Fstl1 Expression Research Articles

THOC7-AS1/OCT1/FSTL1 axis promotes EMT and serves as a therapeutic target in cutaneous squamous cell carcinoma

BackgroundTHOC7-AS1 and FSTL1 expression are frequently upregulated in cutaneous squamous cell carcinoma (cSCC). However, their molecular biological mechanisms remain elusive and their potential as therapeutic targets needs urgent exploration.MethodsHuman tissue samples were used to evaluate clinical parameters. In vitro and in vivo experiments assessed biological functions. Quantitative PCR, western blot, immunohistochemistry, immunocytochemistry, immunoprecipitation, RNA fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, silver staining, chromatin immunoprecipitation, dual luciferase reporter assays etc. were utilized to explore the molecular biological mechanisms.ResultsWe found FSTL1 is an oncogene in cSCC, with high expression in tumor tissues and cells. Its elevated expression closely associates with tumor size and local tissue infiltration. In vitro and in vivo, high FSTL1 expression promotes cSCC proliferation, migration and invasion, facilitating malignant behaviors. Mechanistically, FSTL1 interacts with ZEB1 to promote epithelial-to-mesenchymal transition (EMT) in cSCC cells. Exploring upstream regulation, we found THOC7-AS1 can interact with OCT1, which binds the FSTL1 promoter region and promotes FSTL1 expression, facilitating cSCC progression. Finally, treating tumors with THOC7-AS1 antisense oligonucleotides inhibited cSCC proliferative and migratory abilities, delaying tumor progression.ConclusionsThe THOC7-AS1/OCT1/FSTL1 axis regulates EMT and promotes tumor progression in cSCC. This study provides clues and ideas for cSCC targeted therapy.

COL5A1 Promotes the Progression of Gastric Cancer by Acting as a ceRNA of miR-137-3p to Upregulate FSTL1 Expression.

Simple SummaryThe expression of a variety of microRNAs (miRNAs) and their target genes in gastric cancer is dysregulated and affects the progression of gastric cancer but has not been fully clarified, while bioinformatics is expected to become a method to reveal the relationship and function between them. Thus, through a variety of bioinformatics analyses and experiments, we confirmed that miR-137-3p played a tumor-suppressive role in gastric cancer, and its target gene COL5A1 could reversely sponge miR-137-3p to relieve its targeted inhibition of FSTL1, which may promote the progression of gastric cancer by affecting immune infiltration. These results may provide new ideas for the treatment and future research of gastric cancer.MicroRNAs (miRNAs) and their target genes have been shown to play an important role in gastric cancer but have not been fully clarified. Therefore, our goal was to identify the key miRNA–mRNA regulatory network in gastric cancer by utilizing a variety of bioinformatics analyses and experiments. A total of 242 miRNAs and 1080 genes were screened from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), respectively. Then, survival-related differentially expressed miRNAs and their differentially expressed target genes were screened. Twenty hub genes were identified from their protein–protein interaction network. After weighted gene co-expression network analysis was conducted, we selected miR-137-3p and its target gene, COL5A1, for further research. We found that miR-137-3p was significantly downregulated and that overexpression of miR-137-3p suppressed the proliferation, invasion, and migration of gastric cancer cells. Furthermore, we found that its target gene, COL5A1, could regulate the expression of another hub gene, FSTL1, by sponging miR-137-3p, which was confirmed by dual-luciferase reporter assays. Knockdown of COL5A1 inhibited the proliferation, invasion, and migration of gastric cancer cells, which could be rescued by the miR-137-3p inhibitor or overexpression of FSTL1. Ultimately, bioinformatics analyses showed that the expression of FSTL1 was highly correlated with immune infiltration.

Prediction of Prognosis and Recurrence of Bladder Cancer by ECM-Related Genes.

