Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules α6-integrin, αv-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for α6-integrin, and negative or low αv-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-αvβ3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to αvβ3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation

Transmembrane protein tyrosine phosphatases, such as CD45, can act as both positive and negative regulators of cellular signaling. CD45 positively modulates T cell receptor (TCR) signaling by constitutively priming p56lck through the dephosphorylation of the C-terminal negative regulatory phosphotyrosine site. However, CD45 can also exert negative effects on cellular processes, including events triggered by integrin-mediated adhesion. To better understand these opposing actions of tyrosine phosphatases, the subcellular compartmentalization of CD45 was imaged by using laser scanning confocal microscopy during functional TCR signaling of live T lymphocytes. On antigen engagement, CD45 was first excluded from the central region of the interface between the T cell and the antigen-presenting surface where CD45 would inhibit integrin activation. Subsequently, CD45 was recruited back to the center of the contact to an area adjacent to the site of sustained TCR engagement. Thus, CD45 is well positioned within a supramolecular assembly in the vicinity of the engaged TCR, where CD45 would be able to maintain src-kinase activity for the duration of TCR engagement.

The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway

c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. Recent studies have demonstrated that the Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. We found here that transient expression of Crk induces JNK activation, and this activation was dependent on both the SH2- and SH3-domains of Crk. Expression of p130Cas (Cas), a major binding protein for the Crk SH2-domain, also induced JNK activation, which was blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk was effectively blocked by a dominant-negative form of Rac, suggesting for a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, was efficiently blocked by dominant-negative mutants of Crk. A dominant-negative form of Cas in turn blocked the integrin-, but not epidermal growth factor - nor v-Src-mediated JNK activation. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation.

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1 and α2β1 integrins—through induction of mRNAs encoding the α1 and α2 subunits. In contrast, VEGF did not induce increased expression of the α3β1 integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1 and α2β1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs. In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1 and α2β1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1 and α2β1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Serine Phosphorylation of Focal Adhesion Kinase in Interphase and Mitosis: A Possible Role in Modulating Binding to p130Cas

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas.

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Heterozygous reeler mice (HRM) haploinsufficient for reelin express ≈50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD67)-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD67 down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD67 knockout mice (HG67M). These mice exhibited a down-regulation of GAD67 mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.

Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Urokinase Receptor and Fibronectin Regulate the ERKMAPK to p38MAPK Activity Ratios That Determine Carcinoma Cell Proliferation or Dormancy In Vivo

We discovered that a shift between the state of tumorigenicity and dormancy in human carcinoma (HEp3) is attained through regulation of the balance between two classical mitogen-activated protein kinase (MAPK)-signaling pathways, the mitogenic extracellular regulated kinase (ERK) and the apoptotic/growth suppressive stress-activated protein kinase 2 (p38MAPK), and that urokinase plasminogen activator receptor (uPAR) is an important regulator of these events. This is a novel function for uPAR whereby, when expressed at high level, it enters into frequent, activating interactions with the α5β1-integrin, which facilitates the formation of insoluble fibronectin (FN) fibrils. Activation of α5β1-integrin by uPAR generates persistently high level of active ERK necessary for tumor growth in vivo. Our results show that ERK activation is generated through a convergence of two pathways: a positive signal through uPAR-activated α5β1, which activates ERK, and a signal generated by the presence of FN fibrils that suppresses p38 activity. When fibrils are removed or their assembly is blocked, p38 activity increases. Low uPAR derivatives of HEp3 cells, which are growth arrested (dormant) in vivo, have a high p38/ERK activity ratio, but in spite of a similar level of α5β1-integrin, they do not assemble FN fibrils. However, when p38 activity is inhibited by pharmacological (SB203580) or genetic (dominant negative-p38) approaches, their ERK becomes activated, uPAR is overexpressed, α5β1-integrins are activated, and dormancy is interrupted. Restoration of these properties in dormant cells can be mimicked by a direct re-expression of uPAR through transfection with a uPAR-coding plasmid. We conclude that overexpression of uPAR and its interaction with the integrin are responsible for generating two feedback loops; one increases the ERK activity that feeds back by increasing the expression of uPAR. The second loop, through the presence of FN fibrils, suppresses p38 activity, further increasing ERK activity. Together these results indicate that uPAR and its interaction with the integrin should be considered important targets for induction of tumor dormancy.