +/- 3.24 and 12.18 +/- 1.55 microg/l, P < 0.05). This finding suggests that the various IGFBP-1 isoforms, the degree of phosphorylation and the ratios of these different forms locally may be important during trophoblast invasion and may be implicated in clinical manifestations of impaired placentation later in the second trimester.     

Involvement of insulin-like growth factor-binding protein-related protein 1 in decidualization of human endometrial stromal cells

Uterine decidualization is crucial for successful implantation and the establishment of pregnancy. In the present study, the expression of insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1) in the human uterus and endometrial stromal cells (ESCs) and its physiological significance in decidualization were examined. IGFBP-rP1 protein was localized in the glandular epithelium and stromal cells, and blood vessels in the endometrium. Cultured stromal cells expressed IGFBP-rP1 and secreted it into the medium. IGFBP-rP1 was localized mostly in the cytoplasm near the nucleus. Knocking down the endogenous IGFBP-rP1 expression in stromal cells, by a small interfering (si)RNA, diminished the expression of prolactin and IGFBP-1 which serve as decidual markers. These results suggest that IGFBP-rP1 may play a role in decidualization of ESCs.     

Endocrinology: Insulin-like growth factor binding proteins in a natural pre-ovulatory follicle from a woman with polycystic ovary syndrome

Insulin-like growth factor binding protein (IGFBP) concentrationsin the follicular fluid of ovarian follicles have been shownto correlate with dominance and atresia. IGFBP-2 and IGFBP-4are increased in atresia, and IGFBP-3 is decreased in dominantfollicles. The purpose of this study was to compare the IGFBPconcentration in follicular fluid from a natural pre-ovulatoryfollicle of a woman with polycystic ovarian syndrome (PCOS)with other PCOS follicles and dominant follicles from normallycycling women. Follicular fluid was collected from 5–7mm diameter follicles and a natural pre-ovulatory follicle fromwomen with PCOS, and healthy and atretic follicles from normalwomen. The IGFBP profiles were analysed using Western ligandblotting. The IGFBP concentrations in the 5–7 mm diameterfollicles from the polycystic ovaries containing a pre-ovulatoryfollicle were similar to those in follicles from other womenwith PCOS, and comparable with androgenic cohort follicles fromnormal women. In particular, the IGFBP-2 and IGFBP-4 concentrationswere elevated significantly compared with the oestrogenic cohortfollicles. The concentrations of all IGFBP detected in the follicularfluid from the PCOS pre-ovulatory follicle were significantlyless than those of the 5–7 mm diameter follicles fromthe same subject. The IGFBP concentrations were within the rangeof normal dominant follicles, and IGFBP-2 and IGFBP-3 were atthe lower end of the normal range. The results indicate thatthe PCOS pre-ovulatory follicle contained a normal pattern ofIGFBP expression even though the other follicles exhibited apattern typical of PCOS. These data support the hypothesis thatdecreased concentrations of IGFBP, in particular IGFBP-3, maybe involved in selection of the dominant follicle, and thatwhen a spontaneous pre-ovulatory follicle develops in PCOS,the underlying cause of the polycystic ovaries is not resolvedbut the rest of the ovary remains polycystic.     

Insulin-like growth factor binding protein-6 delays replicative senescence of human fibroblasts

Cellular senescence can be induced by a variety of mechanisms, and recent data suggest a key role for cytokine networks to maintain the senescent state. Here, we have used a proteomic LC-MS/MS approach to identify new extracellular regulators of senescence in human fibroblasts. We identified 26 extracellular proteins with significantly different abundance in conditioned media from young and senescent fibroblasts. Among these was insulin-like growth factor binding protein-6 (IGFBP-6), which was chosen for further analysis. When IGFBP-6 gene expression was downregulated, cell proliferation was inhibited and apoptotic cell death was increased. Furthermore, downregulation of IGFBP-6 led to premature entry into cellular senescence. Since IGFBP-6 overexpression increased cellular lifespan, the data suggest that IGFBP-6, in contrast to other IGF binding proteins, is a negative regulator of cellular senescence in human fibroblasts.     

Localization of insulin-like growth factor-binding protein and endometrial beta-lactoglobulin in cultured decidual and chorionic villus cells

Immunofluorescence staining was used to localize two 'endometrial' proteins, insulin-like growth factor-binding protein and human beta-lactoglobulin homologue, in cultured cells of chorionic villi and decidua. Both cultured cell types were positive for each protein. These results indicate that immunohistochemical detection of these two proteins cannot be used to distinguish between cultured chorionic villus and decidual cells.     