Background Bladder cancer (BLCA) is one of the most common cancers and ranks ninth among all cancers. Extracellular matrix (ECM) genes activate a number of pathways that facilitate tumor development. This study is aimed at providing models to predict BLCA survival and recurrence by ECM genes. Methods Expression data from BLCA samples in GSE32894, GSE13507, GSE31684, GSE32548, and TCGA-BLCA cohorts were downloaded and analyzed. The ECM-related genes were obtained by differentially expressed gene analysis, stage-associated gene analysis, and random forest variable selection. The ECM was constructed in GSE32894 by the hub ECM-related genes and validated in GSE13507, GSE31684, GSE32548, and TCGA-BLCA cohorts. The correlations of the ECM score with cells (T cells, fibroblasts, etc.) and the response to immunotherapeutic drugs were investigated. Four machine learning models were selected and used to construct models to predict the recurrence of BLCA. A total of 15 paired BLCA and normal tissue specimens, human immortalized uroepithelial cell lines, and bladder cancer cell lines were selected for the validation of the difference in expression of FSTL1 between normal tissues and BLCA. Results Six ECM genes (CTHRC1, MMP11, COL10A1, FSTL1, SULF1, and COL5A3) were recognized to be the hub ECM-related genes. The ECM score of each BLCA patient was calculated using these six selected ECM-related genes. BLCA patients with a high ECM score group had significantly lower overall survival rates than patients in the low ECM score group. We found that the ECM score was positively associated with immune cells and fibroblasts and negatively correlated with tumor purity. When treated with immunotherapy, BLCA patients with a high ECM score presented a high response rate and better prognosis. We also found that the combination of FSTL1, stage, age, and gender achieved an AUC value of 0.76 in predicting bladder cancer recurrence. Based on the RT-qPCR results of FSTL1 gene expression, there was an overall decrease in the mRNA expression of FSTL1 in cancer tissues compared to their adjacent normal tissues. Subsequent in vitro validation demonstrated that the FSTL1 expression was downregulated at the gene and protein level compared to that in SVH cells. Conclusion Taken together, our results indicate that ECM-related genes correlate with immune cells, overall survival, and recurrence of BLCA. This study provides a machine learning model for predicting the survival and recurrence of BLCA patients.

Brown adipose tissue involution associated with progressive restriction in progenitor competence.

Human brown adipose tissue (BAT) undergoes progressive involution. This involution process is not recapitulated in rodents, and the underlying mechanisms are poorly understood. Here we show that the interscapular BAT (iBAT) of rabbits whitens rapidly during early adulthood. The transcriptomic remodeling and identity switch of mature adipocytes are accompanied by loss of brown adipogenic competence of progenitors. Single-cell RNA sequencing reveals that rabbit and human iBAT progenitors highly express the FSTL1 gene. When iBAT involutes in rabbits, adipocyte progenitors reduce FSTL1 expression and are refractory to brown adipogenic recruitment. Conversely, FSTL1 is constitutively expressed in mouse iBAT to sustain WNT signaling and prevent involution. Progenitor incompetence and iBAT paucity can be induced in mice by genetic deletion of the Fstl1 gene or ablation of Fstl1+ progenitors. Our results highlight the hierarchy and dynamics of the BAT progenitor compartment and implicate the functional incompetence of FSTL1-expressing progenitors in BAT involution.

Identification of differentially expressed and methylated genes and construction of a co-expression network in age-related macular degeneration

