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Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.

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Mechanistic target of rapamycin complex 1 (mTORC1) is a serine-threonine kinase that is activated by extracellular signals, such as nutrients and growth factors. It plays a key role in the control of various biological processes, such as protein synthesis and energy metabolism by mediating or regulating the phosphorylation of multiple target molecules, some of which remain to be identified. We have here reanalysed a large-scale phosphoproteomics data set for mTORC1 target molecules and identified pre-B cell leukemia transcription factor 2 (PBX2) as such a novel target that is dephosphorylated downstream of mTORC1. We confirmed that PBX2, but not other members of the PBX family, is dephosphorylated in an mTORC1 activity-dependent manner. Furthermore, pharmacological and gene knockdown experiments revealed that glycogen synthase kinase 3 (GSK3) and protein phosphatase 1 (PP1) are responsible for the phosphorylation and dephosphorylation of PBX2, respectively. Our results thus suggest that the balance between the antagonistic actions of GSK3 and PP1 determines the phosphorylation status of PBX2 and its regulation by mTORC1.

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Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of brain tumors, but the role of PRMT1 in medulloblastoma, the most common malignant pediatric brain tumor, remains unexplored. By examining the publicly available databases of pediatric brain tumor collection, we found that PRMT1 was predominantly expressed in medulloblastomas across all the pediatric brain tumors and that the high-level expression of PRMT1 correlated with poor survival of medulloblastoma patients. To determine the role of PRMT1 in medulloblastoma cells, we established an inducible knockdown system and demonstrated that PRMT1 depletion decreased medulloblastoma cell proliferation and induced cell apoptosis. Furthermore, the diamidine compounds, previously shown to exhibit selective PRMT1 inhibition, suppressed medulloblastoma cell viability in a dose-dependent manner. Finally, we observed induction of medulloblastoma cell apoptosis by the potent diamidine compounds at low micromolar concentrations. Together, our results suggest that PRMT1 could be an actionable therapeutic target in medulloblastoma.

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Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction. Protocatechuic aldehyde (PCA) is a major phenolic acid in Chinese herb Danshen (Salvia miltiorrhiza root). This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2 (PKM2) function to protect cardiomyocytes. In rats subjected to isoprenaline, PCA attenuated heart injury and protected cardiomyocytes from apoptosis. Through DARTS and CETSA assays, we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation (OGD). In the nucleus, PCA increased the binding of PKM2 to ß-catenin via preserving PKM2 acetylation, and the complex, in cooperation with T-cell factor 4 (TCF4), was required for transcriptional induction of genes encoding anti-apoptotic proteins, contributing to rescuing cardiomyocyte survival. In addition, PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2. Consistently, PCA increased the binding of PKM2 to ß-catenin, improved heart contractive function, normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation, but the protective effects were lost in Pkm2-deficient heart. Together, we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via ß-catenin/TCF4 signaling cascade, suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.

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The invasive plant Mikania micrantha Kunth (M. micrantha) from South America poses a significant threat to the stability and biodiversity of ecosystems. However, an effective and economical method to control M. micrantha is still lacking. RNA interference (RNAi) has been widely studied and applied in agriculture for trait improvement. Spray-induced gene silencing (SIGS) can produce RNAi silencing effects without introducing heritable modifications to the plant genome and is becoming a novel nontransformation strategy for plant protection. In this study, the genes encoding chlorophyll a/b-binding proteins were selected as targets of RNAi, based on high-throughput sequencing of M. micrantha transcriptome and bioinformatic analyses of sequence specificity. Three types of RNAi molecules, double-stranded RNA, RNAi nanomicrosphere, and short hairpin RNA (shRNA), with their corresponding short interfering RNA sequences were designed and synthesized for SIGS vector construction, from which each RNAi molecule was transcribed and extracted to be sprayed on M. micrantha leaves. Whereas water-treated control leaves remained green, leaves treated with RNAi molecules turned yellow and eventually wilted. Quantitative real-time PCR showed that the expression levels of target genes were significantly reduced in the RNAi-treated groups compared with those of the control, suggesting that all three types of RNAi herbicides effectively silenced the endogenous target genes, which are essential for the growth of M. micrantha. We also found that shRNA showed better silencing efficiency than the other two molecules. Taken together, our study successfully designed three types of RNAi-based herbicides that specifically silenced endogenous target genes and controlled the growth of M. micrantha. Moreover, we identified a gene family encoding chlorophyll a/b-binding proteins that is important for the growth and development of M. micrantha and could serve as potential targets for controlling the spread of M. micrantha.