Effect of anticardiolipin antibodies on prolactin and insulin-like growth factor binding protein-1 production by human decidual cells.

PROBLEM: The effect of anticardiolipin antibodies (ACAs) on basal- and growth factor-stimulated prolactin and insulin-like growth factor (IGF) binding protein (BP)-1 production by cultured human decidual cells was investigated. METHOD OF THE STUDY: Decidual cells were cultured for 24, 48, or 96 hr in medium supplemented with 5% ACA-containing or 5% control serum and increasing concentrations of insulin (1-10 micrograms/mL) or IGF-1 (10-100 ng/mL). RESULTS: No significant increase in prolactin production was observed after addition of increasing doses of insulin and IGF-I in the presence of ACA-containing serum, while a dose-dependent stimulation was seen with control serum. Time-dependent prolactin accumulation was also reduced when cells were cultured in the former conditions. IGF BP-1 release was not affected by insulin and IGF-I in the presence of both sera. However, lower IGF BP-1 levels and a less pronounced time-dependent accumulation were observed in the presence of ACA-positive serum. CONCLUSIONS: Our data suggest that ACAs affect cellular transduction mechanisms regulating critical events, such as decidual cell differentiation. These cellular dysfunctions might be relevant in the induction of some obstetric disorders typical of this syndrome.     

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of differentculture systems on the development of early human embryos invitro. A total of 460 fertilized oocytes from 82 cycles of patientswere transferred into one of four systems: (1) into dropletsof Ham's F10 medium+12% normal human serum (NHS); (2) co-culturedon a human granulosa monolayer; (3) co-cultured with bovineoviductal epithelial cells (BOEC); or (4) co-cultured with bovineuterine epithelial cells (BUEC). The percentage of deavage andthe morphological appearance of embryos were recorded dailyfor 72 h in each system using an inverted phase-contrast microscope.The results showed that the proportions of the fertilized oocyteswhich developed to the four-cell stage 48 h after retrievalwere, by culture system: (1) 70% (84/120); (2) 74% (85/115);(3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval,the proportions of the eight-cell stage were, by culture system:(1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70%(57/82). We conduded that a higher proportion of fertilizedoocytes developed to embryos at the eight-cell stage in systems2, 3 and 4 than in system 1. This indicates the beneficial effectof co-culture of human embryos with granulosa cell, BOEC andBUEC monolayers, which may be due to various factors.     

Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I).

There is evidence that insulin-like growth factor I (IGF-I) is a potent regulator of oestradiol synthesis by human granulosa and luteal cells; however, the question of whether IGF-I regulates progesterone synthesis by these cell types has yet to be answered. As a first step towards this goal, we have compared the effects of IGF-I, follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) on progesterone production by human granulosa cells obtained from individual dominant and cohort follicles, and granulosa luteal cells from preovulatory follicles of patients undergoing in-vitro fertilization (IVF). Granulosa cells from normal, unstimulated follicles cultured in serum-free medium as controls (no additions) produced some progesterone spontaneously. In all cases, FSH stimulated basal progesterone levels (10-fold average increase) and the effect was dose-dependent (ED50 of FSH = 9.1 +/- 3.9 ng/ml). Similar effects were observed when granulosa cells from large follicles were incubated with HCG (ED50 of HCG = 6.9 +/- 2.8 ng/ml). By comparison, the effects of IGF-I on progesterone production were not marked, being absent in 80% of the follicles tested. However, granulosa cells from healthy follicles co-incubated with IGF-I and FSH or HCG produced more progesterone compared with cells treated with the gonadotrophins alone; this effect of IGF-I was dose dependent (ED50 of IGF-I = 10 ng/ml). When the effect of each agonist was tested on IVF granulosa luteal cells, HCG but not FSH or IGF-I stimulated basal progesterone levels but the HCG effect required a two-day lag phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A longitudinal study of maternal plasma insulin-like growth factor binding protein-1 concentrations during normal pregnancy and pregnancies complicated by pre-eclampsia

