Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.

Obesity alters levels of pituitary hormones that govern hepatic immune-metabolic homeostasis, dysregulation of which leads to nonalcoholic fatty liver disease (NAFLD). However, the impact of obesity on intra-pituitary homeostasis is largely unknown. Here, we uncovered a blunted unfolded protein response (UPR) but elevated inflammatory signatures in pituitary glands of obese mice and humans. Furthermore, we found that obesity inflames the pituitary gland, leading to impaired pituitary inositol-requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) UPR branch, which is essential for protecting against pituitary endocrine defects and NAFLD progression. Intriguingly, pituitary IRE1-deletion resulted in hypothyroidism and suppressed the thyroid hormone receptor B (THRB)-mediated activation of Xbp1 in the liver. Conversely, activation of the hepatic THRB-XBP1 axis improved NAFLD in mice with pituitary UPR defect. Our study provides the first evidence and mechanism of obesity-induced intra-pituitary cellular defects and the pathophysiological role of pituitary-liver UPR communication in NAFLD progression.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

A Medium Chain Fatty Acid, 6-hydroxyhexanoic acid (6-HHA), Protects Against Obesity and Insulin Resistance.

Obesity, a worldwide health problem, increases the risk for developing metabolic diseases such as insulin resistance and diabetes. It is well recognized that obesity-associated chronic inflammation plays a key role in the pathogenesis of systemic metabolic dysfunction. Previously, we revealed an anti-inflammatory role for spent culture supernatants isolated from the oral commensal bacterial species Streptococcus gordonii (Sg-SCS). Here, we identified that 6-hydroxyhexanoic acid (6-HHA), a medium chain fatty acid (MCFA), is the one of the key components of Sg-SCS . We found that treatment of 6-HHA in mice fed a high-fat diet (HFD) significantly reduced HFD-mediated weight gain which was largely attributed to a decrease in fat mass. Systemically, 6-HHA improves obesity-associated glucose intolerance and insulin resistance. Furthermore, administration of 6-HHA suppressed obesity-associated systemic inflammation and dyslipidemia. At the cellular level, treatment of 6-HHA ameliorated aberrant inflammatory and metabolic transcriptomic signatures in white adipose tissue of mice with diet-induced obesity (HFD). Mechanistically, we found that 6-HHA suppressed adipocyte-proinflammatory cytokine production and lipolysis, the latter through Gαi-mediated signaling. This work provides direct evidence for the anti-obesity effects of a novel MCFA, which could be a new therapeutic treatment for combating obesity. KEY POINTS: Hydroxyhexanoic medium chain fatty acids (MCFAs) are dietary and bacterial-derived energy sources, however, the outcomes of using MCFAs in treating metabolic disorders are diverse and complex. The MCFA 6-hydroxyhexanoic acid (6-HHA) is a metabolite secreted by the oral bacterial commensal species Streptococcus gordonii; here we investigated its role in modulating high-fat diet (HFD)-induced metabolic dysfunction. In a murine model of obesity, we found 6-HHA-mediated improvement of diet-mediated adiposity, insulin resistance and inflammation were in part due to actions on white adipose tissue (WAT).6-HHA suppressed proinflammatory cytokine production and lipolysis through Gi-mediated signaling in differentiated white adipocytes.

Transcriptome analyses of murine right and left maxilla-mandibular complex.

OBJECTIVE: The objective of the study was to investigate differential gene expression between murine right and left maxilla-mandibular (MxMn) complexes. SETTING AND SAMPLE POPULATION: Wild-type (WT) C57BL/6 embryonic (E) day 14.5 (n = 3) and 18.5 (n = 3) murine embryos. METHODS: The E14.5 and 18.5 embryos were harvested and hemi-sectioned the MxMn complexes into right and left halves in the mid-sagittal plane. We isolated total RNA using Trizol reagent and further purified using the RNA-easy kit (QIAGEN). We confirmed equal expression of house-keeping genes in right and left halves using RT-PCR and then performed paired-end whole mRNA sequencing in LC Sciences (Houston, TX) followed by differential transcript analyses (>1 or <-1 log fold change; p < .05; q < .05; and FPKM >0.5 in 2/3 samples). The Mouse Genome Informatics and Online Mendelian Inheritance in Man databases as well as gnomAD constraint scores were used to prioritize differentially expressed transcripts. RESULTS: There were 19 upregulated and 19 downregulated transcripts at E14.5 and 8 upregulated and 17 downregulated transcripts at E18.5 time-points. These differentially expressed transcripts were statistically significant and shown to be associated with craniofacial phenotypes in mouse models. These transcripts also have significant gnomAD constraint scores and are enriched in biological processes critical for embryogenesis. CONCLUSIONS: We identified significant differential expression of transcripts between E14.5 and 18.5 murine right and left MxMn complexes. These findings when extrapolated to humans, they may provide a biological basis for facial asymmetry. Further experiments are required to validate these findings in murine models with craniofacial asymmetry.

