Pro-proliferative Transcription Factor Research Articles

Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation

We sought to define the mechanism underlying lung microvascular regeneration in a model of severe acute lung injury (ALI) induced by selective lung endothelial cell ablation. Intratracheal instillation of DT in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. Using single-cell RNA sequencing, eight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline and general capillary (gCap) ECs expressing the apelin receptor. At 3 days post-injury, a novel gCap EC population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned at 5 days to proliferative endothelial progenitor-like cells, expressing apelin receptor together with the pro-proliferative transcription factor, Foxm1, and were responsible for the rapid replenishment of all depleted EC populations by 7 days post-injury. Treatment with an apelin receptor antagonist prevented ALI resolution and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. The lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by newly emergent apelin-expressing gCap endothelial stem-like cells that give rise to highly proliferative, apelin receptor-positive endothelial progenitors responsible for the regeneration of the lung microvasculature.

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular response to PARP1 inhibition in ovarian cancer cells as determined by mass spectrometry based proteomics

BackgroundPoly (ADP)-ribose polymerase (PARP) inhibitors have entered routine clinical practice for the treatment of high-grade serous ovarian cancer (HGSOC), yet the molecular mechanisms underlying treatment response to PARP1 inhibition (PARP1i) are not fully understood.MethodsHere, we used unbiased mass spectrometry based proteomics with data-driven protein network analysis to systematically characterize how HGSOC cells respond to PARP1i treatment.ResultsWe found that PARP1i leads to pronounced proteomic changes in a diverse set of cellular processes in HGSOC cancer cells, consistent with transcript changes in an independent perturbation dataset. We interpret decreases in the levels of the pro-proliferative transcription factors SP1 and β-catenin and in growth factor signaling as reflecting the anti-proliferative effect of PARP1i; and the strong activation of pro-survival processes NF-κB signaling and lipid metabolism as PARPi-induced adaptive resistance mechanisms. Based on these observations, we nominate several protein targets for therapeutic inhibition in combination with PARP1i. When tested experimentally, the combination of PARPi with an inhibitor of fatty acid synthase (TVB-2640) has a 3-fold synergistic effect and is therefore of particular pre-clinical interest.ConclusionOur study improves the current understanding of PARP1 function, highlights the potential that the anti-tumor efficacy of PARP1i may not only rely on DNA damage repair mechanisms and informs on the rational design of PARP1i combination therapies in ovarian cancer.

Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity

The release of protons (H+) occurs via the Na+/H+ exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1–dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 μmol/L), SR11302 (1 μmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 μmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 μmol/L, NHE3 inhibitor), concanamycin A (0.1 μmol/L, V-ATPases inhibitor), and Schering (10 μmol/L, H+/K+-ATPase inhibitor). [3H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NH4Cl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (βi) and H+ flux (JH+) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), JH+ (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1–dependent NHE1 activation in MDCK cells.

Abstract 788: Modeling tumor microenvironmental heterogeneity identifies CREBBP as a novel tumor suppressor in breast cancer

Abstract Solid tumors display significant histological, genetic and micro-environmental intra-tumor heterogeneity that can change substantially over the course of their evolutionary trajectory. In particular, changes in the micro-environmental complexity within breast cancer such as hypoxic and nutrient deplete environments are associated with aggressive disease and a poor patient outcome. We sought to identify novel driver alterations in aggressive disease by employing a functional genomics screen in a 3-dimensional model of breast cancer progression that more accurately recapitulates in vivo micro-environmental heterogeneity. Screening of the top 200 recurrently mutated genes in breast cancer in cancer cell line spheroids identified several genes whose silencing impacted growth. A second targeted validation screen in a larger panel of triple negative cell line models showed that silencing of the histone acetyltransferase CREBBP, promoted growth in 3D but had limited effect under traditional 2D culture conditions. Investigation of TCGA and METABRIC datasets showed that CREBBP was more frequently mutated in triple negative breast cancers (TNBCs) and at least a third of TNBCs also displayed gene haploinsufficiency or complete loss of CREBBP. Interrogation of expression and proteomic datasets showed that loss of CREBBP resulted in the upregulation of the pro-proliferative transcription factor FOXM1. Significantly, this conserved FOXM1-driven transcriptional programme was also seen in multiple solid tumors with CREBBP alterations including lung, oesophageal, bladder and endometrial cancers. This was recapitulated in several CREBBP deficient cells where we identified that FOXM1 is driving altered metabolism, allowing cancer cells to grow under nutrient stress conditions. In summary, CREBBP is a bona fide tumor suppressor in up to a third of TNBCs, as well as a wide range of other solid tumors. CREBBP-altered tumors display up-regulation of FOXM1, which alters cancer cell metabolism under nutrient stress conditions. Moreover, CREBBP-altered tumors are selectively sensitive to small molecule inhibitors that target FOXM1 activity, suggesting that this maybe a viable targeted therapeutic approach for CREBBP altered cancers. Citation Format: Barrie Peck, Philip J. Bland, Patty T. Wai, Hannah Cottom, Sarah L. Maguire, Eamonn Morrison, Holly E. Barker, Divya Kriplani, Rebecca Marlow, Kalnisha Naidoo, Gareth Muirhead, Syed Haider, Frances Daley, Frederik Wallberg, Andrew N. Tutt, Rachael C. Natrajan. Modeling tumor microenvironmental heterogeneity identifies CREBBP as a novel tumor suppressor in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 788.

