Novel Roles of Follistatin/Myostatin in Transforming Growth Factor-ß Signaling and Adipose Browning: Potential for Therapeutic Intervention in Obesity Related Metabolic Disorders.

Obesity is a global health problem and a major risk factor for several metabolic conditions including dyslipidemia, diabetes, insulin resistance and cardiovascular diseases. Obesity develops from chronic imbalance between energy intake and energy expenditure. Stimulation of cellular energy burning process has the potential to dissipate excess calories in the form of heat via the activation of uncoupling protein-1 (UCP1) in white and brown adipose tissues. Recent studies have shown that activation of transforming growth factor-ß (TGF-ß) signaling pathway significantly contributes to the development of obesity, and blockade or inhibition is reported to protect from obesity by promoting white adipose browning and increasing mitochondrial biogenesis. Identification of novel compounds that activate beige/brown adipose characteristics to burn surplus calories and reduce excess storage of fat are actively sought in the fight against obesity. In this review, we present recent developments in our understanding of key modulators of TGF-ß signaling pathways including follistatin (FST) and myostatin (MST) in regulating adipose browning and brown adipose mass and activity. While MST is a key ligand for TGF-ß family, FST can bind and regulate biological activity of several TGF-ß superfamily members including activins, bone morphogenic proteins (BMP) and inhibins. Here, we review the literature supporting the critical roles for FST, MST and other proteins in modulating TGF-ß signaling to influence beige and brown adipose characteristics. We further review the potential therapeutic utility of FST for the treatment of obesity and related metabolic disorders.

Circulating myokine levels in different stages of glucose intolerance.

Type 2 diabetes is the fastest growing metabolic disease in the world. Recently, muscle is considered an endocrine organ which secretes various peptides that play an important role in insulin resistance and metabolic syndrome. We assessed 4 different myokines, irisin, interleukin-13 (IL-13), follistatin-related protein-1 (FSTL-1), and fractalkine, in normal, prediabetes, and diabetes patients.A total of 126 participants who visited Gangnam Severance Hospital were enrolled and divided into normal, prediabetes, and diabetes groups based on oral glucose tolerance test and hemoglobin a1c. A cross-sectional study was conducted to measure and compare serum levels of irisin, IL-13, FSTL-1, and fractalkine among the groups.Irisin level showed a tendency to increase in prediabetes group compared to normal group (P < .1) but showed a significant decrease when comparing diabetes from prediabetes group (P < .001). IL-13 decreased in diabetes group compared to prediabetes and normal group (P < .001, P < .05, respectively). FSTL-1 of diabetes group was lower than that of prediabetes group (P < .05), and fractalkine was higher in diabetes group compared to that of prediabetes and normal group (P < .01, P < .01, respectively).Irisin, IL-13, and FSTL-1 levels were reduced in diabetes group compared to normal or prediabetes group while fractalkine showed a progressive increase from normal to diabetes group. Further studies are warranted to study the roles of various myokine in diabetes through a larger prospective study.

4-methylumbelliferone Prevents Liver Fibrosis by Affecting Hyaluronan Deposition, FSTL1 Expression and Cell Localization.

4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models; however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl4-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl4 stimulated expression of Col1a and αSMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL4 treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl4 treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl4-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.

The glycoprotein follistatin-like 1 promotes brown adipose thermogenesis.

OBJECTIVES: The thermogenic brown adipose tissue (BAT) has been proposed as a potential target to prevent or treat obesity and related metabolic diseases. BAT secretes adipokines to regulate the thermogenic program in an autocrine or paracrine manner. Follistatin-like 1 (FSTL1), a glycoprotein involved in adipogenesis and obesity, however, the function of FSTL1 in BAT thermogenesis and in the regulation of systemic energy homeostasis are not fully understood. METHODS: Whole-body ablation Fstl1 heterozygous mice (Fstl1+/-) and its littermates control were injected with CL316,243 to assess energy balance. A series of FSTL1 overexpression and knockdown experiments were carried out to evaluate its function in regulating thermogenic gene expression in brown adipocytes. RESULTS: FSTL1 expression was induced upon BAT activation during cold challenge or ß3-adrenergic activation. FSTL1 haploinsufficiency in mice led to reduced thermogenic gene expression, impaired BAT recruitment, and decreased heat production. FSTL1 cell-autonomously promoted the ß3-adrenergic signaling, which was required to upregulate PPARγ and UCP1 in brown adipocytes. Furthermore, only glycosylated FSTL1 could be secreted from brown adipocytes to induce the ß3-adrenergic activation. CONCLUSIONS: Our results suggest FSTL1 as a novel stimulator of the ß-adrenergic signaling and BAT thermogenesis.