BackgroundAge-related macular degeneration (AMD) is the leading cause of blindness for people over 50 years old worldwide. The purpose of this study was to identify differentially expressed and methylated genes (DEMGs) and construct a co-expression network for AMD.MethodsMicroarray expression (GSE29801 dataset) and DNA methylation (GSE102952 dataset) profiles were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were analyzed between AMD retina tissues and normal retina tissues. A protein-protein interaction (PPI) network was constructed and hub genes were screened, followed by functional enrichment analysis. Then, weighted gene co-expression network analysis (WGCNA) was conducted. The ARPE‐19 cells were maintained in a hypoxic state to construct an AMD cellular model. Enzyme-linked immunosorbent assay (ELISA) and the real-time qPCR (RT-qPCR) were performed for validation.ResultsAfter overlapping, 16 hypermethylated and down-regulated genes and 15 hypomethylated and up-regulated genes were identified for extramacular AMD. A total of 4 hub genes (LMNB2, EMD, HLA-A, and HLA-B) were screened for AMD in the extramacular retina. Furthermore, 13 hypermethylated and down-regulated genes and 31 hypomethylated and up-regulated genes were identified for macular AMD. Among them, 11 hub genes (HLA-A, HLA-B, HLA-DRB1, IFITM3, SAT1, MAOB, CHRDL1, FSTL1, HSPA1A, AR, and YAP1) were considered hub genes. The DEMGs were distinctly related with immune-related biological processes and pathways. A total of 16 co-expression modules were constructed, of which 2 significantly correlated with AMD. The genes in the 2 modules were involved in various crucial signaling pathways. The HIF1α and VEGF levels were significantly up-regulated in cell supernatant of hypoxia-induced ARPE‐19 cells, indicating that the AMD cellular model was successfully established. Hub genes including CHRDL, FSTL1, and IFITM3 displayed significantly higher expression in hypoxia-induced ARPE‐19 cells compared to normal cells. Greater up-regulation of CHRDL, FSTL1, and IFITM3 expression was found in hypoxia-induced ARPE‐19 cells than in normal cells.ConclusionsThese findings offered several key DEMGs and pathways for AMD and constructed AMD-related co-expression modules, deepening understanding of the pathogenesis of AMD.

Follistatin-Like-1 (FSTL1) Is a Fibroblast-Derived Growth Factor That Contributes to Progression of Chronic Kidney Disease.

Our understanding of the mechanisms responsible for the progression of chronic kidney disease (CKD) is incomplete. Microarray analysis of kidneys at 4 and 7 weeks of age in Col4a3-/- mice, a model of progressive nephropathy characterized by proteinuria, interstitial fibrosis, and inflammation, revealed that Follistatin-like-1 (Fstl1) was one of only four genes significantly overexpressed at 4 weeks of age. mRNA levels for the Fstl1 receptors, Tlr4 and Dip2a, increased in both Col4a-/- mice and mice subjected to unilateral ureteral obstruction (UUO). RNAscope® (Advanced Cell Diagnostics, Newark CA, USA) localized Fstl1 to interstitial cells, and in silico analysis of single cell transcriptomic data from human kidneys showed Fstl1 confined to interstitial fibroblasts/myofibroblasts. In vitro, FSTL1 activated AP1 and NFκB, increased collagen I (COL1A1) and interleukin-6 (IL6) expression, and induced apoptosis in cultured kidney cells. FSTL1 expression in the NEPTUNE cohort of humans with focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and IgA nephropathy (IgAN) was positively associated with age, eGFR, and proteinuria by multiple linear regression, as well as with interstitial fibrosis and tubular atrophy. Clinical disease progression, defined as dialysis or a 40 percent reduction in eGFR, was greater in patients with high baseline FSTL1 mRNA levels. FSTL1 is a fibroblast-derived cytokine linked to the progression of experimental and clinical CKD.

FSTL1 increases cisplatin sensitivity in epithelial ovarian cancer cells by inhibition of NF-κB pathway.