Elucidating feed-forward apoptosis signatures in breast cancer datasets: Higher FOS expression associated with a better outcome.

Overstimulation of pro-proliferative pathways and high level expression of pro-proliferative transcription factors (TFs) can lead to apoptosis. This is likely due to TF binding sites for pro-proliferative TFs common to pro-proliferative and pro-apoptosis-effector genes. Certain clinical datasets have indicated that molecular markers associated with higher proliferation rates lead to improved outcomes for patients with cancer. These observations have been extensively assessed on a general basis, however there has been little work dissecting feed-forward apoptosis signaling pathways that may represent specific distinctions between a pro-proliferative mechanism and a pro-apoptotic mechanism in samples from patients with cancer. Using The Cancer Genome Atlas datasets and bioinformatic approaches, the present study reports that higher FOS expression levels, along with higher FOS target apoptosis-effector gene expression, is associated with an increased survival, while higher POU2F1 expression is associated with a reduced survival (average difference of 25.9 months survival). In summary, in the datasets examined FOS represents an apoptosis-driver and high POU2F1 represents a driver mechanism for cancer development.

FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways.

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin – cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome.FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of β-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy.

Alternate transcripts of the Drosophila “activator” E2F are necessary for maintenance of cell cycle exit during development

The E2F family of transcription factors are evolutionarily conserved regulators of the cell cycle that can be divided into two groups based on their ability to either activate or repress transcription. In Drosophila, there is only one “activator” E2F, dE2F1, which provides all of the pro-proliferative activity of E2F during development. Interestingly, the de2f1 gene can be transcribed from multiple promoters resulting in six alternate transcripts. In this study, we sought to investigate the biological significance of the alternate transcriptional start sites. We focused on the de2f1 promoter region where tissue and cell-type specific enhancer activities were observed at the larval stage. While a genomic deletion of this region, de2f1ΔRA, decreased the overall expression level of dE2F1, flies developed normally with no obvious proliferation defects. However, a detailed analysis of the de2f1ΔRA mutant eye imaginal discs revealed that dE2F1 is needed for proper cell cycle exit. We discovered that dE2F1 expression during G1 arrest prior to the differentiation process of the developing eye is important for maintaining cell cycle arrest at a later stage of the eye development. Overall, our study suggests that specific alternate transcripts of “activator” E2F, dE2F1, may have a dual function on cell cycle progression and cannot simply be viewed as a pro-proliferative transcription factor.

Kras(G12D) induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells.

Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic KrasG12D from the endogenous promoter. We show that primary PDECs are susceptible to KrasG12D-driven transformation and form pancreatic ductal adenocarcinomas (PDAC) in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of KrasG12D induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease KrasG12D-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby, explain the crucial role of EGFR for KrasG12D-driven carcinogenesis in the pancreas.

Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAFV600E, the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\\ Pin1 in BRAFV600E-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAFV600E-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.

Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl) were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC) cells that express Lgr5. By 11days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.

Inhibition of cellular STAT3 synergizes with the cytomegalovirus kinase inhibitor maribavir to disrupt infection

Therapeutic strategies controlling human cytomegalovirus (hCMV) infection are limited due to adverse side effects and emergence of antiviral resistance variants. A compound being evaluated for treating hCMV disease is maribavir (MBV) which disrupts replication by inhibiting the viral kinase pUL97. Previous studies have demonstrated that the antiviral activity of MBV is sensitive to the proliferation state of the infected cell. In these studies, we were interested in determining whether inhibition of the pro-proliferative transcription factor, signal transducer and activator of transcription-3 (STAT3), could influence the antiviral activity of MBV. The addition of the STAT3 inhibitor, S3i-201, during infection altered hCMV-mediated changes in cell cycle protein expression. Upon combining S3i-201 with MBV, our data suggest that STAT3 inhibition is acting synergistically with MBV to inhibit infection in vitro. Furthermore, specific concentrations of S3i-201 and MBV induced caspase-dependent death of infected but not uninfected cell. Our studies suggest that treating infection with both S3i-201 and MBV is a novel approach to inhibit hCMV replication.

