Follistatin-like 1 Research Articles

Follistatin and Follistatin Like‐3 Differentially Regulate Adiposity and Glucose Homeostasis

Transforming growth factor-β superfamily ligands, including activin and myostatin, modulate body composition, islet function, and glucose homeostasis. Their bioactivity is controlled by the antagonists follistatin (FST) and FST like-3 (FSTL3). The hypothesis tested was that FST and FSTL3 have distinct roles in regulating body composition, glucose homeostasis, and islet function through regulation of activin and myostatin bioactivity. Three genetic mutant mouse lines were created. FSTL3 knockout (FSTL3 KO), a mouse line producing only the FST288 isoform (FST288-only) and a double mutant (2xM) in which the lines were crossed. FST288-only males were lighter that wild-type (WT) littermates while FSTL3 KO and 2xM males had reduced perigonadal fat pad weights. However, only 2xM mice had increased whole body fat mass and decreased lean mass by quantitative nuclear magnetic resonance (qNMR). Fasting glucose levels in FSTL3 WT and KO mice were lower than FST mice in younger animals but were higher in older mice. Serum insulin and pancreatic insulin content in 2xM mice was significantly elevated over other genotypes. Nevertheless, 2xM mice were relatively insulin resistant and glucose intolerant compared to FST288-only and WT mice. Fractional islet area and proportion of β-cells/islet were increased in FSTL3 KO and 2xM, but not FST288-only mice. Despite their larger size, islets from FSTL3 KO and 2xM mice were not functionally enhanced compared to WT mice. These results demonstrate that body composition and glucose homeostasis are differentially regulated by FST and FSTL3 and that their combined loss is associated with increased fat mass and insulin resistance despite elevated insulin production.

Follistatin-Like 1 in Chronic Systolic Heart Failure

Background— Follistatin-like 1 (FSTL1) is an extracellular glycoprotein found in human serum. Recent work suggests that FSTL1 is secreted in response to ischemic injuries and that its overexpression is protective in the heart and vasculature. Methods and Results— We examined serum FSTL1 levels in patients with chronic heart failure with left ventricular (LV) ejection fraction <40% (n=86). The sample was separated into three tertiles of patients with low, medium, and high FSTL1 levels. Serum FSTL1 was increased 56% above age- and sex-matched healthy controls. Diabetes mellitus, brain natriuretic peptide level, left atrial size, LV posterior wall thickness, LV end-diastolic diameter, and LV mass were significant determinants of FSTL1 serum levels by bivariate analysis. After controlling for significant covariates, FSTL1 levels predicted LV hypertrophy (as measured by LV mass index) by multivariate linear regression analysis ( P <0.001). Unadjusted survival analysis demonstrated increased mortality in patients with increasing FSTL1 levels ( P =0.09). After adjusting for significant parameters, patients with increased FSTL1 remained at the highest risk of death (hazard ratio, 1.028; 95% CI, 0.98 to 1.78; P =0.26). To determine whether elevated FSTL1 levels may be derived from the myocardium, FSTL1 protein expression was measured in explanted failing (n=18) and nonfailing (n=7) human hearts. LV failing hearts showed 2.5-fold higher FSTL1 protein levels over nonfailing control hearts ( P <0.05). Conclusions— Elevated serum FSTL1 in patients with heart failure was associated with LV hypertrophy. Further studies on the role of FSTL1 as a biomarker in chronic systolic heart failure are warranted.

Growth Differentiation Factor 9 (GDF9) Suppresses Follistatin and Follistatin-Like 3 Production in Human Granulosa-Lutein Cells

BackgroundWe have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.MethodologyhGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.Principal FindingsGDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β B-subunit mRNA and inhibin B production were attenuated by adding FST.ConclusionGDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β B-subunit mRNA expression and inhibin B production in hGL cells.

Acute Loading and Aging Effects on Myostatin Pathway Biomarkers in Human Skeletal Muscle After Three Sequential Bouts of Resistance Exercise

To determine the influence of age and resistance exercise on myostatin pathway-related genes, younger (n = 10; 28 ± 5 years) and older (n = 10; 68 ± 6 years) men underwent four testing conditions (T1-T4). A baseline (T1) muscle sample was obtained, whereas the second and third biopsies were obtained 48 hours following the first and second training sessions (T2, T3), and a final biopsy was taken 24 hours following T3. The training sessions consisted of 3 sets of 10 repetitions (80% of one repetition maximum) on leg press, hack squat, and leg extension exercises. Follistatin (FST) messenger RNA was greater in older compared with younger men at T1 and T2 (p < .05). Follistatin-like 3 (FSTL3) messenger RNA was greater in older compared with younger men at T1 and T4 (p < .05). In older men, there was a significant decrease in myostatin (MSTN) messenger RNA at T4 (p < .05). Older men contained less active (Ser-425 phosphorylated) SMAD3 (p-SMAD3) protein than younger men at T3 and T4 (p < .05).Although it is well known that younger individuals possess a greater hypertrophic potential to resistance exercise, it appears that older individuals may paradoxically possess a more favorable resistance exercise response regarding myostatin pathway-related genes and a protein marker of pathway activity. Future research is warranted to examine the physiological significance of this age-dependent mechanism.