To investigate the effects of FSTL1-mediated NF-κB signaling pathway on cisplatin (DDP) sensitivity of EOC cells. FSTL1 expression was determined in epithelial ovarian cancer (EOC) tissues and corresponding adjacent tissues using immunohistochemistry. SKOV3 and SKOV3/DDP cells were transfected and grouped into Blank, Vector, and FSTL1 groups. The sensitivity and 50% inhibitory concentration (IC50) of cells treated with different concentrations of DDP were detected by MTT assay. SKOV3/DDP cells were treated with 20μM DDP, followed by evaluation of cell proliferation, cell apoptosis and determination of NF-κB pathway-related proteins while SKOV3 cells without. FSTL1 expression in EOC tissues and cells was significantly down-regulated, especially decreased in DDP-resistant EOC cells SKOV3/DDP. In SKOV3 cells and SKOV3/DDP cells, the cell viability was reduced and the DDP sensitivity was improved with the decreased IC50 after over-expressing FSTL1. Compared with Blank group, FSTL1 group had declined number of SKOV3 cell colonies and increased cell apoptosis, with obvious up-regulations of FSTL1, Bax/Bcl-2 and cleaved caspase-3 expression and the down-regulations of p-IκBα, p-p65 and survivin expression. Combination of up-regulation of FSTL1 and DDP treatment can also effectively reduce cell colony forming, increase cell apoptosis, and inhibit NF-κB pathway activity of SKOV3/DDP cells. Moreover, this combination can also significantly suppress the growth of subcutaneous xenograft tumors in nude mice. FSTL1 may inhibit NF-κB signaling pathway to suppress the growth and promote the apoptosis of epithelial ovarian cancer cells, and thereby enhancing its DDP sensitivity.

Follistatin-Like 1 Attenuation Suppresses Intervertebral Disc Degeneration in Mice through Interacting with TNF-α and Smad Signaling Pathway.

Background Inflammation plays an important role in intervertebral disc degeneration (IDD). The protein follistatin-like 1 (FSTL1) plays a proinflammatory role in a variety of inflammatory diseases. Objectives The purpose of this study was to investigate whether IDD could be delayed by inhibiting FSTL-1 expression. Methods We established a puncture-induced IDD model in wild-type and FSTL-1+/- mice and collected intervertebral discs (IVDs) from the mice. Safranin O staining was used to detect cartilage loss of IVD tissue, and HE staining was used to detect morphological changes of IVD tissue. We measured the expression of FSTL-1 and related inflammatory indicators in IVD tissues by immunohistochemical staining, real-time PCR, and Western blotting. Results In the age-induced model of IDD, the level of FSTL-1 increased with the exacerbation of degeneration. In the puncture-induced IDD model, FSTL-1-knockdown mice showed a reduced degree of degeneration compared with that of wild-type mice. Further experiments showed that FSTL-1 knockdown also significantly reduced the level of related inflammatory factors in IVD. In vitro experiments showed that FSTL-1 knockdown significantly reduced TNF-α-induced inflammation. Specifically, the expression levels of the inflammatory factors COX-2, iNOS, MMP-13, and ADAMTS-5 were reduced. Knockdown of FSTL-1 attenuated inflammation by inhibiting the expression of P-Smad1/5/8, P-Erk1/2, and P-P65. Conclusion Knockdown of FSTL-1 attenuated inflammation by inhibiting the TNF-α response and Smad pathway activity and ultimately delayed IDD.

Targeting Follistatin like 1 ameliorates liver fibrosis induced by carbon tetrachloride through TGF-\u03b21-miR29a in mice

BackgroundHepatic fibrosis is a pathological response of the liver to a variety of chronic stimuli. Hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. Follistatin like 1 (Fstl1) is a secreted glycoprotein induced by transforming growth factor-β1 (TGF-β1). However, the precise functions and regulation mechanisms of Fstl1 in liver fibrogenesis remains unclear.MethodsHepatic stellate cell (HSC) line LX-2 stimulated by TGF-β1, primary culture of mouse HSCs and a model of liver fibrosis induced by CCl4 in mice was used to assess the effect of Fstl1 in vitro and in vivo.ResultsHere, we found that Fstl1 was significantly up regulated in human and mouse fibrotic livers, as well as activated HSCs. Haplodeficiency of Fstl1 or blockage of Fstl1 with a neutralizing antibody 22B6 attenuated CCl4-induced liver fibrosis in vivo. Fstl1 modulates TGF-β1 classic Samd2 and non-classic JNK signaling pathways. Knockdown of Fstl1 in HSCs significantly ameliorated cell activation, cell migration, chemokines C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 8 (CXCL8) secretion and extracellular matrix (ECM) production, and also modulated microRNA-29a (miR29a) expression. Furthermore, we identified that Fstl1 was a target gene of miR29a. And TGF-β1 induction of Fstl1 expression was partially through down regulation of miR29a in HSCs.ConclusionsOur data suggests TGF-β1-miR29a-Fstl1 regulatory circuit plays a key role in regulation the HSC activation and ECM production, and targeting Fstl1 may be a strategy for the treatment of liver fibrosis.3WAK3Ucq24Ug3VSsxSRHPQVideo Graphical abstract

