Analyzing the Role of αvβ8 Integrin in Glioma-Induced Angiogenesis

In this study we aimed to determine the functional roles for αvβ8 integrin in astrocytoma-induced angiogenesis. These studies originate from our analyses of αvβ8 integrin in developmental brain angiogenesis. αv and β8 knockout (KO) mice develop brain-specific vascular phenotypes that resemble vascular pathologies observed in the malignant astrocytoma, glioblastoma multiforme (GBM). Indeed, a murine xenograft model of astrocytoma suggested a role for the integrin in glioma-induced angiogenesis. Primary mouse astroglia were cultured from wild type (WT) and β8 KO neonates and were immortalized (HPV:E6/E7) and transformed (HRas:G12V). WT and β8 KO transformed astroglia were intracranially injected into athymic mice. WT tumors displayed pathological features of grade III astrocytomas, whereas β8 KO tumors resembled grade IV GBMs. KO tumors contained widespread edema and hemorrhage as well as pathological angiogenesis, as assessed by quantitation of microvascular density and blood vessel morphology. Additionally, exogenous expression of β8 integrin in β8 KO transformed astroglia resolved the pathologies observed in KO tumors giving further credence to the idea that loss of αvβ8 integrin expression correlates with tumorigenic potential of oncogene-transformed astroglia. To compliment our mouse model, several established human glioma cell lines were characterized for expression of αvβ8 integrin protein. Some of the cell lines displayed low expression of αvβ8 integrin, whereas others showed high levels, as compared to non-malignant human astrocytes. Intracranial implantation of high and low β8 integrin-expressing human glioma cell lines resulted in tumors exhibiting similar phenotypes to those observed in the mouse model; low expressers were marked by vascular pathologies indicative of β8 KO mouse tumors. Upon overexpression of β8 integrin in a low β8 integrin-expressing human glioma cell line, angiogenic pathologies were largely resolved. Moreover, intracranially injected αvHI- and αvLO-sorted GBM stem cells (GSCs) resulted in significantly different tumor sizes, where those GSCs endogenously expressing low levels of αv integrin formed two to three fold larger tumors. Furthermore, lentiviral knockdown of β8 integrin in transformed human astrocytes formed tumors that strikingly recapitulated the characteristics of the murine β8-/- tumors, exhibiting a significant increase in microvascular density leading to decreased overall survival. A paracrine mechanism was discovered involving endothelial cell homeostatic control governed by canonical TGFβ signaling initiated by αvβ8 integrin’s role in the latent cytokine’s activation. Diminished TGFβ signaling in tumor-associated endothelial cells promoted increased angiogenesis and decreased overall survival as a result of αvβ8 integrin’s loss on the tumor cell. Collectively, these data suggest an important functional role for αvβ8 integrin in glioma angiogenesis.

ANALYSIS OF β8 INTEGRIN IN NEUROGENESIS AND NEUROVASCULAR HOMEOSTASIS

Neurogenesis in the adult mouse brain occurs within the subventricular zone (SVZ) of the lateral ventricle. In the SVZ, neural stem cells (NSC) reside in a specialized microenvironment, or vascular niche, consisting of blood vessels and their basement membranes. Most NSCs in the SVZ differentiate into progenitor cells, which further differentiate to generate neuroblasts, which then migrate from the SVZ to the olfactory bulbs (OB) along the rostral migratory stream (RMS). ECM-mediated adhesion and signaling within the vascular niche likely contribute to proper NSC self-renewal, survival, differentiation and neuroblast motility. The mechanisms that control these events are poorly understood. Previous studies from our group and others have shown that loss of the ECM receptor, αvβ8 integrin, in NSCs in the embryonic mouse brain leads to severe developmental vascular defects and premature death. Here, the functions of αvβ8 integrin in the adult brain have been examined using mice that have been genetically manipulated to lack a functional β8 integrin gene. This study reveals that loss of β8 integrin leads to widespread defects in homeostasis of the neurovascular unit, including increased intracerebral blood vessels with enhanced perivascular astrogliosis. Additionally, β8 integrin dependent defects in NSC proliferation, survival, and differentiation, as well as neuroblast migration in the RMS were observed both in vivo and in vitro. The defects correlated, in part, with diminished integrin-mediated activation of TGFβ, an ECM ligand of β8 integrin. Collectively, these data identify important adhesion and signaling functions for β8 integrin in the regulation of neural stem and progenitor cells in the SVZ as well as in neuroblast migration along the RMS in the adult brain.

Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling

Band 4.1B is a cytoskeletal adaptor protein that regulates various cellular behavior; however, the mechanisms by which Band 4.1B contributes to intracellular signaling are unclear. This project addresses in vivo and in vitro functions for Band 4.1B in integrin-mediated cell adhesion and signaling. Band 4.1B has been shown to bind to β8 integrin, although cooperative functions of these two proteins have not been determined. Here, functional links between β8 integrin and Band 4.1B were investigated using gene knockout strategies. Ablation of β8 integrin and Band 4.1B genes resulted in impaired cardiac morphogenesis, leading to embryonic lethality by E11.5. These embryos displayed malformation of the outflow tract that was likely linked to abnormal regulation of cardiac neural crest migration. These data indicate the importance of cooperative signaling between β8 integrin and Band 4.1B in cardiac development. The involvement of Band 4.1B in integrin-mediated cell adhesion and signaling was further demonstrated by studying its functional roles in vitro. Band 4.1B is highly expressed in the brain, but its signaling in astrocytes is not understood. Here, Band 4.1B was shown to promote cell spreading likely by interacting with β1 integrin via its band 4.1, ezrin, radixin, and moesin (FERM) domain in cell adhesions. In astrocytes, both Band 4.1B and β1 integrin were expressed in cell-ECM contact sites during early cell spreading. Exogenous expression of Band 4.1B, especially its FERM domain, enhanced cell spreading on fibronectin, an ECM ligand for β1 integrin. However, the increased cell spreading was prohibited by blocking β1 integrin. These findings suggest that Band 4.1B is crucial for early adhesion assembly and/or signaling that are mediated by β1 integrin. Collectively, this study was the first to establish Band 4.1B as a modulator of integrin-mediated adhesion and signaling.