Boom time for neuroscience in China

Summary A decade after the foundation of the Institute of Neuroscience in Shanghai, the field enjoys rapid growth and can attract young investigators back from abroad. Michael Gross reports.

Follistatin-like 1 Suppresses Sensory Afferent Transmission by Activating Na+,K+-ATPase

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Prediction of gestational diabetes mellitus by maternal factors and biomarkers at 11 to 13 weeks

To develop a model for the prediction of gestational diabetes mellitus (GDM) from maternal characteristics and biochemical markers at 11 to 13 weeks' gestation. A prospective screening study on early prediction of pregnancy complications (n = 11, 464), including 297 (2.6%) cases of GDM was used to create the predictive model of GDM based on maternal characteristics. Maternal serum concentrations of adiponectin, follistatin-like-3 (FSTL3) and sex hormone-binding globulin (SHBG) were measured in a case-control study of 80 women who developed GDM and 300 controls. In the screening study, maternal age, body mass index, racial origin, previous history of GDM and macrosomic neonate were significant independent predictors of future GDM. In the GDM group, compared to controls, the median multiple of the normal median adiponectin (0.66; IQR: 0.5-0.9 vs 1.02; IQR: 0.7-1.29) and SHBG (0.81; IQR: 0.6-1.04 vs 1.02; IQR: 0.8-1.2) was lower (p < 0.05), but FSTL3 was not significantly different. In screening for GDM by maternal characteristics, the detection rate was 61.6% at a false-positive rate of 20% and the detection increased to 74.1% by the addition of adiponectin and SHBG. First-trimester screening for GDM can be provided by a combination of maternal characteristics and biomarkers.

Follistatin-like 1 regulates renal IL-1β expression in cisplatin nephrotoxicity

Follistatin-like 1 (FSTL1) is a secreted protein with homology to both Follistatin and the SPARC/BM40 family of matricellular proteins. In this study, we sought to determine the expression patterns of Fstl1 and its cognate receptor Dip2a in the adult, and to assess the consequences of Fstl1 inactivation on development and homeostasis of the kidney. We find that FSTL1 circulates at high levels in both the human and the mouse and that it is also locally expressed in the loop of Henle in the kidney. To begin to understand the in vivo functions of Fstl1, we generated a mouse mutant using a genetrap approach. The hypomorphic Fstl1 genetrap strain displays a strong reduction in FSTL1 expression at the protein level, but it does not show overt developmental defects. FSTL1 has previously been implicated in diverse disease processes as a regulator of inflammatory cytokine expression, and we therefore evaluated the response of the genetrap strain to cisplatin-mediated acute kidney injury, a disease model with highly cytokine-dependent pathology. We find that although TNF-α and Il6 levels are unchanged relative to wild-type, renal Il-1β expression is increased in genetrap mice following cisplatin treatment. Furthermore, histopatological analysis, expression of the tissue injury marker Havcr1, and measurement of serum creatinine demonstrate that reduction of Fstl1 expression sensitizes the kidney to acute cisplatin nephrotoxicity, suggesting a role for FSTL1-mediated Il-1β suppression in protection of the kidney from acute nephrotoxic injury.

DIP2 disco‐interacting protein 2 homolog A (Drosophila) is a candidate receptor for follistatin‐related protein/follistatin‐like 1 – analysis of their binding with TGF‐β superfamily proteins

Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) is a member of the follistatin protein family, all of which share a characteristic structure unit found in follistatin, called the FS domain. Developmental studies have suggested that FRP regulates organ tissue formation in embryos. Immunological studies showed that FRP modifies joint inflammation in arthritic disease, and modulates allograft tolerance. However, the principle physiological function of FRP is currently unknown. To address this issue, we cloned four FRP-associated proteins using a two-hybrid cloning method: disco-interacting protein 2 homolog A from Drosophila (DIP2A), CD14, glypican 1 and titin. Only DIP2A was expected to be a membrane receptor protein with intracellular regions. Over-expression of FLAG epitope-tagged DIP2A augmented the suppressive effect of FRP on FBJ murine osteosarcoma viral oncogene homolog (FOS) expression, and the Fab fragment of IgG to FLAG blocked this effect. Knockdown of Dip2a leaded to Fos gene up-regulation, and this was not affected by exogenous FRP. These in vitro experiments confirmed that DIP2A could be a cell-surface receptor protein and mediate a FOS down-regulation signal of FRP. Moreover, molecular interaction analyses using Biacore demonstrated that FRP bound to DIP2A and CD14, and also with proteins of the TGF-β superfamily, i.e. activin, TGF-β, bone morphogenetic protein 2/4 (BMP-2/4), their receptors and follistatin. FRP binding to DIP2A was blocked by CD14, follistatin, activin and BMP-2. FRP blocked the ligand-receptor binding of activin and BMP-2, but integrated itself with that of BMP-4. This multi-specific binding may reflect the broad physiological activity of FRP.

Brief Communication: Sexual dimorphic expression of myostatin and follistatin like-3 in a rat trans-generational under-nutrition model

The detrimental effects of maternal under-nutrition during gestation on fetal development are well known with an increased propensity of metabolic disorders identified in the adult offspring. Understanding exactly how and by which molecular pathways inadequate nutrition can impact upon offspring phenotype is critical and necessary for the development of treatment methods and ultimately prevention of any negative health effects. Myostatin, a negative regulator of muscle development, has recently been shown to effect glucose homeostasis and fat deposition. The involvement of myostatin in glucose metabolism and adipogenesis thus supports its ability to act in the continued alterations to the postnatal phenotype of the offspring. This hypothesis was examined in the current study using a trans-generational gestationally under-nourished rat model exposed to a high-fat (HF) diet post-weaning. The body weight, body fat, plasma glucose and insulin concentrations of the offspring, both male and female, were investigated in relation to the protein expression of myostatin and its main inhibitor; follistatin like-3 (FSTL-3), in skeletal muscle of mature offspring. Sexual dimorphism was clearly evident in the majority of these measures, including myostatin and FSTL-3 expression. Generally males displayed higher (P < 0.05) myostatin precursor and dimer expression than females, which was especially apparent (P < 0.01) in both chow and HF trans-generationally undernourished (UNAD) groups. In females only, myostatin precursor and dimer expression was altered by both trans-generational under-nutrition and postnatal diet. Overall FSTL-3 expression did not differ between sexes, although difference between sexes within certain treatments and diets were evident. Most notably, HF fed UNAD females had higher (P < 0.05) FSTL-3 expression than HF fed UNAD males. The former group also displayed higher (P < 0.01) FSTL-3 expression compared to all other female groups. In summary, myostatin may prove to be a key mediator of the effects of inadequate prenatal nutrition, independently or in combination with a high-fat postnatal diet on offspring phenotype. Consequently, further study of myostatin may provide a novel therapeutic pathway for the treatment of metabolic disorders; however, it is vital that the influence of nutrition and gender should be taken into consideration.

Abstract 2952: Gene expression profiling in gastric adenomas and carcinomas

Abstract A subset of gastric carcinogenesis is a multistep process, which progresses from chronic gastritis, atrophy, intestinal metaplasia, adenoma and finally leads to gastric carcinoma. To explore the transcriptomic change during the gastric adenoma-carcinoma sequence, a total 26 snap-frozen samples (8 paired normal mucosa, adenoma and carcinoma cases and additional 2 carcinomas) were analyzed by oligonucleotide microarrays. We identified 60 gradually up and down regulated genes according to normal, adenoma and carcinoma sequence. Twenty one genes were gradually decreased from normal mucosa to adenoma to carcinoma and 39 genes were gradually increased. We validated the expression of hydroxyprostaglandin dehydrogenase 15 (HPDG15), follistatin-like 1 (FSTL1), trefoil factor 1 (TFF1) and trefoil factor 2 (TFF2) by real time RT-PCR. The identified gene sets according to adenoma-carcinoma sequence might play critical roles in gastric carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2952.

Placental expression of myostatin and follistatin-like-3 protein in a model of developmental programming

Maternal undernutrition during gestation is known to be detrimental to fetal development, leading to a propensity for metabolic disorders later in the adult lives of the offspring. Identifying possible mediators and physiological processes involved in modulating nutrient transport within the placenta is essential to prevent and/or develop treatments for the effects of aberrant nutrition, nutrient transfer, and detrimental changes to fetal development. A potential role for myostatin as a mediator of nutrient uptake and transport from the mother to the fetus was shown through the recent finding that myostatin acts within the human placenta to modulate glucose uptake and therefore homeostasis. The mRNA and protein expression of myostatin and its inhibitor, follistatin-like-3 (FSTL3), was studied in the placenta and skeletal muscle of a transgenerational Wistar rat model of gestational maternal undernutrition in which the F2 offspring postweaning consumed a high-fat (HF) diet. Alterations in placental characteristics and offspring phenotype, specifically glucose homeostasis, were evident in the transgenerationally undernourished (UNAD) group. Myostatin and FSTL3 protein expression were also higher (P < 0.05) in the placentae of the UNAD compared with the control group. At maturity, UNAD HF-fed animals had higher (P < 0.05) skeletal muscle expression of FSTL3 than control animals. In summary, maternal undernutrition during gestation results in the aberrant regulation of myostatin and FSTL3 in the placenta and skeletal muscle of subsequent generations. Myostatin, through the disruption of maternal nutrient supply to the fetus, may thus be a potential mediator of offspring phenotype.

First-Trimester Follistatin-Like-3 Levels in Pregnancies Complicated by Subsequent Gestational Diabetes Mellitus

OBJECTIVETo determine whether maternal levels of follistatin-like-3 (FSTL3), an inhibitor of activin and myostatin involved in glucose homeostasis, are altered in the first trimester of pregnancies complicated by subsequent gestational diabetes mellitus (GDM).RESEARCH DESIGN AND METHODSThis was a nested case-control study of subjects enrolled in a prospective cohort of pregnant women with and without GDM (≥2 abnormal values on a 100-g glucose tolerance test at ∼28 weeks of gestation). We measured FSTL3 levels in serum collected during the first trimester of pregnancy. Logistic regression analyses were used to determine the risk of GDM.RESULTSWomen who developed GDM (n = 37) had lower first-trimester serum levels of FSTL3 compared with women who did not (n = 127) (median 10,789 [interquartile range 7,013–18,939] vs. 30,670 [18,370–55,484] pg/ml, P < 0.001). When subjects were divided into tertiles based on FSTL3 levels, women with the lowest levels demonstrated a marked increase in risk for developing GDM in univariate (odds ratio 11.2 [95% CI 3.6–35.3]) and multivariate (14.0 [4.1–47.9]) analyses. There was a significant negative correlation between first-trimester FSTL3 levels and ∼28-week nonfasting glucose levels (r = −0.30, P < 0.001).CONCLUSIONSFirst-trimester FSTL3 levels are associated with glucose intolerance and GDM later in pregnancy.

Essential Amino Acids Increase MicroRNA-499, −208b, and −23a and Downregulate Myostatin and Myocyte Enhancer Factor 2C mRNA Expression in Human Skeletal Muscle

Essential amino acids (EAA) stimulate muscle protein synthesis in humans. However, little is known about whether microRNAs (miRNA) and genes associated with muscle growth are expressed differently following EAA ingestion. Our purpose in this experiment was to determine whether miRNA and growth-related mRNA expressed in skeletal muscle are up- or downregulated in humans following the ingestion of EAA. We hypothesized that EAA would alter miRNA expression in skeletal muscle as well as select growth-related genes. Muscle biopsies were obtained from the vastus lateralis of 7 young adult participants (3 male, 4 female) before and 3 h after ingesting 10 g of EAA. Muscle samples were analyzed for muscle miRNA (miR-499, −208b, −23a, −1, −133a, and −206) and muscle-growth related genes [MyoD1, myogenin, myostatin, myocyte enhancer factor C (MEF2C), follistatin-like-1 (FSTL1), histone deacytylase 4, and serum response factor mRNA] before and after EAA ingestion using real-time PCR. Following EAA ingestion, miR-499, −208b, −23a, −1, and pri-miR-206 expression increased (P < 0.05). The muscle-growth genes MyoD1 and FSTL1 mRNA expression increased (P < 0.05), and myostatin and MEF2C mRNA were downregulated following EAA ingestion (P < 0.05). We conclude that miRNA and growth-related genes expressed in skeletal muscle are rapidly altered within hours following EAA ingestion. Further work is needed to determine whether these miRNA are post-transcriptional regulators of growth-related genes following an anabolic stimulus.

Circulating Concentrations of Follistatin-Like 1 in Healthy Individuals and Patients with Acute Coronary Syndrome as Assessed by an Immunoluminometric Sandwich Assay

Follistatin-like 1 (FSTL1) is a 308-amino acid secreted glycoprotein. Tissue levels of FSTL1 are induced in animal models and patients with chronic inflammatory and cardiovascular disease. We hypothesized that FSTL1 can be measured in the human circulation and used as a biomarker in acute coronary syndrome (ACS). We developed an immunoluminometric assay (ILMA), assessed the preanalytic characteristics of FSTL1, and determined circulating FSTL1 concentrations in 120 apparently healthy individuals and 216 patients with ACS. The assay had a limit of detection of 0.17 microg/L, limit of quantification of 1.02 microg/L, intraassay imprecision of < or =12.7%, and interassay imprecision of < or =15.4%. Selectivity was demonstrated with size-exclusion chromatography and lack of cross-reactivity with related proteins. The assay was not appreciably influenced by unrelated biological substances. FSTL1 in serum or whole blood was stable at room temperature for 48 h and was resistant to 4 freeze-thaw cycles. Measured FSTL1 concentrations in citrated plasma and heparin-treated plasma were 18% and 17% lower, respectively, than concentrations measured in serum. Apparently healthy individuals presented with a median FSTL1 serum concentration of 7.18 (range 1.06-18.49) microg/L. Serum FSTL1 concentrations were increased in ACS and related to the risk of all-cause mortality during follow-up. The ILMA permits detection of FSTL1 in human serum and plasma. We expect that the favorable preanalytic characteristics of FSTL1 and the reference limits defined here for apparently healthy individuals will facilitate future studies of FSTL1 as a biomarker in various disease settings, including ACS.

An Immunomodulatory Role for Follistatin‐Like 1 in Heart Allograft Transplantation

Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8(+) T cells and TGFbeta are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8(+) T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFNgamma. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

The Structure of FSTL3·Activin A Complex: DIFFERENTIAL BINDING OF N-TERMINAL DOMAINS INFLUENCES FOLLISTATIN-TYPE ANTAGONIST SPECIFICITY

Transforming growth factor beta family ligands are neutralized by a number of structurally divergent antagonists. Follistatin-type antagonists, which include splice variants of follistatin (FS288 and FS315) and follistatin-like 3 (FSTL3), have high affinity for activin A but differ in their affinity for other ligands, particularly bone morphogenetic proteins. To understand the structural basis for ligand specificity within FS-type antagonists, we determined the x-ray structure of activin A in complex with FSTL3 to a resolution of 2.5 A. Similar to the previously resolved FS.activin A structures, the ligand is encircled by two antagonist molecules blocking all ligand receptor-binding sites. Recently, the significance of the FS N-terminal domain interaction at the ligand type I receptor site has been questioned; however, our data show that for FSTL3, the N-terminal domain forms a more intimate contact with activin A, implying that this interaction is stronger than that for FS. Furthermore, binding studies revealed that replacing the FSTL3 N-terminal domain with the corresponding FS domain considerably lowers activin A affinity. Therefore, both structural and biochemical evidence support a significant interaction of the N-terminal domain of FSTL3 with activin A. In addition, structural comparisons with bone morphogenetic proteins suggest that the interface where the N-terminal domain binds may be the key site for determining FS-type antagonist specificity.

Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis—a differential expression and functional analysis

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G(1) cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1-induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1-transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis.

Activin βA subunit, follistatin and follistatin-like 3 are expressed in the endometrium of ovariectomized rats and regulated by estrogen replacement

Activin A is a growth factor expressed in the endometrium, where it modulates tissue remodeling and enhances decidualization. The effects of activin A are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (FSTL3). We have evaluated the effects of estrogen and progestin on the endometrial expression of activin betaA subunit, follistatin and FSTL3 in ovariectomized rats. Adult female Wistar rats (n = 21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n = 7) or associated with depot medroxyprogesterone acetate (3 mg/kg body weight, i.m. injection, n = 7), or oil vehicle (control group, n = 7). One week later, activin betaA subunit mRNA levels had increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, P < 0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, P < 0.05). This was accompanied by increase of betaA subunit immunostaining in estradiol and estroprogestin treated rats, which was noted only in the surface endometrial epithelium. Follistatin mRNA expression, conversely, showed a significant decrease in the groups treated with estrogen alone and estrogen plus progestin (P < 0.05), and follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprogestin-treated rats compared to controls. FSTL3 expression was similar in the 3 groups. In conclusion, the expression of activin betaA subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin.

Novel Glioblastoma Markers with Diagnostic and Prognostic Value Identified through Transcriptome Analysis

Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II--diffuse astrocytoma, grade III--anaplastic astrocytoma, and grade IV--glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible alpha (GADD45alpha) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45alpha and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45alpha conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. Our study reveals that GADD45alpha and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45alpha overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.