Defining the Role for ZBP-89 in ATM-mediated DNA Damage Response to Irradiation in the Intestine

ZBP-89 (ZNF148, Zfp148) is a zinc-finger transcription factor that inhibits cellular proliferation when overexpressed in cell lines. ZBP-89 forms a protein-protein interaction with p53 and Ataxia-telangiectasia mutated (ATM). However, it is unclear how the interaction modulates the function of these two proteins in vivo. Double strand DNA breakage induced by -irradiation induces ATM phosphoinositol kinase activity, which initiates a cascade of events culminating in cell cycle arrest, DNA repair or apoptosis. Elevated levels of ZBP-89 induce growth arrest and apoptosis in gastrointestinal cell lines. Therefore, we hypothesize that ZBP-89 facilitates cell growth arrest and activation of the DNA repair pathway after ATM activation. To test this hypothesis, we irradiated 4 groups of mice—1) C57BL/6 mice without any genetic deletion; 2) mice with one or both copies of ATM deleted from the intestinal mucosa; 3) mice with the ATM deletion and one copy of Zfp148 deleted; 4) ATM deletion with both copies of Zfp148 deleted. After the intestines were fixed, paraffin-embedded and sectioned, I performed the de-paraffinization step for immunohistochemistry of ZBP-89, p53 and H2AX. To stain for ZBP-89 protein, I performed an antigen retrieval step using sodium citrate, followed by blocking with 3% hydrogen peroxide and serum. I then added the primary antibody then the secondary antibodies. I used diaminobenzidine (DAB), a chromogenic detection method to visualize the antigen-antibody complex. I then counterstained with hematoxylin. We predict that the mice with a deletion of ZBP-89 will exhibit more tissue damage as a result of the irradiation and DNA damage. Results are pending

A role for CITED2, a CBP/p300 interacting protein, in colon cancer cell invasion

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116311/1/feb2s0014579307012264.pd

An isoform of ZBP-89 predisposes the colon to colitis

Alternative splicing enables expression of functionally diverse protein isoforms. The structural and functional complexity of zinc-finger transcription factor ZBP-89 suggests that it may be among the class of alternatively spliced genes. We identified a human ZBP-89 splice isoform (ZBP-89(ΔN)), which lacks amino terminal residues 1–127 of the full-length protein (ZBP-89(FL)). ZBP-89(ΔN) mRNA was co-expressed with its ZBP-89(FL) cognate in gastrointestinal cell lines and tissues. Similarly, ZBP-89(ΔN) protein was expressed. To define its function in vivo, we generated ZBP-89(ΔN) knock-in mice by targeting exon 4 that encodes the amino terminus. Homozygous ZBP-89(ΔN) mice, expressing only ZBP-89(ΔN) protein, experienced growth delay, reduced viability and increased susceptibility to dextran sodium sulfate colitis. We conclude that ZBP-89(ΔN) antagonizes ZBP-89(FL) function and that over-expression of the truncated isoform disrupts gastrointestinal homeostasis

Stress Hematopoiesis Is Regulated by the Krüppel‐Like Transcription Factor ZBP‐89

Previous studies have shown that ZBP‐89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP‐89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89 − / − HSPC also resulted in a myeloid‐to‐B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89 − / − HSPC are reproduced in vitro in the ZBP‐89 ‐silenced multipotent hematopoietic cell line FDCP‐Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA‐1 in ZBP89‐deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP‐89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA‐1 . These data identify an important role for ZBP‐89 in regulating stress hematopoiesis in adult mouse bone marrow. S tem C ells 2014;32:791–801Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106141/1/stem1598.pd

Myeloid-derived suppressor cells in gastrointestinal cancers: A systemic review.

Gastrointestinal (GI) cancers are one of the most common malignancies worldwide, with high rates of morbidity and mortality. Myeloid-derived suppressor cells (MDSCs) are major components of the tumor microenvironment (TME). MDSCs facilitate the transformation of premalignant cells and play roles in tumor growth and metastasis. Moreover, in patients with GI malignancies, MDSCs can lead to the suppression of T cells and natural killer cells. Accordingly, a better understanding of the role and mechanism of action of MDSCs in the TME will aid in the development of novel immune-targeted therapies

Use of Flow Cytometry to Quantify Mouse Gastric Epithelial Cell Populations

Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10–20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50–80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44428/1/10620_2004_Article_224654.pd

ZBP-89 function in colonic stem cells and during butyrate-induced senescence

ZBP-89 (Zfp148, ZNF148) is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP- 89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a Zfp148Cre transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the Zfp148 locus in the colon using the Cdx2NLSCre transgene, reduced the size and number of polyps formed in the Apc-deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the Apc deletion, only when Zfp148 was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed ZNF148 gene expression, inducing CDKN2a (p16 ) gene expression. Collectively, Zfp148 mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the CDKN2A promoter. ERT2 ERT2 Ink4ahttp://deepblue.lib.umich.edu/bitstream/2027.42/168213/2/ZBP-89 function in colonic stem cells and during butyrate-induced senescence.pdfPublished versionDescription of ZBP-89 function in colonic stem cells and during butyrate-induced senescence.pdf : Published versio

Schlafen4+-MDSC in Helicobacter-induced gastric metaplasia reveals role for GTPases

IntroductionMDSCs express SCHLAFEN 4 (SLFN4) in Helicobacter-infected stomachs coincident with spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. We aimed to characterize SLFN4+ cell identity and the role of Slfn4 in these cells.MethodsSingle-cell RNA sequencing was performed on immune cells sorted from PBMCs and stomachs prepared from uninfected and 6-month H. felis-infected mice. Knockdown of Slfn4 by siRNA or PDE5/6 inhibition by sildenafil were performed in vitro. Intracellular ATP/GTP levels and GTPase activity of immunoprecipitated Slfn4 complexes were measured using the GTPase-Glo assay kit. The intracellular level of ROS was quantified by the DCF-DA fluorescent staining, and apoptosis was determined by cleaved Caspase-3 and Annexin V expression. Gli1CreERT2 x Slfn4fl/fl mice were generated and infected with H. felis. Sildenafil was administered twice over 2 weeks by gavaging H. felis infected mice ~4 months after inoculation once SPEM had developed.ResultsSlfn4 was highly induced in both monocytic and granulocytic MDSCs from infected stomachs. Both Slfn4+-MDSC populations exhibited strong transcriptional signatures for type-I interferon responsive GTPases and exhibited T cell suppressor function. SLFN4-containing protein complexes immunoprecipitated from myeloid cell cultures treated with IFNa exhibited GTPase activity. Knocking down Slfn4 or PDE5/6 inhibition with sildenafil blocked IFNa induction of GTP, SLFN4 and NOS2. Moreover, IFNa induction of Slfn+-MDSC function was inhibited by inducing their reactive oxygen species (ROS) production and apoptosis through protein kinase G activation. Accordingly, in vivo disruption of Slfn4 in Gli1CreERT2 x Slfn4fl/fl mice or pharmacologic inhibition by sildenafil after Helicobacter infection also suppressed SLFN4 and NOS2, reversed T cell suppression and mitigated SPEM development.ConclusionTaken together, SLFN4 regulates the activity of the GTPase pathway in MDSCs and precludes these cells from succumbing to the massive ROS generation when they acquire MDSC function

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of \u3e95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days

Epithelial cell turnover in relation to ongoing damage of the gastric mucosa in patients with early gastric cancer: increase of cell proliferation in paramalignant lesions

Gastric cancer is typically an end result of Helicobacter pylori -associated chronic gastritis. The pathogenesis is thought to involve effects on gastric mucosal epithelial cell turnover. In this study, we aimed to compare apoptosis and proliferation in the noncancer-containing mucosa of H. pylori -positive patients with early gastric cancer with these phenomena in H. pylori -positive controls.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41590/1/535_2004_Article_1549.pd