Expression Of Follistatin-like 1 Research Articles

Skeletal muscle-derived FSTL1 starting up angiogenesis by regulating endothelial junction via activating Src pathway can be upregulated by hydrogen sulfide.

Hydrogen sulfide (H2S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H2S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H2S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H2S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H2S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H2S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway.NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H2S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

MiR-125a-3p regulates the expression of FSTL1, a pro-inflammatory factor, during adipogenic differentiation, and inhibits adipogenesis in mice.

Adipogenesis is tightly regulated by various factors, including genes and microRNAs. Excessive fat deposition is the key feature of obesity, which is a low-grade chronic inflammatory disease. Follistatin-like 1 (FSTL1) has been reported to be an important mediator involved in various inflammatory diseases. However, the underlying mechanism of FSTL1 in preadipocyte differentiation and inflammatory response is still unclear. The current study was designed to explore the biological function and potential mechanism of FSTL1 in mouse subcutaneous preadipocyte differentiation. We found that FSTL1 was highly expressed in the early stage of differentiation and subsequently decreased sharply, suggesting that FSTL1 played a possible role in adipogenesis. Meanwhile, the gain- and loss-of-function assays showed that FSTL1 was not only involved in the inflammatory response by inducing the expression of pro-inflammatory factors IL-1β and CCL2 but also significantly attenuated preadipocyte differentiation, as evidenced by the reduction of lipid accumulation and the levels of adipogenic genes, including PPARγ and FABP4. In addition, the target gene prediction and luciferase reporter assay validated that miR-125a-3p targeted the 3' UTR region of FSTL1. These results demonstrated that miR-125a-3p negatively regulated the expression of FSTL1 at the mRNA and protein levels. Furthermore, overexpressing miR-125a-3p in preadipocytes dramatically accelerated adipogenic differentiation and downregulated the levels of IL-1β and CCL2, which were in accordance with the knockdown of FSTL1. On the contrary, treatment with miR-125a-3p inhibitors attenuated adipogenesis but induced the expression of inflammatory genes. In summary, this study suggests a positive function of FSTL1 in adipocyte-induced inflammation and negatively regulates preadipocyte differentiation. Further studies demonstrated that miR-125a-3p could reverse the effect by targeting FSTL1, which might provide a better understanding of treating obesity-related inflammatory diseases.

Effect of the Aerobic and Resistance Training on Follistatin-Like 1 and Leukemia Inhibitory Factor Muscle Gene Expression in Rats Fed With a High-Fat Diet

Background: Follistatin-like 1 (FSTL-1) and leukemia inhibitory factor (LIF) are two myokines that are affected by overweight and have inflammatory and damaging effects. Considering that exercise reduces excess weight, this study aimed to evaluate the effect of aerobic and resistance training on FSTL-1 and LIF muscle gene expression in rats fed with a high-fat diet. Materials and Methods: In this experimental study, 32 rats were randomly divided into healthy control, obese control, obese+aerobic exercise, and obese+resistance exercise groups. The training was performed for 4 weeks at aerobic moderate intensity (50-65% VO2max). For resistance training, rats were also trained to climb the ladder (height 110 cm, slope 80%, and the distance between the bars of the ladder 2 cm), which is based on the determination of one repetition maximum. A high-fat diet was prepared with 40% fat, 13% protein, and 47% carbohydrates and continued until the rats reached the obesity range. The tissue sample was taken from the gluteus muscle. Results: The expression of FSTL-1 and LIF in the obese control group increased significantly compared to the healthy control group (P=0.044 and P=0.039, respectively). The expression of FSTL-1 and LIF in the resistance training group significantly decreased in comparison to the obese control group (P=0.049 and P=0.046, respectively). There was no significant difference between the aerobic exercise group and the obese control group (P=0.053 and P=0.059, respectively). However, a significant difference was observed between aerobic and resistance training groups in terms of FSTL-1 (P=0.042). Conclusion: Resistance exercise seems to have a greater and better effect on FSTL-1 and LIF in the muscles of obese samples compared to aerobic exercise.