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Integrin function in T cell activation recapitulated in tumor growth

An in vitro model using highly purified freshly isolated T cells demonstrated that immobilized ligands for the integrin $\alpha4\beta1$ could cooperate to enhance mitogen signals delivered by coimmobilized anti-CD3 specfic monoclonal antibody OKT3. Costimulation through $\alpha4\beta1$ integrin lead to enhanced proliferation which depended on expression of both IL-2 as well as IL-2 receptor. The transcription factors NF-AT, AP-1, and NF-$\kappa$B, which are involved in the regulation of IL-2 as well as other cytokine genes, were weakly induced by anti-CD3 stimulation alone in electromobility shift assays, but were augmented significantly with $\alpha4\beta1$ costimulation. These results suggested that $\alpha4\beta1$ ligands delivered a growth promoting signal which could synergize with signals induced by engagement of the TCR/CD3 complex, and also suggested a dual function for integrins in both localization and subsequent delivery of a growth promoting signal for T lymphocytes. Integrin involvement in lymphocyte trafficking has been employed as a model for understanding tumor cell metastasis. Therefore we have extended the duality of integrin function in both homing and subsequent delivery of a growth promoting signal to include a role for integrins in providing growth stimulation for tumor cells. Using a gastric derived tumor line, inhibition of adhesion to substrate leads to G0/G1 cell cycle arrest, reduced cyclin A expression, and reduced phospholipid synthesis. This effect could be reversed upon $\alpha2\beta1$ integrin mediated reattachment to collagen. These observations demonstrated a role for an integrin in the growth regulation of a tumor line. The small GTP-binding protein Rho, implicated in phospholipid synthesis, can be inactivated by the ADP-ribosylation exoenzyme C3 from C. botulinum. Addition of C3 to cell cultures inhibited the growth promoting effect due to integrin mediated adhesion. Taken together, these results are consistent with a model for cooperative interaction between integrins and Rho leading to enhanced phospholipid synthesis and mitogen signaling. This model may provide a basis for understanding the phenomena of integrin costimulation in T cell activation. ^

Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Diversity in T cell costimulation by alpha4beta1 integrin

T cell activation requires antigen-specific T cell receptor signals that spatially and temporally coincide with a second costimulatory signal. CD28 and α4β1 integrin both function as T cell costimulators, but their individual mechanisms remain elusive. By directly comparing CD3-dependent functions and signaling pathways employed by these two costimulatory receptors, aspects of their individual signaling mechanisms are explored. We determined that CD28 and α4β1 integrins both use Src-family kinase Lck and MAPK Erk, but to different extents and functional ends. After identifying functional differences between CD28 and integrin costimulatory pathways, the focus of the study turned to integrin signaling in naïve and memory T cell subsets. CD45RO T cells are fully co-activated by natural β1 integrin ligands fibronectin (FN) and VCAM-1, β1 monoclonal antibody 33B6, as well as α4β1 monoclonal antibody 19H8 which binds a combinatorial epitope of the α4β1 heterodimer. While CD28 fully costimulates CD45RA T cells, the degree of activation from integrin ligands varies. FN costimulates CD3-dependent proliferation, IL-2 secretion, and early activation markers CD25 and CD69. However, β1 antibody 33B6, which binds to the same T cell integrins (α4β1 and α5β1) as natural ligand FN, failed to costimulate proliferation or IL-2 in the CD45RA subset, but retained the ability to regulate CD25 and CD69. Unique aspects of 19H8 signaling involve early Erk activation and IL-2 independent proliferation. Signaling defects through 33B6 ligation correlates with poor adhesion under fluid flow conditions, suggesting a cytoskeletal basis for signaling. All together, these data provide evidence for a mechanism of α4β1 integrin signaling and describe functional differences between naïve and memory T cells. ^

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

Beta 1-integrins are required for hippocampal AMPA receptor-dependent synaptic transmission, synaptic plasticity, and working memory.

Integrins comprise a large family of cell adhesion receptors that mediate diverse biological events through cell-cell and cell-extracellular matrix interactions. Recent studies have shown that several integrins are localized to synapses with suggested roles in synaptic plasticity and memory formation. We generated a postnatal forebrain and excitatory neuron-specific knock-out of beta1-integrin in the mouse. Electrophysiological studies demonstrated that these mutants have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent long-term potentiation. Despite the impairment in hippocampal synaptic transmission, the mutants displayed normal hippocampal-dependent spatial and contextual memory but were impaired in a hippocampal-dependent, nonmatching-to-place working memory task. These phenotypes parallel those observed in animals carrying knock-outs of the GluR1 (glutamate receptor subunit 1) subunit of the AMPA receptor. These observations suggest a new function of beta1-integrins as regulators of synaptic glutamate receptor function and working memory.

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^