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The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.

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Limb and CNS expressed 1 like (LIX1L) is over-expressed in several types of tumors. However, the function of LIX1L in glucose metabolism and hepatocellular carcinoma (HCC) progression remains elusive. Here we report that LIX1L is over-expressed in human HCC tissues, which predicts unfavorable prognosis. LIX1L deficiency in vivo significantly attenuated liver cancer initiation in mice. Functional studies indicated that LIX1L overexpression elevated proliferation, migratory, invasive capacities of HCC cells in vitro, and promoted liver cancer growth and metastasis in vivo. LIX1L knockdown up-regulated fructose-1,6-bisphosphatase (FBP1) expression to reduce glucose consumption as well as lactate production. Mechanistically, LIX1L increased miR-21-3p expression, which targeted and suppressed FBP1, thereby promoting HCC growth and metastasis. MiR-21-3p inhibitor could abrogate LIX1L induced enhancement of cell migration, invasion, and glucose metabolism. Inhibition of miR-21-3p suppressed tumor growth in an orthotopic tumor model. Our results establish LIX1L as a critical driver of hepatocarcinogenesis and HCC progression, with implications for prognosis and treatment.

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This study was aimed to design the first dual-target small-molecule inhibitor co-targeting poly (ADP-ribose) polymerase-1 (PARP1) and bromodomain containing protein 4 (BRD4), which had important cross relation in the global network of breast cancer, reflecting the synthetic lethal effect. A series of new BRD4 and PARP1 dual-target inhibitors were discovered and synthesized by fragment-based combinatorial screening and activity assays that together led to the chemical optimization. Among these compounds, 19d was selected and exhibited micromole enzymatic potencies against BRD4 and PARP1, respectively. Compound 19d was further shown to efficiently modulate the expression of BRD4 and PARP1. Subsequently, compound 19d was found to induce breast cancer cell apoptosis and stimulate cell cycle arrest at G1 phase. Following pharmacokinetic studies, compound 19d showed its antitumor activity in breast cancer susceptibility gene 1/2 (BRCA1/2) wild-type MDA-MB-468 and MCF-7 xenograft models without apparent toxicity and loss of body weight. These results together demonstrated that a highly potent dual-targeted inhibitor was successfully synthesized and indicated that co-targeting of BRD4 and PARP1 based on the concept of synthetic lethality would be a promising therapeutic strategy for breast cancer.

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BACKGROUND & AIMS: The paradox of hepatic insulin resistance describes the inability for liver to respond to bioenergetics hormones in suppressing gluconeogenesis whilst maintaining lipid synthesis. Here, we report the deficiency of miR-192-3p in the livers of mice with diabetes and its role in alleviating hepatic steatosis. METHODS: As conventional pre-microRNA (miRNA) stem-loop overexpression only boosts guiding strand (i.e. miR-192-5p) expression, we adopted an artificial AAV(DJ)-directed, RNA Pol III promoter-driven miRNA hairpin construct for star-strand-specific overexpression in the liver. Liver steatosis and insulin resistance markers were evaluated in primary hepatocytes, mice with diabetes, and mice with excessive carbohydrate consumption. RESULTS: Functional loss of miR-192-3p in liver exacerbated hepatic micro-vesicular steatosis and insulin resistance in either mice with diabetes or wild-type mice with excessive fructose consumption. Liver-specific overexpression of miR-192-3p effectively halted hepatic steatosis and ameliorated insulin resistance in these mice models. Likewise, hepatocytes overexpressing miR-192-3p exhibited improved lipid accumulation, accompanied with decreases in lipogenesis and lipid-accumulation-related transcripts. Mechanistically, glucocorticoid receptor (GCR, also known as nuclear receptor subfamily 3, group C, member 1 [NR3C1]) was demonstrated to be negatively regulated by miR-192-3p. The effect of miR-192-3p on mitigating micro-vesicular steatosis was ablated by the reactivation of NR3C1. CONCLUSIONS: The star strand miR-192-3p was an undermined glycerolipid regulator involved in controlling fat accumulation and insulin sensitivity in liver through blockade of hepatic GCR signalling; this miRNA may serve as a potential therapeutic option for the common co-mobility of diabetic mellitus and fatty liver disease. LAY SUMMARY: The potential regulatory activity of star strand microRNA (miRNA) species has been substantially underestimated. In this study, we investigate the role and mechanism of an overlooked star strand miRNA (miR-192-3p) in regulating hepatic steatosis and insulin signalling in the livers of mice with diabetes and mice under excessive carbohydrate consumption.