Computational identification of key transcription factors for embryonic and postnatal Sox2+ dental epithelial stem cell.

While many reptiles can replace their tooth throughout life, human loss the tooth replacement capability after formation of the permanent teeth. It was thought that the difference in tooth regeneration capability depends on the persistence of a specialized dental epithelial structure, the dental lamina that contains dental epithelial stem cells (DESC). Currently, we know very little about DESC such as what genes are expressed or its chromatin accessibility profile. Multiple markers of DESC have been proposed such as Sox2 and Lgr5 . Few single cell RNA-seq experiments have been performed previously, but no obvious DESC cluster was identified in these scRNA-seq datasets, possible due to that the expression level of DESC markers such as Sox2 and Lgr5 is too low or the percentage of DESC is too low in whole tooth. We utilize a mouse line Sox2-GFP to enrich Sox2+ DESC and use Smart-Seq2 protocol and ATAC-seq protocol to generate transcriptome profile and chromatin accessibility profile of P2 Sox2+ DESC. Additionally, we generate transcriptome profile and chromatin accessibility profile of E11.5 Sox2+ dental lamina cells. With transcriptome profile and chromatin accessibility profile, we systematically identify potential key transcription factors for E11.5 Sox2+ cells and P2 Sox2+ cells. We identified transcription factors including Pitx2, Id3, Pitx1, Tbx1, Trp63, Nkx2-3, Grhl3, Dlx2, Runx1, Nfix, Zfp536 , etc potentially formed the core transcriptional regulatory networks of Sox2+ DESC in both embryonic and postnatal stages.

Extracellular vesicle expansion of PMIS-miR-210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism.

BACKGROUND: Colorectal cancer (CRC) has a high mortality rate, and therapeutic approaches to treat these cancers are varied and depend on the metabolic state of the tumour. Profiles of CRC tumours have identified several biomarkers, including microRNAs. microRNA-210 (miR-210) levels are directly correlated with CRC survival. miR-210 expression is higher in metastatic colon cancer cells versus non-metastatic and normal colon epithelium. Therefore, efficient methods to inhibit miR-210 expression in CRC may provide new advances in treatments. METHODS: Expression of miRs was determined in several metastatic and non-metastatic cell lines. miR-210 expression was inhibited using PMIS-miR-210 in transduced cells, which were transplanted into xenograft mice. In separate experiments, CRC tumours were allowed to grow in xenograft mice and treated with therapeutic injections of PMIS-miR-210. Molecular and biochemical experiments identified several new pathways targeted by miR-210 inhibition. RESULTS: miR-210 inhibition can significantly reduce tumour growth of implanted colon cancer cells in xenograft mouse models. The direct administration of PMIS-miR-210 to existing tumours can inhibit tumour growth in both NSG and Foxn1nu/j mouse models and is more efficacious than capecitabine treatments. Tumour cells further transfer the PMIS-miR-210 inhibitor to neighbouring cells by extracellular vesicles to inhibit miR-210 throughout the tumour. miR-210 inhibition activates the cleaved caspase 3 apoptotic pathway to reduce tumour formation. We demonstrate that the long non-coding transcript XIST is regulated by miR-210 correlating with decreased XIST expression in CRC tumours. XIST acts as a competing endogenous RNA for miR-210, which reduces XIST levels and miR-210 inhibition increases XIST transcripts in the nucleus and cytoplasm. The increased expression of NME1 is associated with H3K4me3 and H3K27ac modifications in the NME1 proximal promoter by XIST. CONCLUSION: Direct application of the PMIS-miR-210 inhibitor to growing tumours may be an effective colorectal cancer therapeutic.

A putative long noncoding RNA-encoded micropeptide maintains cellular homeostasis in pancreatic ß cells.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here, we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a ß cell- and neural cell-enriched lncRNA TCL1 Upstream Neural Differentiation-Associated RNA (TUNAR, also known as TUNA, HI-LNC78, or LINC00617). We named this highly conserved 48-amino-acid micropeptide beta cell- and neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localizes at the endoplasmic reticulum (ER) in pancreatic ß cells. Overexpression of BNLN lowered ER calcium levels, maintained ER homeostasis, and elevated glucose-stimulated insulin secretion in pancreatic ß cells. We further assessed the BNLN expression in islets from mice fed a high-fat diet and a regular diet and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic ß cell functions and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.

The unfolded protein response regulates hepatic autophagy by sXBP1-mediated activation of TFEB.

Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyzes of both primary hepatocytes and whole livers further showed that sXBP1 occupies the -743 to -523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction.Abbreviations: AAV8: adeno-associated virus serotype 8; ACTB: actin, beta; ANOVA: analysis of variance; ATF6: activating transcription factor-6; ATG: autophagy related; BECN1: beclin 1; BMI: body mass index; ChIP: chromatin immunoprecipitation; CLEAR: coordinated lysosomal expression and regulation; Cre: cre recombinase; DIO: diet-induced obesity; EBSS: Earle's balanced salt solution; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HFD: high-fat diet; h: hours; HSCs: hepatic stellate cells; INS: insulin; L/A: ammonium chloride and leupeptin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; RD: regular diet; RFP: red fluorescent protein; SERPINA7/TBG: serpin family A member 7; SQSTM1/p62: sequestome 1; sXbp1 LOE: liver-specific overexpression of spliced Xbp1; TFEB: transcription factor EB; TG: thapsigargin; TN: tunicamycin; UPR: unfolded protein response; wks: weeks; WT: wild type; XBP1: X-box binding protein 1; xbp1 LKO: liver-specific Xbp1 knockout.

Pitx2-Sox2-Lef1 interactions specify progenitor oral/dental epithelial cell signaling centers.

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.

Identification and Characterization of Tumor-Initiating Cells in Multiple Myeloma.

BACKGROUND: Treatment failures in cancers, including multiple myeloma (MM), are most likely due to the persistence of a minor population of tumor-initiating cells (TICs), which are noncycling or slowly cycling and very drug resistant. METHODS: Gene expression profiling and real-time quantitative reverse transcription polymerase chain reaction were employed to define genes differentially expressed between the side-population cells, which contain the TICs, and the main population of MM cells derived from 11 MM patient samples. Self-renewal potential was analyzed by clonogenicity and drug resistance of CD24+ MM cells. Flow cytometry (n = 60) and immunofluorescence (n = 66) were applied on MM patient samples to determine CD24 expression. Therapeutic effects of CD24 antibodies were tested in xenograft MM mouse models containing three to six mice per group. RESULTS: CD24 was highly expressed in the side-population cells, and CD24+ MM cells exhibited high expression of induced pluripotent or embryonic stem cell genes. CD24+ MM cells showed increased clonogenicity, drug resistance, and tumorigenicity. Only 10 CD24+ MM cells were required to develop plasmacytomas in mice (n = three of five mice after 27 days). The frequency of CD24+ MM cells was highly variable in primary MM samples, but the average of CD24+ MM cells was 8.3% after chemotherapy and in complete-remission MM samples with persistent minimal residual disease compared with 1.0% CD24+ MM cells in newly diagnosed MM samples (n = 26). MM patients with a high initial percentage of CD24+ MM cells had inferior progression-free survival (hazard ratio [HR] = 3.81, 95% confidence interval [CI] = 5.66 to 18.34, P < .001) and overall survival (HR = 3.87, 95% CI = 16.61 to 34.39, P = .002). A CD24 antibody inhibited MM cell growth and prevented tumor progression in vivo. CONCLUSION: Our studies demonstrate that CD24+ MM cells maintain the TIC features of self-renewal and drug resistance and provide a target for myeloma therapy.

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

Elimination of SOX2/OCT4-Associated Prostate Cancer Stem Cells Blocks Tumor Development and Enhances Therapeutic Response.

SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance. Here, we show that a fluorescent reporter called SORE6 can identify SOX2/OCT4-overexpressing prostate cancer cells. Among tumor cells, the SORE6 reporter identified a small fraction with CSC hallmarks: rapid self-renewal, the capability to form tumors and metastasize, and resistance to chemotherapies. Transcriptome and biochemical analyses identified PI3K/AKT signaling as critical for maintaining the SORE6+ population. Moreover, a SORE6-driven herpes simplex virus thymidine kinase (TK) expression construct could selectively ablate SORE6+ cells in tumors, blocking tumor initiation and progression, and sensitizing tumors to chemotherapy. This study demonstrates a key role of SOX2/OCT4-associated prostate cancer stem cells in tumor development and therapeutic resistance, and identifies the SORE6 reporter system as a useful tool for characterizing CSCs functions in a native tumor microenvironment.

Identification of novel TGF-ß regulated genes with pro-migratory roles.