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown.Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease.Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort.Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function.Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Follistatin-like Protein 1 Inhibits Lung Cancer Metastasis by Preventing Proteolytic Activation of Osteopontin.

Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis, but is downregulated during lung cancer development and progression. The prognostic significance and functional consequences of FSTL1 downregulation in lung cancer are unclear. Here, reduced levels of FSTL1 were detected in various tumors compared with normal tissues and were associated with poor clinical outcome in patients with non-small cell lung cancer, particularly those with lung adenocarcinoma. FSTL1 expression negatively correlated with the metastatic potential of lung cancer cells. Antibody-based neutralization of extracellular FSTL1 increased cellular migration/invasion while addition of recombinant FSTL1 protein diminished the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, treatment with FSTL1 effectively prevented the metastatic progression of lung cancer cells in an orthotopic animal model. Mechanistically, FSTL1 directly bound to the proform of secreted phosphoprotein 1 (SPP1)/osteopontin, restraining proteolytic activation of SPP1, which led to inactivation of integrin/CD44-associated signaling and rearrangement of the actin cytoskeleton. Combined low expression of FSTL1 and high expression of SPP1 predicted a poorer prognosis for patients with lung cancer. This study highlights the novel interaction between FSTL1 and SPP1 and new opportunities to effectively target SPP1-driven metastatic cancers characterized by FSTL1 downregulation. SIGNIFICANCE: These findings describe the novel interaction between FSTL1 and SPP1 and its role in the metastatic progression of lung adenocarcinoma.

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.

Inhibition of MicroRNA-9-5p Protects Against Cardiac Remodeling Following Myocardial Infarction in Mice.

Follistatin-like 1 (Fstl1) protects cardiomyocytes from a broad spectrum of pathologic injuries including myocardial infarction (MI). It is worthy of note that although cardiac Fstl1 is elevated in post-MI microenvironment, its cardioprotective role is still restricted to a limited extent considering the frequency and severity of adverse cardiac remodeling following MI. We therefore propose that intrinsic Fstl1-suppressing microRNA (miRNA) may exist in the heart and its neutralization may further facilitate post-MI recovery. Here, miR-9-5p is predicted as one of the potential Fstl1-targeting miRNAs whose expression is decreased in ischemic myocardium and reversely correlated with Fstl1. Luciferase activity assay further validated Fstl1 as a direct target of miR-9-5p. In addition, forced expression of miR-9-5p in H9c2 cells is concurrent with diminished expression of Fstl1 and vice versa. Importantly, transfection of miR-9-5p mimics in hypoxic H9c2 cells exacerbates cardiac cell death, lactate dehydrogenase release, reactive oxygen species accumulation, and malonyldialdehyde concentration. More importantly, in vivo silencing of miR-9-5p by a specific antagomir in a murine acute MI model effectively preserves post-MI heart function with attenuated fibrosis and inflammatory response. Further studies demonstrated that antagomir treatment stabilizes Fstl1 expression as well as blocks cardiac cell death and reactive oxygen species generation in both ischemia-challenged hearts and hypoxia-treated cardiomyoblasts. Finally, cytoprotection against hypoxic challenge by miR-9-5p inhibitor is partially reversed by knockdown of Fstl1, indicating a novel role of miR-9-5p/Fstl1 axis in survival defense against hypoxic challenge. In summary, these findings identified miR-9-5p as a mediator of hypoxic injury in cardiomyoblasts and miR-9-5p suppression prevents cardiac remodeling after acute MI, providing a potential strategy for early treatment against MI.