The BMP inhibitor follistatin-like 1 (FSTL1) suppresses cervical carcinogenesis.

Follistatin-like 1 (FSTL1) is a cancer-related matricellular secretory protein with contradictory organ-specific roles. Its contribution to the pathogenesis of cervical carcinoma is still not clear. Meanwhile, it is necessary to identify novel candidate genes to understand cervical carcinoma's pathogenesis further and find potential therapeutic targets. We collected cervical carcinoma samples and matched adjacent tissues from patients with the locally-advanced disease and used cervical carcinoma cell lines HeLa and C33A to evaluate the effects of FSTL1 on CC cells. The mRNA transcription and protein expression of FSTL1 in cervical carcinoma tumor biopsy tissues were lower than those of matched adjacent tissues. Patients with a lower ratio of FSTL1 mRNA between the tumor and its matched adjacent tissues showed a correlation with the advanced cervical carcinoma FIGO stages. High expression of FSTL1 markedly inhibited the proliferation, motility, and invasion of HeLa and C33A. Regarding mechanism, FSTL1 plays its role by negatively regulating the BMP4/Smad1/5/9 signaling. Our study has demonstrated the tumor suppressor effect of FSTL1, and these findings suggested a potential therapeutic target and biomarker for cervical carcinoma.

The lncRNA PVT1/miR-590-5p/FSTL1 axis modulates the proliferation and migration of airway smooth muscle cells in asthma

Objective Asthma is a prevalent chronic inflammatory airway disease that is characterised by airway remodelling and airway hyperresponsiveness. Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) contribute to airway remodelling in asthma. However, the molecular mechanism underlying an increased ASMC mass in asthma remains elusive. Herein, we aimed at investigating the regulation of lncRNA PVT1 on ASMCs and focussing on the mechanism in the proliferation and migration. Methods Expression levels of lncRNA PVT1 and miR-590-5p in the serum collected from 24 children with asthma and 10 control children were determined by qRT-PCR. ASMCs proliferation and migration prior to and post platelet-derived growth factor subunit B (PDGF-BB) stimulation were examined by CCK-8 test and transwell assay. Dual-luciferase reporter assay was performed to determine miR-590-5p interaction with lncRNA PVT1 and follistatin-like 1 (FSTL1). Expression of lncRNA PVT1, miR-590-5p, FSTL1, C-Myc, cyclin D1, and cyclin-dependent kinase 1 (CDK1) was tested by quantitative real-time PCR (qRT-PCR) and immunoblotting analysis. Results The expression level of lncRNA PVT1 was higher but the expression level of miR-590-5p was lower in the serum of children with asthma than in control children. The expression level of lncRNA PVT1 was negatively correlated with the expression level of miR-590-5p in asthma. LncRNA PVT1 was upregulated upon PDGF-BB stimulation. LncRNA PVT1 knockdown by its specific shRNA repressed PDGF-BB-induced promotion of proliferation and migration in ASMCs and triggered an elevated miR-590-5p along with declined C-Myc, cyclin D1, and CDK1. The effects of lncRNA PVT1 knockdown on PDGF-BB-induced ASMCs were lost upon miR-590-5p inhibition. MiR-590-5p targeted FSTL1 gene and declined its expression, thus suppressing ASMC proliferation and migration following PDGF-BB stimulation and downregulating C-Myc, cyclin D1, and CDK1 expressions. The effects of miR-590-5p on PDGF-BB-induced ASMCs were lost upon FSTL1 overexpression. Conclusion These results support the notion that the lncRNA PVT1/miR-590-5p/FSTL1 axis modulates ASMCs proliferation and migration following PDGF-BB stimulation, providing a potential therapeutic target to attenuate airway remodelling in asthma.

Follistatin-like 1 deficiency impairs T cell development to promote lung metastasis of triple negative breast cancer.