Transforming growth factor-ß (TGF-ß) signaling plays fundamental roles in the development and homeostasis of somatic cells. Dysregulated TGF-ß signaling contributes to cancer progression and relapse to therapies by inducing epithelial-to-mesenchymal transition (EMT), enriching cancer stem cells, and promoting immunosuppression. Although many TGF-ß-regulated genes have been identified, only a few datasets were obtained by next-generation sequencing. In this study, we performed RNA-sequencing analysis of MCF10A cells and identified 1166 genes that were upregulated and 861 genes that were downregulated by TGF-ß. Gene set enrichment analysis revealed that focal adhesion and metabolic pathways were the top enriched pathways of the up- and downregulated genes, respectively. Genes in these pathways also possess significant predictive value for renal cancers. Moreover, we confirmed that TGF-ß induced expression of MICAL1 and 2, and the histone demethylase, KDM7A, and revealed their regulatory roles on TGF-ß-induced cell migration. We also show a critical effect of KDM7A in regulating the acetylation of H3K27 on TGF-ß-induced genes. In sum, this study identified novel effectors that mediate the pro-migratory role of TGF-ß signaling, paving the way for future studies that investigate the function of MICAL family members in cancer and the novel epigenetic mechanisms downstream TGF-ß signaling.

Hepatic Lysosomal iNOS Activity Impairs Autophagy in Obesity.

BACKGROUND & AIMS: The lysosome is an acidic organelle that is important for maintaining cellular and metabolic homeostasis in hepatocytes. Lysosomal dysfunction and chronic inflammation coexist, and both contribute to obesity-associated hepatic insulin resistance. However, in the context of obesity, the interplay between inflammatory signals and hepatic lysosomal function remains largely unknown. Inducible nitric oxide synthase (iNOS) is a hallmark for inflammation, and is activated in obesity. The aim of this study is to understand the molecular link between iNOS-mediated lysosomal nitric oxide (NO) production, hepatic lysosomal function, and autophagy in the context of obesity-associated insulin resistance. METHODS: The role of iNOS in hepatic autophagy, as related to insulin and glucose homeostasis were studied in mice with diet-induced obesity (DIO). The effects and mechanisms of iNOS-mediated lysosomal NO production on lysosomal function and hepatic autophagy were studied in primary hepatocytes as well as in a mouse model of DIO. RESULTS: We demonstrate that obesity promotes iNOS localization to the lysosome and decreases levels of lysosomal arginine, resulting in an accumulation of NO in hepatic lysosomes. This lysosomal NO production is attenuated by treatment with a NO scavenger, while co-overexpression of mTOR and a lysosomal arginine transporter (SLC38A9) enhances lysosomal NO production and suppresses autophagy. In addition, we show that deletion of iNOS ameliorates lysosomal nitrosative stress in the livers of DIO mice, promotes lysosomal biogenesis by activating transcription factor EB (TFEB), and enhances lysosomal function and autophagy. Lastly, deletion of iNOS in mice with DIO improves hepatic insulin sensitivity, which is diminished by suppression of TFEB or autophagy related 7 (Atg7). CONCLUSIONS: Our studies suggest that lysosomal iNOS-mediated NO signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.

FoxO6 regulates Hippo signaling and growth of the craniofacial complex.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.

Two birds, one stone: hesperetin alleviates chemotherapy-induced diarrhea and potentiates tumor inhibition.

Chemotherapy-induced diarrhea (CID), with clinical high incidence, adversely affects the efficacy of cancer treatment and patients' quality of life. Our study demonstrates that the citrus flavonoid hesperetin (Hst) has a superior potential as a new agent to prevent and alleviate CID. In the animal model for irinotecan (CPT-11) induced CID, Hst could selectively inhibit intestinal carboxylesterase (CES2) and thus reduce the local conversion of CPT-11 to cytotoxic SN-38 which causes intestinal toxicity. Oral administration of Hst manifested an excellent anti-diarrhea efficacy, prohibiting 80% of severe and 100% of mild diarrhea in the CPT-11 administered tumor-bearing mice. In addition, a significant attenuation of intestinal inflammation contributed to the anti-diarrhea effect of Hst. Moreover, Hst was found to work synergistically with CPT-11 in tumor inhibition by suppressing the tumor's STAT3 activity and recruiting tumoricidal macrophages into the tumor microenvironment. The anti-intestinal inflammation and anti-STAT3 properties of Hst would contribute its broad benefits to the management of diarrhea caused by other chemo or targeted agents, and more importantly, enhance and reinforce the anti-tumor effects of these agents, to improve patient outcomes.

Splicing Factor RBM20 Regulates Transcriptional Network of Titin Associated and Calcium Handling Genes in The Heart.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation.

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1-/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.

Mesenchymal MicroRNA Function Branches Out.

Significant amounts of microRNAs (miRs) are detected in exosomes, but their function during fetal development is poorly understood. In this issue of Developmental Cell, Hayashi et al. (2017) show that exosomal miRs secreted by mesenchymal cells can regulate epithelial KIT+ progenitor cell expansion during murine salivary gland organogenesis.