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.

Hepatocyte growth factor/c-Met signalling is important for the selection of transplanted hepatocytes

BackgroundAt present hepatocyte transplantation is a promising option for cellular therapy of end-stage liver diseases. However, the underlying molecular mechanisms need to be better defined in order to translate this...

Identification of Small-Molecule Compounds That Inhibit the PRO-Proliferative Transcription Factor KrüPpel-Like Factor 5 (KLF5) in Colorectal Cancer Cells by Cell-Based High-Throughput Screen (HTS)

Identification of Small-Molecule Compounds That Inhibit the PRO-Proliferative Transcription Factor KrüPpel-Like Factor 5 (KLF5) in Colorectal Cancer Cells by Cell-Based High-Throughput Screen (HTS)

The Fbw7/Human CDC4 Tumor Suppressor Targets Proproliferative Factor KLF5 for Ubiquitination and Degradation through Multiple Phosphodegron Motifs

The proproliferative transcription factor KLF5 plays an important role in promoting cell proliferation and tumorigenesis. KLF5 is a short-lived protein that can be rapidly degraded through the ubiquitin-proteasome pathway in cancer cells. However, the mechanisms regulating protein stability remain poorly understood. In this study, the tumor suppressor Fbw7, a component of the SCF complex (SCF(Fbw7)) E3 ubiquitin ligase, specifically promoted the degradation and ubiquitination of KLF5 but had little effect on the stability of KLF4. Fbw7 interacted with KLF5 in a CDC4 phosphodegron (CPD)-dependent manner. Three CPDs were found in the KLF5 protein. Simultaneous mutation of these CPDs significantly abolished Fbw7-mediated ubiquitination and degradation. Furthermore, Fbw7 deficiency dramatically delayed KLF5 turnover and led to the accumulation of KLF5 protein in cancer cells. Glycogen synthase kinase-3beta could phosphorylate and promote Fbw7-mediated KLF5 degradation. More importantly, Fbw7 negatively regulated the biological activity of KLF5 in gene regulation and cell proliferation. Taken together, these data indicate that Fbw7 is a key negative regulator controlling KLF5-mediated cell proliferation and suggest an additional mechanism linking the loss of Fbw7 function to tumorigenesis.

867a GATA3 is Essential for Proper Morphogenesis of the Pylorus

BACKGROUND: Kruppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is highly expressed in dividing epithelial cells at the crypt base of the intestinal epithelium. KLF5 promotes proliferation In Vitro and In Vivo and is induced bymitogens and various stress stimuli. To study the role of KLF5 in maintaining intestinal homeostasis, we characterized the morphology of mice with conditional deletion of Klf5 in the gut using the villin-Cre (VilCre) recombinase system. METHODS: Mice carrying floxed alleles of Klf5 (Klf5fl/fl) were crossed with Vil-Cre;Klf5fl/+ mice to generate Vil-Cre;Klf5fl/fl mice. Klf5fl/fl littermates served as controls. Mice were sacrificed at 8 weeks of age and the small intestines and colon removed for histological examination. Immunohistochemical staining was used to examine the expression of Klf5, the proliferation marker, Ki67 and various markers of differentiation. RESULTS: Immunohistochemical staining of Klf5 in the small and large bowels of the VilCre;Klf5fl/fl mice showed that deletion of the floxed Klf5 alleles was variable, with the most complete deletion occurring in the distal small intestine and proximal colon. Initial characterization of the mice indicated that the Vil-Cre;Klf5fl/fl mice were leaner than the Klf5fl/fl littermates, weighing 18% less than genderand age-matched controls (n=5, P = 0.01). Histological staining of intestinal and colonic tissues from Vil-Cre;Klf5fl/fl mice revealed a disruption of normal crypt architecture and a regenerative phenotype, particularly in the colon. Staining with the proliferation marker, Ki67, showed proliferating cells scattered throughout the epithelia of the small and large intestines, with a phenotype similar to that of EphB2/EphB3-null mice. Paneth cells likewise migrated upward from the crypt base. Immunohistochemical staining for EphB2 and EphB3 in Klf5-deleted tissues revealed reduced expression of both receptors and loss of a normal expression gradient within the crypts. Reduction of EphB2 and EphB3 correlated with reduced expression of the differentiation markers intestinal alkaline phosphatase and carbonic anhydrase I. CONCLUSION: Deletion of Klf5 in the gut epithelium results in dramatic changes in morphology that correlate with reduced expression of Wnt targets, EphB2 and EphB3. The boundary between proliferating and differentiation zones are lost, resulting in scattering of proliferating cells throughout the epithelium and reduced differentiation. Thus, KLF5 is critical for maintenance of normal crypt architecture in the intestinal mucosa due to its effects on epithelial cell positioning and differentiation.

Inflammatory response underlies Pulmonary Hypertension

We have shown upregulation of IL‐6 mRNA, IL‐6 bioactivity and activation of PY‐STAT3, a proproliferative transcription factor in MCT‐induced pulmonary hypertension (PH), accompanied by a loss of endothelial caveolin‐1, a potent immunomodulator. To investigate the role of inflammation in hypoxia‐induced PH and compare with the MCT model, rats (¡Ö200 g) were subjected to hypobaric hypoxia or given MCT (sc, 60 mg/kg). Hemodynamic data, expression of caveolin‐1, eNOS, PY‐STAT3 were examined at 48h, 1 and 2 wks. Both models revealed progressive PH and right ventricular hypertrophy. Progressive PY‐STAT3 activation was observed at 48h, i.e. before the onset of PH (MCT, control [C], 100¡À0 vs 48h, 134¡À10*, hypoxia, C, 100¡À vs 48h, 453¡À70 *, *= P&lt;0.05 vs C). Transient increase in eNOS expression at 1 wk was observed in both models. Unlike the MCT model, hypoxia‐induced PH did not exhibit loss of caveolin‐1. Caveolin‐1, known to inhibit PY‐STAT3 activation may be dysfunctional in the hypoxia model. In conclusion, dissimilar models of PH show activation of PY‐STAT3, indicating that inflammatory response may have a significant role in the pathogenesis of PH. (Supported in part by MF children¡ s Hosp. Res. Fund).

Haploinsufficiency of Krüppel-Like Factor 5 Rescues the Tumor-Initiating Effect of the ApcMin Mutation in the Intestine

Inactivation of the tumor suppressor adenomatous polyposis coli, with the resultant activation of beta-catenin, is the initiating event in the development of a majority of colorectal cancers. Krüppel-like factor 5 (KLF5), a proproliferative transcription factor, is highly expressed in the proliferating intestinal crypt epithelial cells. To determine whether KLF5 contributes to intestinal adenoma formation, we examined tumor burdens in Apc(Min/+) mice and Apc(Min/+)/Klf5(+/-) mice. Compared with Apc(Min/+) mice, Apc(Min/+)/Klf5(+/-) mice had a 96% reduction in the number of intestinal adenomas. Reduced tumorigenicity in the Apc(Min/+)/Klf5(+/-) mice correlated with reduced levels and nuclear localization of beta-catenin as well as reduced expression of two beta-catenin targets, cyclin D1 and c-Myc. In vitro studies revealed a physical interaction between KLF5 and beta-catenin that enhanced the nuclear localization and transcriptional activity of beta-catenin. Thus, KLF5 is necessary for the tumor-initiating activity of beta-catenin during intestinal adenoma formation in Apc(Min/+) mice, and reduced expression of KLF5 offsets the tumor-initiating activity of the Apc(Min) mutation by reducing the nuclear localization and activity of beta-catenin.

Mechanism of shortened bones in mucopolysaccharidosis VII

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease in which deficiency in β-glucuronidase results in glycosaminoglycan (GAG) accumulation in and around cells, causing shortened long bones through mechanisms that remain largely unclear. We demonstrate here that MPS VII mice accumulate massive amounts of the GAG chondroitin-4-sulfate (C4S) in their growth plates, the cartilaginous region near the ends of long bones responsible for growth. MPS VII mice also have only 60% of the normal number of chondrocytes in the growth plate and 55% of normal chondrocyte proliferation at 3 weeks of age. We hypothesized that this reduction in proliferation was due to C4S-mediated overactivation of fibroblast growth factor receptor 3 (FGFR3). However, MPS VII mice that were FGFR3-deficient still had shortened bones, suggesting that FGFR3 is not required for the bone defect. Further study revealed that MPS VII growth plates had reduced tyrosine phosphorylation of STAT3, a pro-proliferative transcription factor. This was accompanied by a decrease in expression of leukemia inhibitory factor (LIF) and other interleukin 6 family cytokines, and a reduction in phosphorylated tyrosine kinase 2 (TYK2), Janus kinase 1 (JAK1), and JAK2, known activators of STAT3 phosphorylation. Intriguingly, loss of function mutations in LIF and its receptor leads to shortened bones. This suggests that accumulation of C4S in the growth plate leads to reduced expression of LIF and reduced STAT3 tyrosine phosphorylation, which results in reduced chondrocyte proliferation and ultimately shortened bones.