Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

KLF5 and p53 comprise an incoherent feed-forward loop directing cell-fate decisions following stress.

In response to stress, cells make a critical decision to arrest or undergo apoptosis, mediated in large part by the tumor suppressor p53. Yet the mechanisms of these cell fate decisions remain largely unknown, particularly in normal cells. Here, we define an incoherent feed-forward loop in non-transformed human squamous epithelial cells involving p53 and the zinc-finger transcription factor KLF5 that dictates responses to differing levels of cellular stress from UV irradiation or oxidative stress. In normal unstressed human squamous epithelial cells, KLF5 complexes with SIN3A and HDAC2 repress TP53, allowing cells to proliferate. With moderate stress, this complex is disrupted, and TP53 is induced; KLF5 then acts as a molecular switch for p53 function by transactivating AKT1 and AKT3, which direct cells toward survival. By contrast, severe stress results in KLF5 loss, such that AKT1 and AKT3 are not induced, and cells preferentially undergo apoptosis. Thus, in human squamous epithelial cells, KLF5 gates the response to UV or oxidative stress to determine the p53 output of growth arrest or apoptosis.

Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

A GATA6-centred gene regulatory network involving HNFs and ΔNp63 controls plasticity and immune escape in pancreatic cancer.

OBJECTIVE: Molecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme. DESIGN: We combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO). RESULTS: This comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype. CONCLUSIONS: Our work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.

Inorganic arsenic promotes luminal to basal transition and metastasis of breast cancer.

Inorganic arsenic (iAs/As2 O32- ) is an environmental toxicant found in watersheds around the world including in densely populated areas. iAs is a class I carcinogen known to target the skin, lungs, bladder, and digestive organs, but its role as a primary breast carcinogen remains controversial. Here, we examined a different possibility: that exposure to iAs promotes the transition of well-differentiated epithelial breast cancer cells characterized by estrogen and progesterone receptor expression (ER+/PR+), to more basal phenotypes characterized by active proliferation, and propensity to metastasis in vivo. Our results indicate two clear phenotypic responses to low-level iAs that depend on the duration of the exposure. Short-term pulses of iAs activate ER signaling, consistent with its reported pseudo-estrogen activity, but longer-term, chronic treatments for over 6 months suppresses both ER and PR expression and signaling. In fact, washout of these chronically exposed cells for up to 1 month failed to fully reverse the transcriptional and phenotypic effects of prolonged treatments, indicating durable changes in cellular physiologic identity. RNA-seq studies found that chronic iAs drives the transition toward more basal phenotypes characterized by impaired hormone receptor signaling despite the conservation of estrogen receptor expression. Because treatments for breast cancer patients are largely designed based on the detection of hormone receptor expression, our results suggest greater scrutiny of ER+ cancers in patients exposed to iAs, because these tumors may spawn more aggressive phenotypes than unexposed ER+ tumors, in particular, basal subtypes that tend to develop therapy resistance and metastasis.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature.

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.

Variant anatomy of the biliary system as a cause of pancreatic and peri-ampullary cancers.

BACKGROUND: The cause of most pancreatic and periampullary cancers (PAC) is unknown. Recently, anatomic variations such as pancreatobiliary maljunction have been recognized as risk factors, similar to Barrett-related gastro-esophageal cancers. METHODS: Pre-operative MRI from 860 pancreatic/biliary resections, including 322 PACs, were evaluated for low-union (cystic duct joining the common hepatic duct inside of the pancreas or within 5 mm of the pancreatic border) RESULTS: Low-union, seen <10% of the population, was present in 44% of PACs (73% distal bile duct/cholangiocarcinoma, 42% pancreatic head, and 34% ampullary). It was significantly lower(11%) in conditions without connection to the ductal system (thus not exposed to the ductal/biliary tract contents), namely mucinous cystic neoplasms and intrahepatic cholangiocarcinomas(p < 0.0001). Intra-pancreatic type low-union was seen in 16% of PACs versus 2% of controls(p < 0.0001). DISCUSSION: This study establishes an association between low-union and PACs, and points to an anatomy-induced chemical/bilious carcinogenesis. This may explain why most pancreas cancers are in the head. It is possible that the same chemical milieu, caused by conditions other than low-union/insertion, may also play a role in the remaining half of PACs. This opens various treatment opportunities including milieu modifications (chemoprevention), focused screening of at-risk patients, and early detection with possible corrective actions.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFß and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFß- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Disruption of FOXP3-EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation.

Background & Aims: Forkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function. Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn's disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant+, interleukin 6 (IL6)-treated or CD-CD4+ T cells to assess FOXP3-EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants. Results: EZH2 binding was abrogated by inflammatory bowel disease-associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3-EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria-derived CD4+T cells from CD patients, we observed decreased FOXP3-EZH2 interaction. Conclusions: FOXP3-C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3-EZH2 interaction. Studies in lesion-derived CD4+ T cells have shown that reduced FOXP3-EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3-EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation.

