HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Biovalorization of mango byproduct through enzymatic extraction of dietary fiber.

Mango is considered one of the most important tropical fruits worldwide in terms of its consumption and consumer acceptability. Its processing generates huge quantities of mango byproducts, which is often discarded unscrupulously into the environment and, therefore, needs effective waste management practices. The extraction of mango peels' dietary fiber using enzymatic method can be a useful valorization strategy for management of mango by-products. In the present investigation, dietary fiber (soluble and insoluble fraction) was extracted by enzymatic hydrolysis using α-amylase, protease, and amyloglucosidase. Highest yield of dietary fiber (67.5%, w/w) was obtained at 60 °C temperature using recommended enzyme concentrations including α-amylase (40 µL), protease (110 µL), and amyloglucosidase (200 µL) after a treatment time of 60 min. SEM analysis indicated the increased porosity of dietary fiber samples caused due to the hydrolytic effect of enzymes on its surface structure, whereas FTIR analysis confirmed the functional groups present in dietary fiber. The coexistence of crystalline and amorphous nature of polymers present in soluble and insoluble fractions of dietary fiber was assessed by XRD analysis. Further, the analysis of functional properties including WHC, OHC, and SC revealed the suitability of using extracted mango peel's dietary fiber in the food systems.

Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

Determination of Sexual Dimorphism of the Human Sacrum Based on Receiver Operating Characteristic Curve Analysis of Morphometric Parameters.

Background Sex estimation of unidentified incomplete skeletons poses a challenge to paleoanthropologists and forensic experts. The sacrum is a part of the axial skeleton and contributes to the formation of the pelvic girdle. It is a significant bone for the identification of the sex in the human skeletal system due to associated functional differences of the pelvic bones in males and females. However, there is a lack of cognizance of different morphometric parameters of the sacrum which may be crucial for determining sex, particularly when a part of the bone is available. This study aimed to recognize the best morphometric parameters for the identification of the sex of the sacrum even when fragmented bones were available and compare the various parameters for sexual dimorphism in different populations. Methodology The study was conducted on 110 dry adult human sacra in the anatomy department. Out of these, 42 sacra were female and 68 were male. Morphometric measurements were performed with the help of a digital vernier caliper. Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Morphometric measurements of male and female sacra were compared using Student's t-test. The receiver operating characteristic (ROC) curve analysis was performed to establish the most appropriate cut-off values for each parameter. Results The mean sacral length measured from the promontory to the apex of the sacrum was higher in males compared to females (p < 0.001), whereas the sacral index was higher in female sacra in comparison to male sacra (p < 0.001). Furthermore, the mean height of the first posterior sacral foramina (PSF) was higher in male sacra bilaterally (p < 0.05). On ROC analysis, the area under the curve was 0.994 for the sacral index and 0.862 for the sacral length. Conclusions In this study, the sacral index was noted to be the most important morphometric parameter for the identification of the sex of the sacra. Additionally, the height of the S2 body, the height of the first anterior sacral foramina, and the height of the first PSF can be contemplated with an accuracy of 60-70% if only a part of the sacrum is available for determining the sex. Hence, this study emphasizes the significance of morphometric parameters of the sacrum in the determination of sex, especially in forensic cases when the skull and pelvis are fragmented or unavailable.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

SOX9 reprograms endothelial cells by altering the chromatin landscape.

The transcription factor SOX9 is activated at the onset of endothelial-to-mesenchymal transition (EndMT) during embryonic development and in pathological conditions. Its roles in regulating these processes, however, are not clear. Using human umbilical vein endothelial cells (HUVECs) as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal genes and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 displays features of a pioneer transcription factor, such as opening of chromatin and leading to deposition of active histone modifications at silent chromatin regions, guided by SOX dimer motifs and H2A.Z enrichment. We further observe highly transient and dynamic SOX9 binding, possibly promoted through its eviction by histone phosphorylation. However, while SOX9 binding is dynamic, changes in the chromatin landscape and cell fate induced by SOX9 are persistent. Finally, our analysis of single-cell chromatin accessibility indicates that SOX9 opens chromatin to drive EndMT in atherosclerotic lesions in vivo. This study provides new insight into key molecular functions of SOX9 and mechanisms of EndMT and highlights the crucial developmental role of SOX9 and relevance to human disease.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Role of inflammatory markers and their trends in predicting the outcome of medical expulsive therapy for distal ureteric calculus.