FSTL1 enhances chemoresistance and maintains stemness in breast cancer cells via integrin ß3/Wnt signaling under miR-137 regulation.

FSTL1 is a protein coding gene associated with cell signaling pathway regulation and the progression of a variety of disorders. In this study, we hypothesized that FSTL1 increases oncogenesis in breast cancer by enhancing stemness and chemoresistance. RT-PCR and IHC revealed significantly higher FSTL1 mRNA and protein levels in TNBC than in non-TNBC specimens and in breast cancer cell lines. We then found that FSTL1 levels were significantly increased in chemoresistant cells. LIVE/DEAD, MTT cell viability and colony formation assays did in fact demonstrate that FSTL1 is required for CDDP and DOX chemoresistance in breast cancer cell lines. FSTL1 overexpression caused significant elevation of stem cell biomarkers, as well as breast cancer cell proliferation. To determine whether the Wnt/ß-catenin signaling pathway is involved in the observed effects of FSTL1, we assessed levels of pathway target. TOP/FOP flash, colony formation, and tumor sphere formation assays indicated that FSTL1 activates Wnt/ß-catenin signaling through integrin ß3. We then sought to identify a microRNA (miRNA) that regulates FSTL1 activity. Luciferase assays demonstrated that miR-137 reduces FSTL1 mRNA and protein levels. Ultimately, our findings indicate that there is an miR-137/FSTL1/integrin ß3/Wnt/ß-catenin signaling axis in breast cancer cells that regulates stemness and chemoresistance.

BBS4 regulates the expression and secretion of FSTL1, a protein that participates in ciliogenesis and the differentiation of 3T3-L1.

Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity.

Up-Regulation of Follistatin-Like 1 By the Androgen Receptor and Melanoma Antigen-A11 in Prostate Cancer.

BACKGROUND: High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC). METHODS: Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft. RESULTS: Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen. CONCLUSION: AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to promote growth and progression of CRPC. Prostate 77:505-516, 2017. © 2016 Wiley Periodicals, Inc.

A genetic polymorphism affects the risk and prognosis of renal cell carcinoma: association with follistatin-like protein 1 expression.

Few single nucleotide polymorphisms (SNPs) associated with the risk of renal cell carcinoma (RCC) have been identified, yet genetic predisposition contributes significantly to this malignancy. We previously showed that follistatin-like 1 (FSTL1) was significantly down-regulated in clear cell RCC (ccRCC), in particular metastatic ccRCC. In the present study, we systemically investigated the associations of the 6 SNPs within FSTL1-coding genomic region with RCC risk and postoperative prognosis. Age- and gender-matched case-control study (417 vs 855) indicated that rs1259293 variant genotype CC was significantly associated with an increased risk of RCC, with an odds ratio of 2.004 (95% confidence internal [CI] = 1.190-3.375). Multivariate Cox regression analysis in 309 of 417 cases showed that rs1259293 genotype (CC vs TT + CT) independently predicted an unfavorable prognosis, with a hazard ratio of 2.531 (95% CI = 1.052-6.086). Expression of FSTL1 was significantly higher in adjacent renal tissues than in tumors, and significantly higher in the tissues with rs1259293 TT genotype than in those with rs1259293 TC+CC genotypes. rs1259293 C allele might generate a CTCF binding site that blocks trans-activation of FSTL1 expression. Our results indicate that rs1259293 is associated with an increased risk and unfavorable postoperative prognosis of RCC, possibly by down-regulating FSTL1 expression in renal tissues.

[mRNA expression of Follistatin and follistatin-like 3, bone morphogenetic protein-4 antagonists in lung tissues of hypoxic mice].

OBJECTIVE: To observe the expression changes of Follistatin (FSN) and follistatin-like 3 (FSRP) mRNA, both of bone morphogenetic protein-4(BMP4) antagonists, in mice lung tissue under different hypoxic time and its relation with BMP4. METHODS: Thirty BMP4+/+ C57BL/6J wild type mice were randomly divided into normoxic control group, and 4 groups including 1 day, 3 days, 7 days and 21 days under hypoxic condition. The hypoxic animal model was established by exposing the mice to hypoxic condition for different time. The expressing level of FSN and FSRP mRNA in lung tissues were measured by Quantitative Real-Time PCR. RESULTS: FSN and FSRP mRNA increased in hypoxic 1 day group as (28.0 ± 9.4) and (2.0 ± 0.4), in hypoxic 3 day group, FSN mRNA increased by (6.3 ± 3.2) and FSRP mRNA by (1.67 ± 0.7) (P < 0. 05). Compared with the normoxic group (1.77 ± 0.36) and (1.22 ± 0.15) (P < 0. 01), FSN and FSRP mRNA up-regulated in hypoxic 7 day group. The positive degree of FSN and FSRP mRNA in hypoxic 21 day group were (1.04 ± 0.27) and (0.85 ± 0.10) (P < 0. 05). In normoxic 1 day group, FSN mRNA of lung tissues of BMP4+/- C57BL/6J mice was (0.95 ± 0.05); FSRP mRNA was (1.11 ± 0.03) (P < 0. 05). In BMP4+/- C57BL/6J mice lung tissues, FSN mRNA were (1.10 ± 0.40) (P < 0. 05); FSRP mRNA were (0.85 ± 0.18). CONCLUSIONS: The expression of FSN and FSRP mRNA in hypoxic 1;3;7 day mice lung tissues increased obviously, and FSN may play an important role in the hypoxic pulmonary hypertension through BMP4.

Follistatin-like 5 is expressed in restricted areas of the adult mouse brain: Implications for its function in the olfactory system.

Follistatin-like 5 (Fstl5), a member of the follistatin family of genes, encodes a secretory glycoprotein. Previous studies revealed that other members of this family including Fstl1 and Fstl3 play an essential role in development, homeostasis, and congenital disorders. However, the in vivo function of Fstl5 is poorly understood. To gain insight into the function of Fstl5 in the mouse central nervous system, we examined the Fstl5 expression pattern in the adult mouse brain. The results of in situ hybridization analysis showed a highly restricted pattern of Fstl5, namely, with localization in the olfactory system, hippocampal CA3 area and granular cell layer of the cerebellum. Restricted expression in the olfactory system suggests a possible role for Fstl5 in maintaining odor perception.

State-dependent signaling by Cav1.2 regulates hair follicle stem cell function.

The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Cav1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Cav1.2, they lack detectable voltage-dependent calcium currents. Cav1.2(TS) acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Cav1.2 to induce anagen and overcome the TS phenotype. Cav1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration.

'See-saw' expression of microRNA-198 and FSTL1 from a single transcript in wound healing.

Post-transcriptional switches are flexible effectors of dynamic changes in gene expression. Here we report a new post-transcriptional switch that dictates the spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. Expression of primate-specific exonic microRNA-198 (miR-198), located in the 3'-untranslated region of follistatin-like 1 (FSTL1) messenger RNA, switches to expression of the linked open reading frame of FSTL1 upon wounding in a human ex vivo organ culture system. We show that binding of a KH-type splicing regulatory protein (KSRP, also known as KHSRP) to the primary transcript determines the fate of the transcript and is essential for the processing of miR-198: transforming growth factor-ß signalling switches off miR-198 expression by downregulating KSRP, and promotes FSTL1 protein expression. We also show that FSTL1 expression promotes keratinocyte migration, whereas miR-198 expression has the opposite effect by targeting and inhibiting DIAPH1, PLAU and LAMC2. A clear inverse correlation between the expression pattern of FSTL1 (pro-migratory) and miR-198 (anti-migratory) highlights the importance of this regulatory switch in controlling context-specific gene expression to orchestrate wound re-epithelialization. The deleterious effect of failure of this switch is apparent in non-healing chronic diabetic ulcers, in which expression of miR-198 persists, FSTL1 is absent, and keratinocyte migration, re-epithelialization and wound healing all fail to occur.

Heart failure-induced skeletal myopathy in spontaneously hypertensive rats.

BACKGROUND: Although skeletal muscle atrophy and changes in myosin heavy chain (MyHC) isoforms have often been observed during heart failure, their pathophysiological mechanisms are not completely defined. In this study we tested the hypothesis that skeletal muscle phenotype changes are related to myogenic regulatory factors and myostatin/follistatin expression in spontaneously hypertensive rats (SHR) with heart failure. METHODS: After developing tachypnea, SHR were subjected to transthoracic echocardiogram. Pathological evidence of heart failure was assessed during euthanasia. Age-matched Wistar-Kyoto (WKY) rats were used as controls. Soleus muscle morphometry was analyzed in histological sections, and MyHC isoforms evaluated by electrophoresis. Protein levels were assessed by Western blotting. STATISTICAL ANALYSIS: Student'st test and Pearson correlation. RESULTS: All SHR presented right ventricular hypertrophy and seven had pleuropericardial effusion. Echocardiographic evaluation showed dilation in the left chambers and left ventricular hypertrophy with systolic and diastolic dysfunction in SHR. Soleus weight and fiber cross sectional areas were lower (WKY 3615 ± 412; SHR 2035 ± 224 µm(2); P<0.001), and collagen fractional volume was higher in SHR. The relative amount of type I MyHC isoform was increased in SHR. Myogenin, myostatin, and follistatin expression was lower and MRF4 levels higher in SHR. Myogenin and follistatin expression positively correlated with fiber cross sectional areas and MRF4 levels positively correlated with I MyHC isoform. CONCLUSION: Reduced myogenin and follistatin expression seems to participate in muscle atrophy while increased MRF4 protein levels can modulate myosin heavy chain isoform shift in skeletal muscle of spontaneously hypertensive rats with heart failure.

Therapeutic impact of follistatin-like 1 on myocardial ischemic injury in preclinical models.

BACKGROUND: Acute coronary syndrome is a leading cause of death in developed countries. Follistatin-like 1 (FSTL1) is a myocyte-derived secreted protein that is upregulated in the heart in response to ischemic insult. Here, we investigated the therapeutic impact of FSTL1 on acute cardiac injury in small and large preclinical animal models of ischemia/reperfusion and dissected its molecular mechanism. METHODS AND RESULTS: Administration of human FSTL1 protein significantly attenuated myocardial infarct size in a mouse or pig model of ischemia/reperfusion, which was associated with a reduction of apoptosis and inflammatory responses in the ischemic heart. Administration of FSTL1 enhanced the phosphorylation of AMP-activated protein kinase in the ischemia/reperfusion-injured heart. In cultured cardiac myocytes, FSTL1 suppressed apoptosis in response to hypoxia/reoxygenation and lipopolysaccharide-stimulated expression of proinflammatory genes through its ability to activate AMP-activated protein kinase. Ischemia/reperfusion led to enhancement of bone morphogenetic protein-4 expression and Smad1/5/8 phosphorylation in the heart, and FSTL1 suppressed the increased phosphorylation of Smad1/5/8 in ischemic myocardium. Treating cardiac myocytes with FSTL1 abolished the bone morphogenetic protein-4-stimulated increase in apoptosis, Smad1/5/8 phosphorylation, and proinflammatory gene expression. In cultured macrophages, FSTL1 diminished lipopolysaccharide-stimulated expression of proinflammatory genes via activation of AMP-activated protein kinase and abolished bone morphogenetic protein-4-dependent induction of proinflammatory mediators. CONCLUSIONS: Our data indicate that FSTL1 can prevent myocardial ischemia/reperfusion injury by inhibiting apoptosis and inflammatory response through modulation of AMP-activated protein kinase- and bone morphogenetic protein-4-dependent mechanisms, suggesting that FSTL1 could represent a novel therapeutic target for post-myocardial infarction, acute coronary syndrome.

FSTL5 is a marker of poor prognosis in non-WNT/non-SHH medulloblastoma.

PURPOSE: Integrated genomics approaches have revealed at least four distinct biologic variants of medulloblastoma: WNT (wingless), SHH (sonic hedgehog), group C, and group D. Because of the remarkable clinical heterogeneity of group D tumors and the dismal prognosis of group C patients, it is vital to identify molecular biomarkers that will allow early and effective treatment stratification in these non-WNT/non-SHH tumors. PATIENTS AND METHODS: We combined transcriptome and DNA copy-number analyses for 64 primary medulloblastomas. Bioinformatic tools were used to discover marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the screening cohort. The prognostic power of follistatin-like 5 (FSTL5) immunopositivity was tested for 235 nonoverlapping medulloblastoma samples on two independent tissue microarrays. RESULTS: Comprehensive analyses of transcriptomic and genetic alterations delineate four distinct variants of medulloblastoma. Stable subgroup separation was achieved by using the 300 transcripts that varied the most. Distinct expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time polymerase chain reaction. Immunopositivity of FSTL5 identified a large cohort of patients (84 of 235 patients; 36%) at high risk for relapse and death. Importantly, more than 50% of non-WNT/non-SHH tumors displayed FSTL5 negativity, delineating a large patient cohort with a good prognosis who would otherwise be considered intermediate or high-risk on the basis of current molecular subgrouping. CONCLUSION: FSTL5 expression denoted a dismal prognosis both within and across medulloblastoma subgroups. The addition of FSTL5 immunohistochemistry to existing molecular stratification schemes constitutes a reliable and cost-effective tool for prognostication in future clinical trials of medulloblastoma.

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

BACKGROUND: We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin ß(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. METHODOLOGY: hGL 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 ß(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 FINDINGS: GDF9 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 ß(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 ß(B)-subunit mRNA and inhibin B production were attenuated by adding FST. CONCLUSION: GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin ß(B)-subunit mRNA expression and inhibin B production in hGL cells.

Activin-A and myostatin response and steroid regulation in human myometrium: disruption of their signalling in uterine fibroid.

CONTEXT: Investigation of activin-A (A) and myostatin (M) in human myometrium (HM) and leiomyoma (HL) will explain their involvement in human myometrial pathophysiology. OBJECTIVE: We aimed to investigate A and M response and steroid regulation in HM. We also evaluated A and M expression and response in HL. DESIGN: Tissues were analyzed and cultured. PATIENTS: Patients included fertile (in proliferative phase) and menopausal women undergoing hysterectomy. INTERVENTIONS: HM explant cultures were treated with A and M (for Smad-7 mRNA quantification) or estrogen and progesterone (for A and M mRNA quantification). A and M expression levels were also evaluated in menopausal (physiological absence of steroids) HM specimens. A and M and their receptors were evaluated in HL (n = 8, diameter 5-8 cm) compared with their matched HM. HL explants cultures were treated with A and M (for Smad7 mRNA quantification), and, to explain the absence of response, the levels of follistatin, follistatin-related gene (FLRG), and Cripto were evaluated. RESULTS: A and M increased Smad7 expression in HM explants. A and M mRNAs were both reduced after estradiol treatment, unchanged after progesterone treatment, but were higher in menopausal than fertile (in proliferative phase) specimens. A, M, and FLRG were expressed at higher levels in HL compared with adjacent HM, whereas the receptors, follistatin, and Smad7 mRNAs resulted unchanged. Cripto mRNA was expressed only in HL. CONCLUSIONS: A and M act on human HM and are regulated by steroids. In HL there is an increase of A, M, FLRG, and Cripto expression.

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.

Follistatin-like protein 1 promotes arthritis by up-regulating IFN-gamma.

Follistatin-like protein-1 (FSTL-1) is a poorly characterized protein that is up-regulated in the early stage of collagen-induced arthritis and that exacerbates arthritis when delivered by gene transfer. The current study was designed to determine the mechanism by which FSTL-1 promotes arthritis. FSTL-1 was injected into mouse paws, resulting in severe paw swelling associated with up-regulation of IFN-gamma transcript and the IFN-gamma-induced chemokine, CXCL10. Mice depleted of T cells were protected. A central role for IFN-gamma was confirmed by the finding that mice deficient in IFN-gamma failed to exhibit paw swelling in response to injection of FSTL-1. Furthermore, IFN-gamma secretion from mouse spleen cells exposed to a weak TCR signal was increased 5-fold in the presence of FSTL-1. FSTL-1 could be induced by innate immune signals, including TLR4 agonists and the arthritogenic cytokine, IL-1beta, via an NFkappaB pathway. Finally, FSTL-1 was found to be overexpressed in human arthritis and its neutralization inhibited murine collagen-induced arthritis and suppressed IFN-gamma and CXCL10 production in arthritic joints. These findings demonstrate that FSTL-1 plays a critical role in arthritis by enhancing IFN-gamma signaling pathways and suggest a mechanism by which FSTL-1 bridges innate and adaptive immune responses.

Gene expression profiling of human hibernating myocardium: increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart.

A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.