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Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy.

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We have previously shown that high expression of the nucleic acid binding factor YB-1 is strongly associated with poor prognosis in a variety of cancer types. The 3-dimensional protein structure of YB-1 has yet to be determined and its role in transcriptional regulation remains elusive. Drug targeting of transcription factors is often thought to be difficult and there are very few published high-throughput screening approaches. YB-1 predominantly binds to single-stranded nucleic acids, adding further difficulty to drug discovery. Therefore, we have developed two novel screening assays to detect compounds that interfere with the transcriptional activation properties of YB-1, both of which may be generalizable to screen for inhibitors of other nucleic acid binding molecules. The first approach is a cell-based luciferase reporter gene assay that measures the level of activation of a fragment of the E2F1 promoter by YB-1. The second approach is a novel application of the AlphaScreen system, to detect interference of YB-1 interaction with a single-stranded DNA binding site. These complementary assays examine YB-1 binding to two discrete nucleic acid sequences using two different luminescent signal outputs and were employed sequentially to screen 7360 small molecule compounds leading to the identification of three putative YB-1 inhibitors.

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BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in migraine pathophysiology. CGRP acts primarily by activating a receptor composed of 3 proteins: calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP). We tested the hypothesis that sex differences exist in protein levels of two key components of this CGRP receptor: CLR and RCP. METHODS: We used specific antibodies to assess baseline protein levels of CLR and RCP in the spinal trigeminal nucleus caudalis (SpVc) and upper cervical spinal cord of both male and female rats. We also tested if manipulations that knock-down the expression of RCP in SpVc, using locally-mediated gene transfer of short hairpin RNA (shRNA), ameliorate pain in an animal model of intracranial migraine-like pain induced by chemical noxious stimulation of the meninges. To assess pain, we used tests of ongoing pain (rat face grimace test and freezing behavior) and tests of facial mechanical hypersensitivity and allodynia. RESULTS: There was no difference in CLR levels between male and female animals (p > 0.11) in SpVc and the upper cervical cord. However, female animals exhibited greater baseline levels of RCP (up to 3-fold higher) compared to males (p < 0.002). The knock-down of RCP expression in SpVc attenuated mechanical facial allodynia induced by chemical noxious stimulation of the meninges, but had little effect on ongoing pain behaviors in female and male animals. CONCLUSIONS: RCP is an integral component of the CGRP receptor and may play a key role in mediating CGRP induced central sensitization after noxious stimulation of the meninges. RCP expression in the SpVc and upper cervical cord is sexually dimorphic, with higher levels of expression in females. This dimorphism may be related to the increased incidence of migraines in females-a hypothesis that should be tested in the future.

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Background & Aims: Recent studies have shown that cancers arise as a result of the positive selection of driver somatic events in tumor DNA, with negative selection playing only a minor role, if any. However, these investigations were concerned with alterations at nonrepetitive sequences and did not take into account mutations in repetitive sequences that have very high pathophysiological relevance in the tumors showing microsatellite instability (MSI) resulting from mismatch repair deficiency investigated in the present study. Methods: We performed whole-exome sequencing of 47 MSI colorectal cancers (CRCs) and confirmed results in an independent cohort of 53 MSI CRCs. We used a probabilistic model of mutational events within microsatellites, while adapting pre-existing models to analyze nonrepetitive DNA sequences. Negatively selected coding alterations in MSI CRCs were investigated for their functional and clinical impact in CRC cell lines and in a third cohort of 164 MSI CRC patients. Results: Both positive and negative selection of somatic mutations in DNA repeats was observed, leading us to identify the expected true driver genes associated with the MSI-driven tumorigenic process. Several coding negatively selected MSI-related mutational events (n = 5) were shown to have deleterious effects on tumor cells. In the tumors in which deleterious MSI mutations were observed despite the negative selection, they were associated with worse survival in MSI CRC patients (hazard ratio, 3; 95% CI, 1.1-7.9; P = .03), suggesting their anticancer impact should be offset by other as yet unknown oncogenic processes that contribute to a poor prognosis. Conclusions: The present results identify the positive and negative driver somatic mutations acting in MSI-driven tumorigenesis, suggesting that genomic instability in MSI CRC plays a dual role in achieving tumor cell transformation. Exome sequencing data have been deposited in the European genome-phenome archive (accession: EGAS00001002477).

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BACKGROUND & AIMS: Oncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines. METHODS: RNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated ß-galactosidase activity assays identified cellular senescence. RESULTS: E47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated ß-galactosidase activity and altered expression of senescence markers. CONCLUSIONS: E47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327.

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BACKGROUND & AIMS: Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP- and SNX9-dependent pathway. METHODS: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and Citrobacter rodentium strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions. RESULTS: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction. CONCLUSIONS: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.

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Rheumatoid arthritis (RA) is the most common complex multifactorial joint related autoimmune inflammatory disease with unknown etiology accomplished with increased cardiovascular risks. RA is characterized by the clinical findings of synovial inflammation, autoantibody production, and cartilage/bone destruction, cardiovascular, pulmonary and skeletal disorders. Pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and IL-10 were responsible for the induction of inflammation in RA patients. Drawbacks such as poor efficacy, higher doses, frequent administration, low responsiveness, and higher cost and serious side effects were associated with the conventional dosage forms for RA treatment. Nanomedicines were recently gaining more interest towards the treatment of RA, and researchers were also focusing towards the development of various anti-inflammatory drug loaded nanoformulations with an aid to both actively/passively targeting the inflamed site to afford an effective treatment regimen for RA. Alterations in the surface area and nanoscale size of the nanoformulations elicit beneficial physical and chemical properties for better pharmacological activities. These drug loaded nanoformulations may enhances the solubility of poorly water soluble drugs, improves the bioavailability, affords targetability and may improve the therapeutic activity. In this regimen, the present review focus towards the novel nanoparticulate formulations (nanoparticles, nanoemulsions, solid lipid nanoparticles, nanomicelles, and nanocapsules) utilized for the treatment of RA. The recent advancements such as siRNA, peptide and targeted based nanoparticulate systems for RA treatment were also discussed. Special emphasis was provided regarding the pathophysiology, prevalence and symptoms towards the development of RA.

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Bone represents a common site of metastasis from several solid tumours, including breast, prostate and lung malignancies. The onset of bone metastases (BM) is associated not only with serious skeletal complications, but also shortened overall survival, owing to the lack of curative treatment options for late-stage cancer. Despite the diagnostic advances, BM detection often occurs in the symptomatic stage, underlining the need for novel strategies aimed at the early identification of high-risk patients. To this purpose, both bone turnover and tumour-derived markers are being investigated for their potential diagnostic, prognostic and predictive roles. In this review, we summarize the pathogenesis of BM in breast, prostate and lung tumours, while exploring the current research focused on the identification and clinical validation of BM biomarkers.

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The pregnane X receptor (PXR) plays an important and diverse role in mediating xenobiotic induction of drug-metabolizing enzymes and transporters. Several protein isoforms of PXR exist, and they have differential transcriptional activity upon target genes; transcript variants 3 (PXR3) and 4 (PXR4) do not induce target gene expression, whereas transcript variants 1 (PXR1) and 2 (PXR2) respond to agonist by activating target gene expression. PXR protein variants also display differences in protein-protein interactions; PXR1 interacts with p53, whereas PXR3 does not. Furthermore, the transcript variants of PXR that encode these protein isoforms are differentially regulated by methylation and deletions in the respective promoters of the variants, and their expression differs in various human cancers and also in cancerous tissue compared to adjacent normal tissues. PXR1 and PXR4 mRNA are downregulated by methylation in cancerous tissue and have divergent effects on cellular proliferation when ectopically overexpressed. Additional detailed and comparative mechanistic studies are required to predict the effect of PXR transcript variant expression on carcinogenesis, therapeutic response, and the development of toxicity.

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The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 - namely, the transmembrane receptor form ST2L - prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated. Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L. The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.

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BACKGROUND & AIMS: The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function of LGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail. METHODS: We interrogated the function and expression of LGR family members using human pluripotent stem cell-derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice. RESULTS: We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human-mouse species-specific differences at later time points of embryonic development. CONCLUSIONS: Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.