Insulin-like growth factor binding protein-1 (IGFBP-1) is synthesized by the decidual stroma, and is thought to act locally to inhibit IGF activity and so limit trophoblast invasion. Cross-sectional studies have reported conflicting data on maternal circulating concentrations of IGFBP-1 in early pregnancy before the development of pre-eclampsia. A longitudinal study was performed in 10 women who went on to develop pre-eclampsia and a group of 12 normal pregnant controls, chosen to be similar for maternal age, booking body mass index (BMI) and gestational age. Maternal IGFBP-1 concentrations were measured in plasma obtained at 16, 20, 24, 28, 32 and 36 weeks. Plasma IGFBP-1 concentrations were unchanged over this period in normal pregnancy. In contrast, the concentrations in women who developed pre-eclampsia increased progressively. At 16, 20, and 24 weeks the concentrations were significantly lower compared to normal pregnancy, at 28 and 32 weeks, similar, but by 36 weeks the concentrations were significantly greater than the normal controls. The data show that circulating IGFBP-1 concentrations are lower in early pregnancy before the development of pre-eclampsia. Thus, it is suggested that IGFBP-1-induced inhibition of IGF activity is unlikely to be responsible for the impaired trophoblast invasion observed in pre-eclampsia.     

Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells

In a prospective randomized study, excess embryos from 100 womenundergoing in-vitro fertilization were cultured from the 2-cellto the hatched-blastocyst stage in the presence or absence ofa confluent monolayer of Vero cells. The frequencies of fragmentation,developmental arrest, multi-nucleation and blastocyst formationwere observed for 254 embryos over 7 days in culture. The numberof nucleated cells, and fine structure of trophectoderm andinner cell mass were analysed at the expanded blastocyst stageon day 5.5 post-insemination. The frequency of hatching fromthe zona pellucida was determined between days 6 and 7 post-insemination.With respect to these developmental parameters, the findingsindicate that no overt or statistically significant improvementin early human embryogenesis occurs in the co-culture system.     

Insulin-like growth factor family in malignant haemangiopericytomas: the expression and role of insulin-like growth factor I receptor

Haemangiopericytoma is a rare soft tissue tumour originating from the contractile pericapillary cells. Relatively little is known about its molecular pathogenesis. To address this issue, the insulin-like growth factor family (IGFs) was analysed in 19 tumours collected from a human tumour bank network. Seven of the tumours were associated with severe hypoglycaemia. Of these, six were retroperitoneal and one was located in the leg. 3 out of the 19 tumours (15·8 per cent) were positive for insulin-like growth factor I (IGF I) mRNA and 11 were positive for IGF II mRNA (57·9 per cent). Almost 90 per cent of haemangiopericytomas expressed IGF I receptor (IGF IR) mRNA (17 out of 19), five (26·3 per cent) expressed IGF binding protein 1 (IGF BP1), three (15·8 per cent) expressed IGF BP2, and four (21 per cent) exhibited IGF BP3 mRNA. All of the 14 haemangiopericytomas examined with regard to specific receptor binding were IGF IR positive, ranging from 1·2 to 16·2 per cent. Binding was much higher in IGF I/IGF IR positive tumours (15·3±0·7) than in IGF I negative/IGF IR positive tumours (5·1±3·3). The potential role of IGF IR as a growth promoting factor in malignant haemangiopericytoma was studied using antisense oligonucleotides and monoclonal antibody αIR3 that specifically inhibit IGF IR synthesis or activity. 10 µM IGF IR antisense oligonucleotides significantly inhibited the growth of haemangiopericytoma cells in culture, by around 50 per cent; monoclonal antibody against IGF IR (αIR3) also significantly inhibited proliferation. The data suggest that IGF IR may play an important role in the genesis and progression of malignant haemangiopericytomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Role of insulin-like growth factor binding proteins (IGFBPs) in breast cancer proliferation and metastasis

Cancers of the breast, prostate, and lung commonly metastasize to the bone resulting in osteolysis, pathologic fracture, pain and significant clinical morbidity. To date, the reason for such selectivity in the site of metastasis remains largely unknown. The bone is a rich source of many chemokines and growth factors, including: insulin-like growth factor (IGF) I and II, transforming growth factor-β (TGF-β), interleukins, and tumour necrosis factor-α (TNF-α) [1]. We propose that exposure of breast cancer cells to the bone microenvironment results in alterations in gene expression that favour the growth and proliferation of tumour cells in the bone. To investigate this hypothesis, MDA-MB-231 breast carcinoma cells were exposed to bone-derived conditioned media (BDCM) generated by culturing fetal rat calvaria for 24 h under serum free conditions. Using cDNA microarray technology, we have identified the insulin-like growth factor family of binding proteins (IGFBPs) as genes whose expression profiles are consistently and significantly altered with exposure to this simulated bone environment in vitro, when compared to untreated controls. Our data suggests that the upregulation of IGFBP-3 seen with exposure to the bone microenvironment is directly linked to an increase in TGF-β mediated cell proliferation. Furthermore, this process appears to be functioning through an IGF-independent mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.