Co-occurrence of a maternally inherited DNMT3A duplication and a paternally inherited pathogenic variant in EZH2 in a child with growth retardation and severe short stature: atypical Weaver syndrome or evidence of a DNMT3A dosage effect?

Overgrowth syndromes are a clinically heterogeneous group of disorders characterized by localized or generalized tissue overgrowth and varying degrees of developmental and intellectual disability. An expanding list of genes associated with overgrowth syndromes include the histone methyltransferase genes EZH2 and NSD1, which cause Weaver and Sotos syndrome, respectively, and the DNA methyltransferase (DNMT3A) gene that results in Tatton-Brown-Rahman syndrome (TBRS). Here, we describe a 5-year-old female with a paternally inherited pathogenic mutation in EZH2 (c.2050C>T, p.Arg684Cys) and a maternally inherited 505-kb duplication of uncertain significance at 2p23.3 (encompassing five genes, including DNMT3A) who presented with intrauterine growth restriction, slow postnatal growth, short stature, hypotonia, developmental delay, and neuroblastoma diagnosed at the age of 8 mo. Her father had tall stature, dysmorphic facial features, and intellectual disability consistent with Weaver syndrome, whereas her mother had short stature, cognitive delays, and chronic nonprogressive leukocytosis. It has been previously shown that EZH2 directly controls DNA methylation through physical association with DNMTs, including DNMT3A, with concomitant H3K27 methylation and CpG promoter methylation leading to repression of EZH2 target genes. Interestingly, NSD1 is involved in H3K36 methylation, a mark associated with transcriptional activation, and exhibits exquisite dosage sensitivity leading to overgrowth when deleted and severe undergrowth when duplicated in vivo. Although there is currently no evidence of dosage effects for DNMT3A, the co-occurrence of a duplication involving this gene and a pathogenic alteration in EZH2 in a patient with severe undergrowth is suggestive of a similar paradigm and further study is warranted.

Resolution of Cochlear Inflammation: Novel Target for Preventing or Ameliorating Drug-, Noise- and Age-related Hearing Loss.

A significant number of studies support the idea that inflammatory responses are intimately associated with drug-, noise- and age-related hearing loss (DRHL, NRHL and ARHL). Consequently, several clinical strategies aimed at reducing auditory dysfunction by preventing inflammation are currently under intense scrutiny. Inflammation, however, is a normal adaptive response aimed at restoring tissue functionality and homeostasis after infection, tissue injury and even stress under sterile conditions, and suppressing it could have unintended negative consequences. Therefore, an appropriate approach to prevent or ameliorate DRHL, NRHL and ARHL should involve improving the resolution of the inflammatory process in the cochlea rather than inhibiting this phenomenon. The resolution of inflammation is not a passive response but rather an active, highly controlled and coordinated process. Inflammation by itself produces specialized pro-resolving mediators with critical functions, including essential fatty acid derivatives (lipoxins, resolvins, protectins and maresins), proteins and peptides such as annexin A1 and galectins, purines (adenosine), gaseous mediators (NO, H2S and CO), as well as neuromodulators like acetylcholine and netrin-1. In this review article, we describe recent advances in the understanding of the resolution phase of inflammation and highlight therapeutic strategies that might be useful in preventing inflammation-induced cochlear damage. In particular, we emphasize beneficial approaches that have been tested in pre-clinical models of inflammatory responses induced by recognized ototoxic drugs such as cisplatin and aminoglycoside antibiotics. Since these studies suggest that improving the resolution process could be useful for the prevention of inflammation-associated diseases in humans, we discuss the potential application of similar strategies to prevent or mitigate DRHL, NRHL and ARHL.

The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD).

Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a pivotal role in maintaining immunologic self-tolerance. We and others have shown previously that EZH2 is recruited to the FOXP3 promoter and its targets in Treg cells. To further address the role for EZH2 in Treg cellular function, we have now generated mice that lack EZH2 specifically in Treg cells (EZH2Δ/ΔFOXP3+). We find that EZH2 deficiency in FOXP3+ T cells results in lethal multiorgan autoimmunity. We further demonstrate that EZH2Δ/ΔFOXP3+ T cells lack a regulatory phenotype in vitro and secrete proinflammatory cytokines. Of special interest, EZH2Δ/ΔFOXP3+ mice develop spontaneous inflammatory bowel disease. Guided by these results, we assessed the FOXP3 and EZH2 gene networks by RNA sequencing in isolated intestinal CD4+ T cells from patients with Crohn's disease. Gene network analysis demonstrates that these CD4+ T cells display a Th1/Th17-like phenotype with an enrichment of gene targets shared by FOXP3 and EZH2. Combined, these results suggest that the inflammatory milieu found in Crohn's disease could lead to or result from deregulation of FOXP3/EZH2-enforced T cell gene networks contributing to the underlying intestinal inflammation.

