Silica nanoparticle releases SIRT6-induced epigenetic silencing of follistatin.

Follistatin (FST) plays a protective role during silica nanoparticle (SiO2 NP) exposure. SiO2 NP treatment induces FST transcription with an unknown mechanism. We herein reported that SIRT6, one of the sirtuin family members, induced epigenetic silencing of FST. The expression of FST was elevated after SIRT6 knockdown while reduced after SIRT6 overexpression. Chromatin immunoprecipitation revealed a direct interaction between SIRT6 with FST promoter. Knockdown of SIRT6 increased both Ac-H3K9 level and Ac-H3K56 level at FST promoter region. SiO2 NP treatment de-stabilized SIRT6 mRNA and reduced SIRT6 expression, leading to the activation of FST transcription. Finally, over-expression of SIRT6 increased SiO2 NP-induced apoptosis. Collectively, this study provided evidence that SIRT6 is a negative regulator of FST transcription and participates in the regulation of cell survival during silica nanoparticle exposure.

Fingolimod induces neuronal-specific gene expression with potential neuroprotective outcomes in maturing neuronal progenitor cells exposed to HIV.

Fingolimod (FTY720), a structural analogue of sphingosine, targets sphingosine-1-phosphate receptor signaling and is currently an immunomodulatory therapy for multiple sclerosis. Fingolimod accesses the central nervous system (CNS) where its active metabolite, fingolimod phosphate (FTY720-P), has pleotropic neuroprotective effects in an inflammatory microenvironment. To investigate potential neuronal-specific mechanisms of fingolimod neuroprotection, we cultured the human neuronal progenitor cell line, hNP1, in differentiation medium supplemented with HIV- or Mock-infected supernatants, with or without FTY720-P. Gene expression was investigated using microarray and functional genomics. FTY720-P treatment increased differentially expressed (DE) neuronal genes by 33% in HIV-exposed and 40% in Mock-exposed cultures. FTY720-P treatment broadened the functional profile of DE genes in HIV-exposed versus Mock-exposed neurons, including not only immune responses but also transcriptional regulation and cell differentiation, among others. FTY720-P treatment downregulated the gene for follistatin, the antagonist of activin signaling, in all culture conditions. FTY720-P treatment differentially affected both glycolysis-related and immune response genes in Mock- or HIV-exposed cultures, significantly upregulating 11 glycolysis-related genes in HIV-exposed neurons. FTY720-P treatment also differentially upregulated genes related to innate immune responses and antigen presentation in Mock-exposed and more so in HIV-exposed neurons. However, in HIV-exposed neurons, FTY720-P depressed the magnitude of differential expression in almost half the genes, suggesting an anti-inflammatory potential. Moreover, in HIV-exposed neurons, FTY720-P reduced expression of the amyloid precursor protein (APP) gene, resulting in reduced expression of the APP protein. This study provides new evidence that fingolimod alters neuronal gene expression in inflammatory, viral-infected microenvironments, with the potential for neuroprotective effects.

Follistatin as potential therapeutic target in prostate cancer.

Follistatin is a single-chain glycosylated protein whose primary function consists in binding and neutralizing some members of the transforming growth factor-ß superfamily such as activin and bone morphogenic proteins. Emerging evidence indicates that this molecule may also play a role in the malignant progression of several human tumors including prostate cancer. In particular, recent findings suggest that, in this tumor, follistatin may also contribute to the formation of bone metastasis through multiple mechanisms, some of which are not related to its specific activin or bone morphogenic proteins' inhibitory activity. This review provides insight into the most recent advances in understanding the role of follistatin in the prostate cancer progression and discusses the clinical and therapeutic implications related to these findings.

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.

Nucleolar follistatin promotes cancer cell survival under glucose-deprived conditions through inhibiting cellular rRNA synthesis.

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

Silencing of FLRG, an antagonist of activin, inhibits human breast tumor cell growth.

Activin, a member of the transforming growth factor beta (TGFbeta) superfamily, regulates diverse processes, such as cellular growth and differentiation. There is increasing evidence that TGFbeta and its signaling effectors are key determinants of tumor cell behavior. Loss of sensitivity to TGFbeta-induced growth arrest is an important step toward malignancy. We previously characterized FLRG as an extracellular antagonist of activin. Here, we show that activin-induced growth inhibition is altered in FLRG-expressing breast cancer lines. Silencing FLRG induced growth inhibition, which is reversible upon addition of exogenous FLRG. We showed that FLRG silencing effects resulted from restoration of endogenous activin functions as shown by increased levels of phosphorylated smad2 and up-regulation of activin target gene transcripts. Furthermore, the growth inhibition induced by FLRG silencing was reversible by treatment with a soluble form of type II activin receptor. Finally, a strong expression of FLRG was observed in invasive breast carcinomas in contrast with the normal luminal epithelial cells in which FLRG was not detected. Our data provide strong evidence that endogenous FLRG contributes to tumor cell proliferation through antagonizing endogenous activin effects.

Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin.

Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.

Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis.

Wnt4(-/-) XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFbeta superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary.