Sebastian Brenner

NAD(P)H Oxidase 1, a Product of Differentiated Colon Epithelial Cells, Can Partially Replace Glycoprotein 91phox in the Regulated Production of Superoxide by Phagocytes

Reactive oxygen species (ROS) serve several physiological functions; in some settings they act in host defense, while in others they function in cellular signaling or in biosynthetic reactions. We studied the expression and function of a recently described source of ROS, NAD(P)H oxidase 1 or Nox1, which has been associated with cell proliferation. In situ hybridization in mouse colon revealed high Nox1 expression within the lower two-thirds of colon crypts, where epithelial cells undergo proliferation and differentiation. Human multitumor tissue array analysis confirmed colon-specific Nox1 expression, predominantly in differentiated epithelial tumors. Differentiation of Caco2 and HT29 cells with 1α,25-dihydroxyvitamin D3 or IFN-γ enhances Nox1 expression and decreases cell proliferation, suggesting that Nox1 does not function as a mitogenic oxidase in colon epithelial cells. Transduction with retrovirus encoding Nox1 restored activation and differentiation-dependent superoxide production in gp91phox-deficient PLB-985 cells, indicating close functional similarities to the phagocyte oxidase (phox). Furthermore, coexpression of cytosolic components, p47phox and p67phox, augments Nox1 activity in reconstituted K562 cells. Finally, Nox1 partially restores superoxide production in neutrophils differentiating ex vivo from gp91phox-deficient CD34+ peripheral blood-derived stem cells derived from patients with X-linked chronic granulomatous disease. These studies demonstrate a significant functional homology (cofactor-dependent and activation-regulated superoxide production) between Nox1 and its closest homologue, gp91phox, suggesting that targeted up-regulation of Nox1 expression in phagocytic cells could provide a novel approach in the molecular treatment of chronic granulomatous disease.

S1P1 overexpression stimulates S1P-dependent chemotaxis of human CD34+ hematopoietic progenitor cells but strongly inhibits SDF-1/CXCR4-dependent migration and in vivo homing

The CXC chemokine receptor 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1) regulate egress and homing of hematopoietic stem cells. Activation of sphingosine-1-phosphate (S1P) receptors (S1P(1-5)) modulates chemokine-induced migration of lymphocytes and hematopoietic stem cells. To analyze the influence of S1P(1) on SDF-1-dependent chemotaxis and trafficking, we overexpressed S1P(1) in CD34+ mobilized peripheral blood progenitor cells (PBPCs). Using a gamma-retroviral vector, transgene overexpression was achieved in more than 90% of target cells. S1P(1) transgene positive PBPCs showed enhanced chemotaxis towards S1P. S1P(1) overexpression resulted in reduced CXCR4 surface expression levels and strong inhibition of SDF-1-dependent ERK1/2 phosphorylation and Ca(2+) flux. Furthermore, SDF-1-dependent migration of S1P(1) overexpressing PBPCs or Jurkat cells was reduced up to 10-fold. Sublethally irradiated NOD/SCID mice were transplanted with 6-day cultured PBPCs overexpressing either S1P(1)-IRES-GFP or GFP alone. Screening for GFP positive human cells in the mouse bone marrow 20h after transplantation revealed an eightfold reduction in bone marrow homing of S1P(1) transgene expressing cells. Our data suggest that S1P(1) acts as an inhibitor of CXCR4-dependent migration of hematopoietic cells to sites of SDF-1 production.

Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells

OBJECTIVE The Notch signaling pathway has been shown to play a role in bone marrow-derived stromal cell differentiation, however, the precise outcome of Notch activation remains controversial. The aim of this study was to evaluate the effect of Notch signaling in primary human bone marrow-derived stromal cells (hBMSCs). MATERIALS AND METHODS hBMSCs were transduced to >90% with lentiviral vectors containing either human notch1 intracellular domain (NICD), jagged1, or dominant negative mastermind1. Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiation was quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney (ALPL) activity and expression of adipogenic or osteogenic marker genes. RESULTS NICD and jagged1 transgene-expressing hBMSCs demonstrated enhanced mineralization, nodule formation, and ALPL activity in osteogenic differentiation media. These findings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL. In contrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation and reduced peroxisome proliferator-activated receptor-gamma, fatty acid binding protein 4, and adiponectin precursor gene expression. Co-overexpression of dominant negative mastermind1 and NICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, high endogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPL activity compared to a group of samples with low ALPL activity. CONCLUSION In summary, our data suggest that induction of Notch signaling enhances the osteogenic differentiation of hBMSCs while inhibiting the adipogenic fate.

Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy

Over the past dozen years, the majority of clinical gene therapy trials for inherited genetic diseases and cancer therapy have been performed using murine onco-retrovirus as the gene delivery vector. The earliest systems used were relatively inefficient in both the rates of transduction and expression of the transgene. Formidable obstacles inherent in the cell biology and/or the immunology of the target cell systems limited the efficacy of gene therapy for many target diseases. Development of novel retrovirus gene transfer systems that are in progress have begun to overcome these obstacles. Evidence of this progress is the recent successful functional correction of the immune T and B lymphocyte deficiency in patients with X-linked severe combined immunodeficiency (X-SCID) and adenosine deaminase (ADA)-deficient SCID following onco-retrovirus vector ex vivo transduction of autologous marrow stem cells [Science 296 (2002) 2410; Science 288 (2000) 669; N. Engl. J. Med. 346 (2002) 1185]. These achievements of prolonged clinical benefit from gene therapy were tempered by the finding of insertional mutageneses in two of the treated X-SCID patients [N. Engl. J. Med. 348 (2003) 255].

The histone demethylase UTX regulates stem cell migration and hematopoiesis.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Patients with Chronic Granulomatous Disease Have a Reduced Peripheral Blood Memory B Cell Compartment

In this study, we have identified an altered B cell compartment in patients with chronic granulomatous disease (CGD), a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH activity. This is characterized by an expansion of CD5-expressing B cells, and profound reduction in B cells expressing the memory B cell marker, CD27. Both findings were independent of the age, genotype, and clinical status of the patients, and were not accompanied by altered CD5 and CD27 expression on T cells. Focusing on CD27-positive B cells, considered to be memory cells based on somatically mutated Ig genes, we found that the reduction was not caused by CD27 shedding or abnormal retention of CD27 protein inside the cell. Rather, it was determined that CD27-negative B cells were, appropriately, CD27 mRNA negative, consistent with a naive phenotype, whereas CD27-positive B cells contained abundant CD27 mRNA and displayed somatic mutations, consistent with a memory B cell phenotype. Thus, it appears that CGD is associated with a significant reduction in the peripheral blood memory B cell compartment, but that the basic processes of somatic mutation and expression of CD27 are intact. X-linked carriers of CGD revealed a significant correlation between the percentage of CD27-positive B cells and the percentage of neutrophils with normal NADPH activity, reflective of the degree of X chromosome lyonization. These results suggest a role for NADPH in the process of memory B cell formation, inviting further exploration of secondary Ab responses in CGD patients.

Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness.

Impact of CXCR4 inhibition on FLT3-ITD−positive human AML blasts

OBJECTIVE Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD-positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition. MATERIALS AND METHODS We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34(+) hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD. RESULTS We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor-1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD-positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area-forming cells compared to FLT3-wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area-forming cells and proliferation of FLT3-ITD-positive cells, but did not affect FLT3-wild-type cells-highlighting the critical interaction between CXCR4 and FLT3-ITD. CONCLUSION CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD-positive AML.

Combining SDF-1/CXCR4 antagonism and chemotherapy in relapsed acute myeloid leukemia

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mRNA transfection of CXCR4-GFP fusion--simply generated by PCR-results in efficient migration of primary human mesenchymal stem cells.

We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.