Cell type specific expression of Follistatin-like 1 (Fstl1) in mouse embryonic lung development.

Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-β1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα+ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.

TGF-β1 induces Fstl1 via the Smad3-c-Jun pathway in lung fibroblasts.

Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial profibrotic glycoprotein that is upregulated in fibrotic lung tissues, and it promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 upregulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional and translational levels. Although TGF-β1 rapidly activated the Smad, MAPK, and Akt pathways in lung fibroblasts, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. Analysis of the luciferase reporter activity identified a functional c-Jun transcription site in the Fstl1 promoter. Our results suggested a critical role for the Smad3-c-Jun pathway in the regulation of Fstl1 expression by TGF-β1 during fibrogenesis.

Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

Abstract Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including autoimmunity and IL-17 driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17 mediated cytokine production is unknown. To examine the role of FSTL-1 in IL-17 signaling, we utilized parallel in vitro bone marrow stromal cell models of FSTL-1 attenuation. Microarray identified FSTL-1 regulated genes that could influence IL-17 dependent production of IL-6 and G-CSF, wherein we discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. In FSTL-1 KO BMSCs, ectopic Il17rc expression rescued IL-17 stimulated cytokine, and ectopic Fstl1 expression rescued Il17rc expression as well as IL-17 stimulated cytokines. Mechanisms influencing FSTL-1 dependent Il17rc transcript abundance were examined using Actinomycin D and nascent mRNA labeling approaches and identified that FSTL-1 regulates Il17rc gene transcription. Taken together, this work identifies a mechanism by which FSTL-1 influences IL-17 driven inflammation and reveals a novel function for FSTL-1 as a modulator of gene expression. Thus, enhanced understanding of the interplay between FSTL-1 and IL-17 mediated inflammation may provide insight into potential therapeutic targets of IL-17 mediated diseases.

Abstract A24: Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling

Abstract Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis. However, the prognostic significance and functional consequence of FSTL1 downregulation remains unclear in lung cancer. Here we show that the reduced levels of FSTL1 are widely detected in tumors compared to normal tissues and reflect a poor clinical outcome in non-small cell lung cancer, especially lung adenocarcinoma. Moreover, we found that FSTL1 expression negatively correlates with the metastatic potential of lung cancer cells in vitro and in vivo. Whereas the antibody-based neutralization of extracellular FSTL1 enhances the cellular migration/invasion ability, the inclusion of recombinant FSTL1 protein diminishes the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, the administration of FSTL1 was capable of effectively preventing the metastatic progression of lung cancer cells in an orthotropic animal model. The in vitro protein-protein interaction (PPI) assay demonstrated that FSTL1 directly binds to the pro-form of secreted phosphoprotein 1 (SPP1), also named osteopontin (OPN), and thereby restrains the proteolytic activation of SPP1/OPN, which leads to the inactivation of integrin/CD44-associated signaling cascades and actin cytoskeleton. Runx2 and osterix transcription factors are required for the expression of SPP1/OPN. Thus, FSTL1 compromised the SPP1/OPN related translation control activity through PPI mechanism to reverse the phenotype in lung cancer. The signature of combining low-level FSTL1 with high-level SPP1 predicts a worse prognosis in lung cancer patients. This study is the first to document the PPI between FSTL1 and SPP1/OPN and our findings provide a novel and effective strategy to combat SPP1/OPN-driven metastatic cancers with FSTL1 downregulation. Citation Format: Jean Chiou, Yuan-Feng Lin, Michael Hsiao. Translation control of tumor metastasis via FSTL1 binding with SPP1/OPN to modulated cytoskeleton remodeling. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A24.