Gold Nanoparticle Reprograms Pancreatic Tumor Microenvironment and Inhibits Tumor Growth.

Altered tumor microenvironment (TME) arising from a bidirectional crosstalk between the pancreatic cancer cells (PCCs) and the pancreatic stellate cells (PSCs) is implicated in the dismal prognosis in pancreatic ductal adenocarcinoma (PDAC), yet effective strategies to disrupt the crosstalk is lacking. Here, we demonstrate that gold nanoparticles (AuNPs) inhibit proliferation and migration of both PCCs and PSCs by disrupting the bidirectional communication via alteration of the cell secretome. Analyzing the key proteins identified from a functional network of AuNP-altered secretome in PCCs and PSCs, we demonstrate that AuNPs impair secretions of major hub node proteins in both cell types and transform activated PSCs toward a lipid-rich quiescent phenotype. By reducing activation of PSCs, AuNPs inhibit matrix deposition, enhance angiogenesis, and inhibit tumor growth in an orthotopic co-implantation model in vivo. Auto- and heteroregulations of secretory growth factors/cytokines are disrupted by AuNPs resulting in reprogramming of the TME. By utilizing a kinase dead mutant of IRE1-α, we demonstrate that AuNPs alter the cellular secretome through the ER-stress-regulated IRE1-dependent decay pathway (RIDD) and identify endostatin and matrix metalloproteinase 9 as putative RIDD targets. Thus, AuNPs could potentially be utilized as a tool to effectively interrogate bidirectional communications in the tumor microenvironment, reprogram it, and inhibit tumor growth by its therapeutic function.

"The molecule's the thing:" the promise of molecular modeling and dynamic simulations in aiding the prioritization and interpretation of genomic testing results.

Clinical genomics is now a reality and lies at the heart of individualized medicine efforts. The success of these approaches is evidenced by the increasing volume of publications that report causal links between genomic variants and disease. In spite of early success, clinical genomics currently faces significant challenges in establishing the relevance of the majority of variants identified by next generation sequencing tests. Indeed, the majority of mutations identified are harbored by proteins whose functions remain elusive. Herein we describe the current scenario in genomic testing and in particular the burden of variants of uncertain significance (VUSs). We highlight a role for molecular modeling and molecular dynamic simulations as tools that can significantly increase the yield of information to aid in the evaluation of pathogenicity. Though the application of these methodologies to the interpretation of variants identified by genomic testing is not yet widespread, we predict that an increase in their use will significantly benefit the mission of clinical genomics for individualized medicine.

Krüppel-like factor KLF10 deficiency predisposes to colitis through colonic macrophage dysregulation.

Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-ß1 signaling in both CD8(+) and CD4(+) T lymphocytes. In the present study, we demonstrate a novel role for KLF10 in the regulation of TGFßRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10(-/-) bone marrow-derived macrophages into wild-type (WT) mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of proinflammatory LyC6(+)MHCII(+) cells and a reciprocal decrease of the anti-inflammatory LyC6(-)MHCII(+) subset. Additionally, the anti-inflammatory CD11b(+)CX3CR1(hi) subset of colonic macrophages is significantly decreased in KLF10(-/-) compared with WT mice under inflammatory conditions. Molecularly, CD11b(+) colonic macrophages from KLF10(-/-) mice exhibit a proinflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFßRII expression in colonic macrophages and found that, in the absence of KLF10, macrophages express lower levels of TGFßRII and display an attenuated Smad-2 phosphorylation following TGF-ß1 stimulation. We further show that KLF10 directly binds to the TGFßRII promoter in macrophages, leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFßRII expression in macrophages and the acquisition of a "regulatory" phenotype that contributes to intestinal mucosal homeostasis.

Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.

Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.

Krüppel-like factor KLF10 regulates transforming growth factor receptor II expression and TGF-ß signaling in CD8+ T lymphocytes.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-ß1 (TGF-ß1) signaling in CD4(+) T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-ß receptor II (TGF-ßRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8(+) T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10(-/-) CD8(+) T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8(+) T cells from the spleen of adult mice express lower levels of surface TGF-ßRII (TßRII). Congruently, in vitro activation of KLF10-deficient CD8(+) T cells upregulate TGF-ßRII to a lesser extent compared with wild-type (WT) CD8(+) T cells, which results in attenuated Smad2 phosphorylation following TGF-ß1 stimulation compared with WT CD8(+) T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-ßRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-ßRII among viral-specific KLF10(-/-) CD8(+) T cells and a higher percentage of IFN-γ-producing CD8(+) T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-ßRII in CD8(+) T cells. Thus, KLF10 regulation of TGF-ßRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-ß1/TGF-ßRII signaling pathway is crucial.

Membrane-to-nucleus signaling links insulin-like growth factor-1- and stem cell factor-activated pathways.

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3ß inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes.