Urrutia, Raúl

We have previously shown that amino acid changes in the human Kruppel-Like Factor (KLF) 11 protein is associated with the development of maturity onset diabetes of the young VII, whereas complete inactivation of this pathway by the −331 human insulin mutation causes neonatal diabetes mellitus. Here, we report that Klf11−/− mice have decreased circulating insulin levels, alterations in the control of blood glucose and body weight, as well as serum dyslipidemia, but do not develop diabetes. Functional assays using ex vivo liver tissue sections demonstrate that Klf11−/− mice display increased insulin sensitivity. Genome-wide experiments validated by pathway-specific quantitative PCR arrays reveal that the Klf11−/− phenotype associates to alterations in the regulation of gene networks involved in lipid metabolism, in particular those regulated by peroxisome proliferator-activated receptor-γ. Combined, these results demonstrate that the major phenotype given by the whole-body deletion of Klf11 in mouse is not diabetes but increased insulin sensitivity, likely due to altered transcriptional regulation in target tissues. The absence of diabetes in the Klf11−/− mouse either indicates an interspecies difference for the role of this transcription factor in metabolic homeostasis between mouse and humans, or potentially highlights the fact that other molecular factors can compensate for its absence. Nevertheless, the data of this study, gathered at the whole-organism level, further support a role for KLF11 in metabolic processes like insulin sensitivity, which regulation is critical in several forms of diabetes.

Abstract Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor. Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP. Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis. Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle. Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2015-2087

We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1‐like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology‐based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG‐like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53‐response elements at positions −37 and −7, respectively. Experiments using reporter assays combined with site‐directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well‐validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53‐induced gene, which negatively regulates the protumoral factor NUPR1. J. Cell. Physiol. 230: 2936–2950, 2015. © 2015 Wiley Periodicals, Inc.

Objective— Gestational diabetes mellitus (GDM) produces fetal hyperglycemia with increased lifelong risks for the exposed offspring of cardiovascular and other diseases. Epigenetic mechanisms induce long-term gene expression changes in response to in utero environmental perturbations. Moreover, microRNAs (miRs) control the function of endothelial cells (ECs) under physiological and pathological conditions and can target the epigenetic machinery. We investigated the functional and expressional effect of GDM on human fetal ECs of the umbilical cord vein (HUVECs). We focused on miR-101 and 1 of its targets, enhancer of zester homolog-2 (EZH2), which trimethylates the lysine 27 of histone 3, thus repressing gene transcription. EZH2 exists as isoforms α and β. Approach and Results— HUVECs were prepared from GDM or healthy pregnancies and tested in apoptosis, migration, and Matrigel assays. GDM-HUVECs demonstrated decreased functional capacities, increased miR-101 expression, and reduced EZH2- β and trimethylation of histone H3 on lysine 27 levels. MiR-101 inhibition increased EZH2 expression and improved GDM-HUVEC function. Healthy HUVECs were exposed to high or normal d -glucose concentration for 48 hours and then tested for miR-101 and EZH2 expression. Similar to GDM, high glucose increased miR-101 expression. Chromatin immunoprecipitation using an antibody for EZH2 followed by polymerase chain reaction analyses for miR-101 gene promoter regions showed that both GDM and high glucose concentration reduced EZH2 binding to the miR-101 locus in HUVECs. Moreover, EZH2-β overexpression inhibited miR-101 promoter activity in HUVECs. Conclusions— GDM impairs HUVEC function via miR-101 upregulation. EZH2 is both a transcriptional inhibitor and a target gene of miR-101 in HUVECs, and it contributes to some of the miR-101-induced defects of GDM-HUVECs.

Abstract Cancer-associated fibroblasts (CAFs), the most abundant cells in the tumor microenvironment (TME), are a key source of the extracellular matrix (ECM) that constitutes the desmoplastic stroma. Through remodeling of the reactive tumor stroma and paracrine actions, CAFs regulate cancer initiation, progression, and metastasis, as well as tumor resistance to therapies. The CAFs found in stroma-rich primary hepatocellular carcinomas (HCC) and liver metastases of primary cancers of other organs predominantly originate from hepatic stellate cells (HSTC), which are pericytes associated with hepatic sinusoids. During tumor invasion, HSTCs transdifferentiate into myofibroblasts in response to paracrine signals emanating from either tumor cells or a heterogeneous cell population within the hepatic tumor microenvironment. Mechanistically, HSTC-to-myofibroblast transdifferentiation, also known as, HSTC activation, requires cell surface receptor activation, intracellular signal transduction, gene transcription, and epigenetic signals, which combined ultimately modulate distinct gene expression profiles that give rise to and maintain a new phenotype. The current review defines a paradigm that explains how HSTCs are activated into CAFs to promote liver metastasis. Furthermore, a focus on the most relevant intracellular signaling networks and epigenetic mechanisms that control HSTC activation is provided. Finally, we discuss the feasibility of targeting CAF/activated HSTCs, in isolation or in conjunction with targeting cancer cells, which constitutes a promising and viable therapeutic approach for the treatment of primary stroma-rich liver cancers and liver metastasis. Mol Cancer Res; 13(4); 604–12. ©2014 AACR.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-β1 (TGF-β1) signaling in CD4+ T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-β receptor II (TGF-βRII) 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-βRII (TβRII). Congruently, in vitro activation of KLF10-deficient CD8+ T cells upregulate TGF-βRII to a lesser extent compared with wild-type (WT) CD8+ T cells, which results in attenuated Smad2 phosphorylation following TGF-β1 stimulation compared with WT CD8+ T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-βRII 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-βRII 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-βRII in CD8+ T cells. Thus, KLF10 regulation of TGF-βRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-β1/TGF-βRII signaling pathway is crucial.

SummaryChronic obesity leads to inflammation, tissue dysfunction, and cellular senescence. It was proposed that cellular senescence during obesity and aging drives inflammation and dysfunction. Consistent with this, clearance of senescent cells increases healthspan in progeroid mice. Here, we show that the protein Deleted in Breast Cancer‐1 (DBC1) regulates cellular senescence during obesity. Deletion of DBC1 protects preadipocytes against cellular senescence and senescence‐driven inflammation. Furthermore, we show protection against cellular senescence in DBC1 KO mice during obesity. Finally, we found that DBC1 participates in the onset of cellular senescence in response to cell damage by mechanism that involves binding and inhibition of HDAC3. We propose that by regulating HDAC3 activity during cellular damage, DBC1 participates in the fate decision that leads to the establishment of cellular senescence and consequently to inflammation and tissue dysfunction during obesity.

Inducible gene expression, which requires chromatin remodeling on gene promoters, underlies the epigenetically inherited differentiation program of most immune cells. However, chromatin-mediated mechanisms that underlie these events in T regulatory cells remain to be fully characterized. Here, we report that inducibility of FOXP3, a key transcription factor for the development of T regulatory cells, depends upon Kruppel-like factor 10 (KLF10) interacting with two antagonistic histone-modifying systems. We utilized chromatin immunoprecipitation, genome-integrated reporter assays, and functional domain KLF10 mutant proteins, to characterize reciprocal interactions between this transcription factor and either the Sin3-histone deacetylase complex or the histone acetyltransferase, p300/CBP-associated factor (PCAF). We characterize a Sin3-interacting repressor domain on the NH2 terminus of KLF10, which works to limit the activating function of this transcription factor. Indeed, inactivation of this Sin3-interacting domain renders KLF10 able to physically associate with PCAF as to induce FOXP3 gene transcription. We show that this biochemical data derived from studying our genome-integrated reporter cell system are recapitulated in primary murine lymphocytes. Collectively, these results advance our understanding of how a single transcription factor, namely KLF10, functions as a toggle to integrate antagonistic signals regulating FOXP3 and, thus, immune activation.

Abstract Cancer-associated inflammation is a molecular key feature in pancreatic ductal adenocarcinoma. Oncogenic KRAS in conjunction with persistent inflammation is known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood. Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in pancreatic epithelial cells to promote KrasG12D-driven carcinogenesis. NFATc1 activation is induced by inflammation and itself accelerates inflammation-induced carcinogenesis in KrasG12D mice, whereas genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 complexes with STAT3 for enhancer–promoter communications at jointly regulated genes involved in oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1–STAT3 cooperativity is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer. Together, these studies unravel new mechanisms of inflammatory-driven pancreatic carcinogenesis and suggest beneficial effects of chemopreventive strategies using drugs that are currently available for targeting these factors in clinical trials. Significance: Our study points to the existence of an oncogenic NFATc1–STAT3 cooperativity that mechanistically links inflammation with pancreatic cancer initiation and progression. Because NFATc1–STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of inflammation and cancer, our study has significant relevance for potentially managing pancreatic cancer and other inflammatory-driven malignancies. Cancer Discov; 4(6); 688–701. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 621