Our study aims to detect the underlying mechanism of the suppressive effect of Follistatin-like 1 (FSTL1) on lung metastasis of triple negative breast cancer (TNBC). We found that FSTL1 had no effect on the proliferation and metastasis of 4T1 cells in vitro, while in the tumor-bearing Fstl1 heterozygous (Fstl1+/-) mice, the number of anti-tumor T lymphocytes in the lung was significantly reduced with the increase in lung metastasis. Impaired development of T cells can cause dysfunction of adaptive immune system, which promotes cancer metastasis. Therefore the effect of FSTL1 on T cell development was further investigated.Lower population of T cells in periphery and decreased proliferation of CD4- CD8- double negative (DN) thymocytes and impairment development of T cells were found in Fstl1+/- mice. Furthermore, high expression of FSTL1 in medullary thymus epithelial (mTEC) cells and decreased mRNA expression of inducible costimulator on activated T-cell ligand (Icosl) in mTECsh Fstl1 were detected. Combining other studies that the generation of ICOSL by mTEC cells promotes CD4+ single positive (SP) thymocytes to produce IL-2, which promotes T cell development. Our results indicate FSTL1 deficiency in mTEC cells impairs T cell development to promote the lung metastasis of TNBC.

Follistatin-like 1 (FSTL1) is a prognostic biomarker and correlated with immune cell infiltration in gastric cancer

BackgroundFollistatin-like 1 (FSTL1) plays a central role in the progression of tumor and tumor immunity. However, the effect of FSTL1 on the prognosis and immune infiltration of gastric cancer (GC) remains to be elucidated.MethodsThe expression of FSTL1 data was analyzed in Oncomine and TIMER databases. Analyses of clinical parameters and survival data were conducted by Kaplan-Meier plotter and immunohistochemistry. Western blot assay and real-time quantitative PCR (RT-qPCR) were used to analyze protein and mRNA expression, respectively. The correlations between FSTL1 and cancer immune infiltrates were analyzed by Tumor Immune Estimation Resource (TIME), Gene Expression Profiling Interactive Analysis (GEPIA), and LinkedOmics database.ResultsThe expression of FSTL1 was significantly higher in GC tissues than in normal tissues, and bioinformatic analysis and immunohistochemistry (IHC) indicated that high FSTL1 expression significantly correlated with poor prognosis in GC. Moreover, FSTL1 was predicted as an independent prognostic factor in GC patients. Bioinformatics analysis results suggested that FSTL1 mainly involved in tumor progression and tumor immunity. And significant correlations were found between FSTL1 expression and immune cell infiltration in GC.ConclusionsThe study effectively revealed useful information about FSTL1 expression, prognostic values, potential functional networks, and impact of tumor immune infiltration in GC. In summary, FSTL1 can be used as a biomarker for prognosis and evaluating immune cell infiltration in GC.

Dynamic resistance exercise increases skeletal muscle-derived FSTL1 inducing cardiac angiogenesis via DIP2A–Smad2/3 in rats following myocardial infarction

PurposeThe aim of this study was to investigate the potential of dynamic resistance exercise to generate skeletal muscle-derived follistatin like-1 (FSTL1), which may induce cardioprotection in rats following myocardial infarction (MI) by inducing angiogenesis. MethodsMale, adult Sprague-Dawley rats were randomly divided into 5 groups (n = 12 in each group): sham group (S), sedentary MI group (MI), MI + resistance exercise group (MR), MI + adeno-associated virus (AAV)–FSTL1 injection group (MA), and MI + AAV–FSTL1 injection + resistance exercise group (MAR). The AAV–FSTL1 vector was prepared by molecular biology methods and injected into the anterior tibialis muscle. The MI model was established by ligation of the left anterior descending coronary artery. Rats in the MR and MAR groups underwent 4 weeks of dynamic resistance exercise training using a weighted climbing-up ladder. Heart function was evaluated by hemodynamic measures. Collagen volume fraction of myocardium was observed and analyzed by Masson's staining. Human umbilical vein vessel endothelial cells culture and recombinant human FSTL1 protein or transforming growth factor-β receptor 1 (TGFβR1) inhibitor treatment were used to elucidate the molecular signaling mechanism of FSTL1. Angiogenesis, cell proliferation, and disco interacting protein 2 homolog A (DIP2A) location were observed by immunofluorescence staining. The expression of FSTL1, DIP2A, and the activation of signaling pathways were detected by Western blotting. Angiogenesis of endothelial cells was observed by tubule experiment. One-way analysis of variance and Student's t test were used for statistical analysis. ResultsResistance exercise stimulated the secretion of skeletal muscle FSTL1, which promoted myocardial angiogenesis, inhibited pathological remodeling, and protected cardiac function in MI rats. Exercise facilitated skeletal muscle FSTL1 to play a role in protecting the heart. Exogenous FSTL1 promoted the human umbilical vein vessel endothelial cells proliferation and up-regulated the expression of DIP2A, while TGFβR1 inhibitor intervention down-regulated the phosphorylation level of Smad2/3 and the expression of vascular endothelial growth factor-A, which was not conducive to angiogenesis. FSTL1 bound to the receptor, DIP2A, to regulate angiogenesis mainly through the Smad2/3 signaling pathway. FSTL1–DIP2A directly activated Smad2/3 and was not affected by TGFβR1. ConclusionDynamic resistance exercise stimulates the expression of skeletal muscle-derived FSTL1, which could supplement the insufficiency of cardiac FSTL1 and promote cardiac rehabilitation through the DIP2A–Smad2/3 signaling pathway in MI rats.

312-OR: Functional Decline in Brown Adipose Progenitors during BAT Remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune-deficient mice. RNA-sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down-regulated, while genes involved in lipid and amino acid metabolism were up-regulated. Single-cell RNA sequencing discovered a unique population of stromal vascular fraction cells specifically expressing genes regulating muscle and mitochondrial function. Knocking down these genes in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans. Disclosure Z. Huang: None. Z. Zhang: None. H. Ruan: None. Funding American Diabetes Association (1-18-IBS-167 to H-B.R.)

Functional decline in brown adipose progenitors during BAT remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune‐deficient mice. RNA‐sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down‐regulated, while genes involved in lipid and amino acid metabolism were up‐regulated. Knocking down genes regulating muscle and mitochondrial function in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes in vitro and in vivo. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans.Support or Funding InformationAmerican Diabetes Association (18‐IBS‐167)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FSTL1 suppresses tumor cell proliferation, invasion and survival in non-small cell lung cancer.

Follistatin like-1 (FSTL1) is a secreted glycoprotein involved in a series of physiological and pathological processes. However, its contribution to the development of cancer, especially the pathogenesis of NSCLC, remains to be elucidated. We explored the expression, function, and molecular mechanism of FSTL1 in NSCLC. In this study, we detected the expression of FSTL1 in a panel of NSCLC cell lines and lung normal epithelial cell line by qRT-PCR and western blot analysis and found that FSTL1 was downregulated in NSCLC cells compared with normal control. Knockdown of FSTL1 with different shRNA sequences result in increased cell proliferation and cell migration, invasion and reduced cell apoptosis in A549 cell line with high FSTL1 endogenous level. FSTL1 overexpression in H446 cell line with low FSTL1 endogenous level suppressed cell proliferation and migration, invasion and increased cell apoptosis. Knockdown and overexpression of FSTL1 caused altered cell cycle. Reduced cell apoptosis was revealed in FSTL1 knockdown cells accompanied by increased FAS expression and decreased FASL, cleaved caspase-3 and −7 expression. By contrast, overexpression of FSTL1 caused reduced FAS level and increased activated caspase-3 and −7 expression, which may lead to increased cell apoptosis. Moreover, the changed migration and invasion ability in FSTL1 sufficient or deficient cells may be caused by alterations in MMP2, MMP3 and MMP9 expression. Altogether, our results revealed the critical tumor-suppression function of FSTL1 in NSCLC progression, suggesting that FSTL1 might be an important factor in NSCLC progression.

Effects of FSTL1 on cell proliferation in breast cancer cell line MDA‑MB‑231 and its brain metastatic variant MDA‑MB‑231‑BR.

In the past decades, altered Follistatin-like 1 (FSTL1) expression has been documented in a variety of cancers, while its functional roles are poorly understood. Particularly in breast cancer, the expression of FSTL1 and its signaling pathway remain to be determined. In the present study, an elevated FSTL1 expression and a supressed cell proliferation were detected in a specific brain metastatic cell line MDA-MB-231-BR (231-BR), compared with its parental cell line MDA-MB-231. However, this protein was hardly detected in the other three breast cancer cell lines. Next, lentiviral vectors encoding FSTL1 or FSTL1 specific shRNAs were used to overexpress or knock down FSTL1 in MDA-MB-231 or 231-BR, respectively (MDA-MB-231FSTL1 or 231-BRsh FSTL1). Results showed that overexpression of FSTL1 inhibited MDA-MB-231 cell proliferation, while knockdown of FSTL1 in 231-BR cells promotes cell proliferation, compared with their corresponding control groups. These results were further confirmed in nude mouse xenografts. The tumor volume in 231-BR cell-bearing mice was significantly smaller than that of MDA-MB-231 group, and reduction of tumor volume was detected in MDA-MB-231FSTL1 cell-bearing mice compared with the control group. Previous studies revealed that TGF-β-Smad2/3 signaling pathway was activated in 231-BR and MDA-MB-231FSTL1 cells, which may contribute to the inhibited cell proliferation. In addition, Smad3 knockdown could restore the inhibition of cell proliferation induced by FSTL1 overexpression in MDA-MB-231FSTL1 cells, indicating that the anti-proliferative effect of FSTL1 overexpression may be associated with Smad3 involved TGF-β signaling pathway regulation. This study identified FSTL1 as an inhibitor of cell proliferation in MDA-MB-231 and 231-BR cell lines, which may provide new insights into the development and management of breast cancer.

FSTL1 contributes to tumor progression via attenuating apoptosis in a AKT/GSK-3β - dependent manner in hepatocellular carcinoma.

Several investigations have demonstrated that follistatin-like 1 (FSTL1) is implicated in the initiation and progression of diverse cancers. It remains unclear whether FSTL1 acted as a cancer-promoting gene through its overexpression in HCC. We detected FSTL1 protein expression in 210 consecutive HCC cases curatively resected in our hospital between 2004 and 2007. The correlation between FSTL1 expression in HCC tissues and post-surgical prognosis of HCCs was analyzed. The in vitro experiments including apoptosis assessment, MTT, BrdU incorporation ELISA assay, Western immunoblotting, and qRT-PCR were performed to determine the impact of FSTL1 on apoptosis and proliferation abilities of HCC cells and the relevant mechanisms. FSTL1 protein was found aberrantly increased in 172 of 210 HCC tissues (81.9%) compared to adjacent liver tissues. FSTL1 overexpression was apparently associated with larger tumor size, advanced TNM staging, portal vein invasion, intra-hepatic metastases. Patients with higher FSTL1 expression in tumors suffered from the worse overall survival rate as assessed by comparison of Kaplan-Meier survival curves. Higher FSTL1 expression in HCC tissues was identified as a independent poor post-surgical prognostic predictor for HCC. Silencing FSTL1 by siRNA promoted cell apoptosis and leaded to suppression of cell viability and proliferation in MHCC97h cells. Furthermore, enforced expression of FSTL1 obtained the opposite results in Huh7 cells. Mechanistic investigation showed that FSTL1 repressed HCC cell apoptosis through AKT/GSK-3β/Bcl2/BAX/Bim signaling. These data proved that FSTL1 contributed to unfavorable post-surgical outcome of HCC patients via inhibiting cell apoptosis.