Follistatin-like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with nitrofen-induced congenital diaphragmatic hernia

Background/purposePulmonary hypoplasia (PH), characterized by incomplete alveolar development, remains a major therapeutic challenge associated with congenital diaphragmatic hernia (CDH). Follistatin-like 1 (Fstl1) is a crucial regulator of alveolar formation and maturation, which is strongly expressed in distal airway epithelium. Fstl1-deficient mice exhibit reduced airspaces, impaired alveolar epithelial cell differentiation, and insufficient production of surfactant proteins similar to PH in human CDH. We hypothesized that pulmonary Fstl1 expression is decreased during alveolarization in the nitrofen-induced CDH model. MethodsTimed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 and divided into control−/nitrofen-exposed specimens. Alveolarization was assessed using morphometric analysis techniques. Pulmonary gene expression of Fstl1 was determined by qRT-PCR. Immunofluorescence-double-staining for Fstl1 and alveolar epithelial marker surfactant protein C (SP-C) was performed to evaluate protein expression/localization. ResultsRadial alveolar count was significantly reduced in hypoplastic lungs of nitrofen-exposed fetuses with significant down regulation of Fstl1 mRNA expression on D18 and D21 compared to controls. Confocal-laser-scanning-microscopy revealed strikingly diminished Fstl1 immunofluorescence and SP-C expression in distal alveolar epithelium of nitrofen-exposed fetuses with CDH-associated PH on D18 and D21 compared to controls. ConclusionsDecreased expression of Fstl1 in alveolar epithelium may disrupt alveolarization and pulmonary surfactant production, thus contributing to the development of PH in the nitrofen-induced CDH model. Level of evidence2b (Centre for Evidence-Based Medicine, Oxford).

A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth.

SPARC is a matricellular protein that is involved in both pancreatic cancer and diabetes. It belongs to a wider family of proteins that share structural and functional similarities. Relatively little is known about this extended family, but evidence of regulatory interactions suggests the importance of a holistic approach to their study. We show that Hevin, SPOCKs, and SMOCs are strongly expressed within islets, ducts, and blood vessels, suggesting important roles for these proteins in the normal pancreas, while FSTL-1 expression is localised to the stromal compartment reminiscent of SPARC. In direct contrast to SPARC, however, FSTL-1 expression is reduced in pancreatic cancer. Consistent with this, FSTL-1 inhibited pancreatic cancer cell proliferation. The complexity of SPARC family proteins is further revealed by the detection of multiple cell-type specific isoforms that arise due to a combination of post-translational modification and alternative splicing. Identification of splice variants lacking a signal peptide suggests the existence of novel intracellular isoforms. This study underlines the importance of addressing the complexity of the SPARC family and provides a new framework to explain their controversial and contradictory effects. We also demonstrate for the first time that FSTL-1 suppresses pancreatic cancer cell growth.

Follistatin-like 1 promotes cardiac fibroblast activation and protects the heart from rupture.

Follistatin‐like 1 (Fstl1) is a secreted protein that is acutely induced in heart following myocardial infarction (MI). In this study, we investigated cell type‐specific regulation of Fstl1 and its function in a murine model of MI. Fstl1 was robustly expressed in fibroblasts and myofibroblasts in the infarcted area compared to cardiac myocytes. The conditional ablation of Fstl1 in S100a4‐expressing fibroblast lineage cells (Fstl1‐cfKO mice) led to a reduction in injury‐induced Fstl1 expression and increased mortality due to cardiac rupture during the acute phase. Cardiac rupture was associated with a diminished number of myofibroblasts and decreased expression of extracellular matrix proteins. The infarcts of Fstl1‐cfKO mice displayed weaker birefringence, indicative of thin and loosely packed collagen. Mechanistically, the migratory and proliferative capabilities of cardiac fibroblasts were attenuated by endogenous Fstl1 ablation. The activation of cardiac fibroblasts by Fstl1 was mediated by ERK1/2 but not Smad2/3 signaling. This study reveals that Fstl1 is essential for the acute repair of the infarcted myocardium and that stimulation of early fibroblast activation is a novel function of Fstl1.