OBJECTIVE: Symptomatic ureteric stones cause surrounding inflammation-promoting obstruction. C-reactive protein (CRP), white blood cell count (WC), and neutrophil percentage (NP) tend to rise after inflammatory response. Monitoring response during the course of medical expulsive therapy (MET) may help in deciding early intervention, thereby decreasing morbidity. We assessed the role and trends of these markers in predicting the outcome of MET. MATERIALS AND METHODS: One hundred and ninety-two patients with distal ureteric calculus of size >5 mm were included in this prospective study from April 2017 to March 2018 after ethical committee approval. CRP, NP, and WC were measured on day 1, 7, and 14 of MET, and analysis was done. RESULTS: On univariate analysis, stone size and mean values of CRP, WC, and NP on day 1, 7 and 14 in stone nonpassers were significantly higher compared to stone passers (P < 0.05). Receiver operator curve analysis showed area under the curve value of 0.798 (P = 0.001) for CRP and cut off value determined was 1.35 mg/dL. Multivariate analysis of different variables showed significant association of higher CRP (>1.35 mg/dL) and larger stone size (>7 mm) with MET failure. Decreasing trend of CRP was seen in both groups, but values were higher in stone nonpassers. WC and NP showed decreasing trend in stone passers but persistently high in stone nonpassers. CONCLUSIONS: Higher CRP and larger stone size were associated with failure of MET. WC and NP showed decreasing trend in stone passers and persistently higher in nonpassers, which may potentially predict failure of MET, however, their role need to be further studied.

Current noise of a protein-selective biological nanopore.

1/f current noise is ubiquitous in protein pores, porins, and channels. We have previously shown that a protein-selective biological nanopore with an external protein receptor can function as a 1/f noise generator when a high-affinity protein ligand is reversibly captured by the receptor. Here, we demonstrate that the binding affinity and concentration of the ligand are key determinants for the nature of current noise. For example, 1/f was absent when a protein ligand was reversibly captured at a much lower concentration than its equilibrium dissociation constant against the receptor. Furthermore, we also analyzed the composite current noise that resulted from mixtures of low-affinity and high-affinity ligands against the same receptor. This study highlights the significance of protein recognition events in the current noise fluctuations across biological membranes.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

Association of circulating monocyte chemoattractant protein-1 levels with polycystic ovary syndrome: A meta-analysis.

PROBLEM: Polycystic ovary syndrome (PCOS) is a common hormonal disorder that has a huge impact on the human infertility. Increased levels of various circulating inflammatory cytokines have been observed in PCOS patients, which can contribute to the pathogenesis of PCOS. Monocyte chemoattractant protein-1 (MCP-1), a secretory chemokine, is a potent chemotactic factor that recruits monocytes/macrophages to inflammatory foci. Several previous studies comparing the circulating MCP-1 levels between non-PCOS and PCOS patients have yielded contradictory results. Therefore, the aim of this meta-analysis was to investigate whether circulating MCP-1 levels vary between non-PCOS and PCOS patients. METHODS: Research articles published before November 11, 2020, were screened to identify eligible studies. Heterogeneity, sensitivity, and publication bias were analyzed using STATA software. Standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated by the STATA software using a random-effects model. RESULTS: 11 studies were included in this meta-analysis involving 897 individuals: 368 non-PCOS patient and 529 PCOS patients. Our pooled meta-analysis results show that circulating MCP-1 levels were significantly higher in PCOS patients than in non-PCOS patients (SMD = 0.84, 95% CI = [0.37, 1.31], Z = 3.50, p < 0.01). However, due to the limited number of studies included in this meta-analysis, subgroup analysis determined that circulating MCP-1 levels were not significantly varied between obese non-PCOS and obese PCOS patients (SMD = 0.42, 95% CI = [-0.65, 1.49], Z = 0.77, p = 0.442) as well as between non-PCOS and PCOS patients without obesity (SMD = 2.04, 95% CI = [-0.84, 4.93], Z = 1.39, p = 0.166). In addition, circulating MCP-1 levels were also not significantly different between obese and non-obese PCOS patients (SMD = -0.04, 95% CI = [-0.68, 0.60], Z = 0.11, p = 0.909). CONCLUSION: Our findings reveal that circulating MCP-1 levels are upregulated in women with PCOS and are associated with an increased risk of PCOS.

Protein Ligand-Induced Amplification in the 1/f Noise of a Protein-Selective Nanopore.

Previous studies of transmembrane protein channels have employed noise analysis to examine their statistical current fluctuations. In general, these explorations determined a substrate-induced amplification in the Gaussian white noise of these systems at a low-frequency regime. This outcome implies a lack of slowly appearing fluctuations in the number and local mobility of diffusing charges in the presence of channel substrates. Such parameters are among the key factors in generating a low-frequency 1/f noise. Here, we show that a protein-selective biological nanopore exhibits a substrate-induced amplification in the 1/f noise. The modular composition of this biological nanopore includes a hydrophilic transmembrane protein pore fused to a water-soluble binding protein on its extramembranous side. In addition, this protein nanopore shows an open substate populated by a high-frequency current noise because of the flickering of an engineered polypeptide adaptor at the tip of the pore. However, the physical association of the protein ligand with the binding domain reversibly switches the protein nanopore from a high-frequency noise substate into a quiet substate. In the absence of the protein ligand, our nanopore shows a low-frequency white noise. Remarkably, in the presence of the protein ligand, an amplified low-frequency 1/f noise was detected in a ligand concentration-dependent fashion. This finding suggests slowly occurring equilibrium fluctuations in the density and local mobility of charge carriers under these conditions. Furthermore, we report that the excess in 1/f noise is generated by reversible switches between the noisy ligand-released substate and the quiet ligand-captured substate. Finally, quantitative aspects of the low-frequency 1/f noise are in accord with theoretical predictions of the current noise analysis of protein channel-ligand interactions.

TGF-ß1 induces VEGF expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is one of the most serious and iatrogenic complications that can occur during in vitro fertilization treatment. Although the pathogenesis of OHSS is not fully understood, vascular endothelial growth factor (VEGF) has been recognized as an important mediator of the development of OHSS. Transforming growth factor-beta-1 (TGF-ß1) is known to regulate various ovarian functions. However, whether VEGF can be regulated by TGF-ß1 in human granulosa cells has not been determined. In addition, the role of TGF-ß1 in the pathogenesis of OHSS remains unknown. In the present study, we demonstrate that TGF-ß1 stimulates VEGF expression in and secretion from both immortalized human granulosa-lutein (hGL) cells and primary hGL cells. Our results demonstrate that the SMAD2/3, ERK1/2, and p38 MAPK signaling pathways are involved in TGF-ß1-induced VEGF expression and secretion. Using a mouse OHSS model, we show that the expression levels of TGF-ß1 and VEGF are increased in the ovaries of OHSS mice. Blocking TGF-ß1 signaling inhibits the development of OHSS by attenuating VEGF expression. Moreover, clinical results reveal that the protein levels of TGF-ß1 and VEGF are increased in the follicular fluid of patients with OHSS, and that the levels of these two proteins in the follicular fluid are positively correlated. The results of this study help to elucidate the mechanisms by which VEGF expression is regulated in hGL cells, which could lead to the development of alternative therapeutic approaches for treating OHSS.

Single-Molecule Protein Detection in a Biofluid Using a Quantitative Nanopore Sensor.

Protein detection in complex biological fluids has wide-ranging significance across proteomics and molecular medicine. Existing detectors cannot readily distinguish between specific and nonspecific interactions in a heterogeneous solution. Here, we show that this daunting shortcoming can be overcome by using a protein bait-containing biological nanopore in mammalian serum. The capture and release events of a protein analyte by the tethered protein bait occur outside the nanopore and are accompanied by uniform current openings. Conversely, nonspecific pore penetrations by nontarget components of serum, which take place inside the nanopore, are featured by irregular current blockades. As a result of this unique peculiarity of the readout between specific protein captures and nonspecific pore penetration events, our selective sensor can quantitatively sample proteins at single-molecule precision in a manner distinctive from those employed by prevailing methods. Because our sensor can be integrated into nanofluidic devices and coupled with high-throughput technologies, our approach will have a transformative impact in protein identification and quantification in clinical isolates for disease prognostics and diagnostics.

Repressive Epigenetic Signatures Safeguard the Liver.

To date, how epigenetic changes are regulated during liver regeneration remains unclear. In this issue of Developmental Cell, Wang and colleagues (2019) employed transcriptomic and epigenomic profiling to explore how Uhrf1, an epigenetic regulator of DNA methylation, functions in liver regeneration using a mouse model of partial hepatectomy.

Factors Associated with the Development of Secondary Multidrug-resistant Tuberculosis.

BACKGROUND: Spread of multidrug-resistant tuberculosis (TB) is a threat to India's TB control program. We conducted this study with the objective to determine the risk factors for the development of secondary multidrug-resistant TB. METHODS: We conducted an unmatched case-control study involving 247 multidrug-resistant TB patients as "cases" and 494 individuals who were declared as "cured" after category I DOTS treatment as "controls." Data were collected through face-to-face interviews and review of treatment records. Multivariable logistic regressions were used to analyze the collected data. RESULTS: The mean duration for which cases took first-line anti-TB drug was 19.7 months. The mean duration between initial diagnosis of TB and diagnosis of multi-drug resistant TB (MDR-TB) was 28.3 months. In our study, 26.7%, 50.2%, and 23.1% of MDR-TB cases had one, two, or more previous episodes of TB before being diagnosed as MDR-TB. In multivariable analysis, low or no formal education (album-oriented rock [AOR] =1.63 [confidence interval (CI) = 1.03-3.11]), labor occupation (AOR = 2.15 [CI = 1.18-3.90]), smoking (AOR = 2.56 [CI = 1.19-3.26]), having HIV (AOR = 9.45 [CI = 6.80-15.9]), migration for job (AOR = 3.70 [CI = 1.96-5.67]), stopping TB treatment due to comorbid conditions (AOR = 8.86 [CI = 5.45-11.2]), and having type 2 diabetes (AOR = 3.4 [CI = 1.96-5.16]) were associated with MDR-TB. CONCLUSIONS: Government of India should devise strategy to prevent interruption of treatment to stop the emergence and spread of MDR-TB. We need to better integrate TB control activities with diabetes and tobacco control programs for better health outcome among patients.

Hepatocyte Nuclear Factor 4-Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver.

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.

Real-time measurement of protein-protein interactions at single-molecule resolution using a biological nanopore.

Protein-protein interactions (PPIs) are essential for many cellular processes. However, transient PPIs are difficult to measure at high throughput or in complex biological fluids using existing methods. We engineered a genetically encoded sensor for real-time sampling of transient PPIs at single-molecule resolution. Our sensor comprises a truncated outer membrane protein pore, a flexible tether, a protein receptor and a peptide adaptor. When a protein ligand present in solution binds to the receptor, reversible capture and release events of the receptor can be measured as current transitions between two open substates of the pore. Notably, the binding and release of the receptor by a protein ligand can be unambiguously discriminated in a complex sample containing fetal bovine serum. Our selective nanopore sensor could be applied for single-molecule protein detection, could form the basis for a nanoproteomics platform or might be adapted to build tools for protein profiling and biomarker discovery.

YAP transcriptionally regulates ErbB2 to promote liver cell proliferation.

The Hippo signaling pathway is implicated in regulation of liver size and dysregulation of this pathway contributes to tumorigenesis. The transcriptional targets and downstream pathways of the Hippo pathway effector YAP that contribute to liver growth have yet to be well-characterized. We examined the liver transcriptome in response to YAP overexpression and identify the ErbB signaling pathway as a mediator of cell growth downstream of YAP. ErbB2 is transcriptionally regulated by YAP in both the mouse liver and in HepG2 human hepatoma cells. Knockdown of YAP or pharmacological inhibition with verteporfin reduced ERBB2 levels in HepG2 cells. Analysis of ChIP-seq data revealed enrichment of the transcription factor TEAD4 at the ERBB2 promoter. Using luciferase reporter and chromatin immunoprecipitation assays, we show that YAP and TEAD4 directly bind to and activate a regulatory element in the ErbB2 promoter in both the mouse liver and HepG2 cells. Functionally, knockdown of YAP reduced EGF-induced ERBB2-mediated HepG2 cell proliferation and PI3K/AKT activation. Our findings highlight a mechanism by which YAP exerts its effects on liver cell proliferation through the ErbB signaling pathway by directly regulating the transcription of ErbB2.

S1P Stimulates Proliferation by Upregulating CTGF Expression through S1PR2-Mediated YAP Activation.

Dysregulation of the Hippo pathway in the liver results in overgrowth and eventually tumorigenesis. To date, several upstream mechanisms have been identified that affect the Hippo pathway, which ultimately regulate YAP, the major downstream effector of the pathway. However, upstream regulators of the Hippo pathway in the liver remain poorly defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been shown to stimulate hepatocellular carcinoma (HCC) cell proliferation, but whether the Hippo pathway is involved in S1P-stimulated HCC cell proliferation remains to be determined. Here it is demonstrated that S1P activates YAP and that the S1P receptor 2 (S1PR2/S1P2) mediates S1P-induced YAP activation in both human and mouse HCC cells. S1P promotes YAP-mediated upregulation of cysteine-rich protein 61 and connective tissue growth factor (CTGF), and stimulates HCC cell proliferation. By using siRNA-mediated knockdown approaches, only CTGF was required for S1P-stimulated cell proliferation. Of note, S1P activates YAP in a MST1/2-independent manner suggesting that the canonical Hippo kinase is not required for S1P-mediated proliferation in liver. The upregulation of CTGF and S1P2 were also observed in liver-specific YAP overexpression transgenic mouse hepatocytes. Moreover, YAP regulated liver differentiation-dependent gene expression by influencing the chromatin binding of HNF4α based on ChIP-seq analysis. Finally, results using gain- and loss-of-function approaches demonstrate that HNF4α negatively regulated S1P-induced CTGF expression.Implications: These findings reveal a role for S1P in stimulating HCC cell proliferation by upregulating CTGF expression through S1P2-mediated YAP activation. Mol Cancer Res; 16(10); 1543-55. ©2018 AACR.