Autophagy plays a role in FSTL1-induced epithelial mesenchymal transition and airway remodeling in asthma.

Asthma is a chronic disease related to airway hyperresponsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1 (FSTL1) promotes EMT and airway remodeling by intensifying autophagy. With the use of transmission electron microscopy (TEM), double-membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in patients with asthma and OVA-challenged mice compared with healthy controls. The expression of FSTL1 and beclin-1 was upregulated in the airways of patients with asthma and OVA-challenged mice, accompanied by airway EMT and remodeling. In OVA-challenged Fstl1+/- mice, the degree of airway remodeling and autophagy was decreased compared with control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY-294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells, as measured by E-cadherin, N-cadherin, and vimentin expression. In line herewith, administration of LY-294002 reduced the expression of autophagy, EMT, and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway, which may be beneficial to patients with refractory asthma.

Epigenetic inactivation of follistatin-like 1 mediates tumor immune evasion in nasopharyngeal carcinoma.

Follistatin like-1 (FSTL1) is a secreted glycoprotein involved in a series of physiological and pathological processes. However, its contribution to the development of cancer, especially the pathogenesis of nasopharyngeal carcinoma (NPC), remains to be elucidated. We aimed to investigate the dysregulation of FSTL1 and its possible function in NPC. FSTL1 was frequently downregulated in NPC cell lines and primary tumor biopsies by promoter hypermethylation. Ectopic expression of FSTL1 significantly suppressed the colony formation, proliferation, migration and invasion ability of NPC cells and induced cell apoptosis. Overexpression of FSTL1 decreased the tumorigenicity of NPC cells in vivo. In addition, the proliferation of NPC cells in vitro was inhibited by treatment with soluble recombinant FSTL1 protein. The protein level of FSTL1 was decreased in primary NPC tumors and was associated with downregulated interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). Furthermore, recombinant human FSTL1 protein induced secretion of IL-1β and TNF-α in macrophage cultures, therefore FSTL1 might activate macrophages and attenuate the immune evasion of NPC cells. In conclusion, the epigenetic downregulation of FSTL1 may suppress the proliferation and migration of NPC cells, leading to dysfunctional innate responses in surrounding macrophages.

Follistatin-like 1 attenuates apoptosis via disco-interacting protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in rats.

Follistatin-like 1 (FSTL1), an extracellular glycoprotein, has been reported to decrease apoptosis in ischemic cardiac diseases, but its effect in ischemic stroke has not been examined. We hypothesized that recombinant FSTL1 attenuates neuronal apoptosis through its receptor disco-interacting protein 2 homolog A (DIP2A) and the Akt pathway after middle cerebral artery occlusion (MCAO) in rats. One hundred forty male Sprague-Dawley rats were subjected to 2 hours of MCAO followed by reperfusion. In a subset of animals, the time course and location of FSTL1 and DIP2A were detected by Western blot and immunofluorescence double staining. Another set of animals were intracerebroventricularly given either recombinant FSTL1 1 hour after reperfusion or FSTL1-small interfering RNA (siRNA) 48 hours before reperfusion. Additionally, DIP2A was knockdown by siRNA in some animals. Infarction volume and neurological deficits were measured, and the expression of FSTL1, DIP2A, phosphorylated Akt, cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling were quantified using Western blot. The expression of FSTL1 and DIP2A was increased in neurons and peaked 24 hours after MCAO. Recombinant FSTL1 reduced brain infarction and improved neurological deficits 24 and 72 hours after MCAO via activation of its receptor DIP2A and downstream phosphorylation of Akt. These effects were reversed by DIP2A-siRNA and FSTL1-siRNA. Recombinant FSTL1 decreases neuronal apoptosis and improves neurological deficits through phosphorylation of Akt by activation of its receptor DIP2A after MCAO in rats. Thus, FSTL1 may have potentials as a treatment for patients with ischemic stroke.

‘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-β 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.

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.

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.

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.