The Rho small GTPase: functions in health and disease.

Cell shape changes, contractility, adhesion, migration, gene transcription, cytokinesis, membrane trafficking, and growth, require Rho small GTPase function. The basis for this is that Rho regulates actin filament assembly, and serum response factor (SRF)-mediated gene transcription. Upon activation by serum or cell adhesion, Rho stimulates a distinct signal transduction pathway that induces cytoskeletal and transcriptional responses through diverse effectors. Rho activity is tightly controlled by guanine nucleotide exchange factors, GTPase activating proteins, and guanine dissociation inhibitors. Dysregulation of the Rho pathway is implicated in multiple pathological conditions including cancer and metastasis, cardiovascular disease, bacterial and viral pathogenesis, hepatic disease, and developmental disorders.

Physical and functional interactions of Galphaq with Rho and its exchange factors.

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.

A rho exchange factor mediates thrombin and Galpha(12)-induced cytoskeletal responses.

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106). The involvement of the G(12) family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Galpha(12) or Galpha(13) induced cell rounding, and antibodies to Galpha(12) or Galpha(13) blocked the response to thrombin. In contrast, activation or inhibition of Galpha(q) function had relatively little effect. The cytoskeletal response to Galpha(12) was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Galpha(12). The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Galpha(12) protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.

Activation of the Lbc Rho exchange factor proto-oncogene by truncation of an extended C terminus that regulates transformation and targeting.

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted alpha-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the alpha-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

The membrane-bound isoform of stem cell factor synergizes with soluble flt3 ligand in supporting early hematopoietic cells in long-term cultures of normal and aplastic anemia bone marrow.

The hematopoietic growth factors stem cell factor (SCF) and flt3 ligand (flt3L) are produced within the hematopoietic microenvironment in a membrane-bound and soluble isoform. To elucidate the relevance of the two isoforms in the network of early-acting cytokines, we examined the interaction of membrane-bound SCF with the soluble forms of SCF and flt3L in long-term cultures of human bone marrow cells. Feeder layers of the murine SCF-deficient Steel stromal cell line transfected with human cDNA stably expressing SCF as a transmembrane molecule were used to support growth of mononuclear cells and CD34+ progenitors derived from normal human bone marrow or from hypoplastic marrow of patients with aplastic anemia (AA). The output of nonadherent progenitor cells representing colony-forming units (CFU) and high-proliferative potential colony-forming cells (HPP-CFC) was scored weekly in secondary methylcellulose cultures; the number of colonies derived from long-term culture-initiating cells (LTC-IC) was determined in nonadherent and adherent cells at 5 weeks. Membrane-bound SCF expressed in the stromal layer was more effective than soluble SCF and soluble flt3L in maintaining clonogenic progenitors. Furthermore, the transmembrane form of SCF effectively synergized with both exogenously supplied factors, although the effect of flt3L was superior to the effect of soluble SCF. In cultures of normal bone marrow cells, addition of flt3L enhanced the total number of CFU and HPP-CFC-type progenitors, primarily of the granulocyte/macrophage lineage, by six- to ninefold after 3 weeks and of LTC-IC-derived colonies by 13-fold after 5 weeks of culture. In cultures of AA cells, both the number and the survival rate of clonogenic precursors were severely impaired even in the presence of flt3L, which, however, yielded a two- to sixfold enhancement of CFU and HPP-CFC numbers at 1 to 2 weeks. In comparison with the hematopoietic function of human Dexter-type stroma cultures, murine feeders expressing high levels of membrane-associated human SCF were equivalent in supporting hematopoiesis during the initial 3 to 4 culture weeks when supplemented with flt3L. These results demonstrate that soluble flt3L interacts with membrane-bound SCF in supporting the long-term growth of bone marrow progenitor cells. The hypothesis that SCF and flt3L function synergistically during the very early stages of human hematopoiesis is thereby reinforced.

Distinct roles for DH and PH domains in the Lbc oncogene.

Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which specifically acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent fibroblasts and Rho-dependent actin stress fiber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress fibers and for efficient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.

Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway.

Constitutive activation of growth factor receptor signaling pathways leads to uncontrolled growth, but why tumor cells become anchorage independent is less clear. The fact that integrins transmit signals required for cell growth suggests that constitutive activation of steps downstream from integrins mediates anchorage independence. Since the small GTPase Rho may mediate integrin signal transduction, the effects of serum and the Rho nucleotide exchange factor oncogenes dbl and lbc on cell growth and signaling pathways were examined. Our data show that these oncogenes induce anchorage-independent but serum-dependent growth and stimulation of signaling pathways. These results show, therefore, that anchorage-independent growth results from constitutive activation of integrin-dependent signaling events. They also support the view that Rho is a functionally important mediator of integrin signaling.

Involvement of the small GTPase rho in integrin-mediated activation of mitogen-activated protein kinase.

Engagement and clustering of integrins triggers a number of intracellular signaling events, including activation of the mitogen-activated protein (MAP) kinases Erk1 and Erk2. To investigate the mechanism by which integrins mediate the activation of MAP kinases upon binding of NIH 3T3 cells to fibronectin, we assessed the effects of both inhibiting and activating the small GTPase Rho. We observed that inhibition of Rho by the Rho-specific inhibitor C3 exoenzyme or by a dominant negative Rho A (RhoN19) inhibited MAP kinase activation. Conversely, activation of Rho by expression of an activated Rho A mutant (RhoQ63L), or the Rho-specific guanine nucleotide exchange factor lbc, enhanced and partially mimicked activation of Erk2 by plating on fibronectin. These results therefore show that Rho is involved in the integrin-dependent activation of MAP kinase.

Direct involvement of the small GTP-binding protein Rho in lbc oncogene function.

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco-lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

Novel human oncogene lbc detected by transfection with distinct homology regions to signal transduction products.

In order to isolate transforming genes involved in leukemias, DNA from a CML acute phase sample was transfected into NIH-3T3 cells and found to be tumorigenic in nude mice. Partial genomic cloning using human repeat sequence as probe followed by cDNA cloning of this oncogene, termed lbc, was undertaken. The lbc cDNA sequence shows no identity to known proteins and codes for a predicted hydrophilic protein product of 47 kD, which contains several consensus kinase phosphorylation sites. The N-terminus encodes a consensus E-F hand motif followed by a region of homology to the transforming human oncogene dbl associated with regulatory activity for the ras superfamily of small G proteins, while the C-terminus contains homology with pleckstrin and rac protein kinase in a region which overlaps with the recently defined PH (pleckstrin homology) domain. Lbc expression is restricted to human hematopoietic cells and skeletal muscle, lung and heart. Transfection of 3T3 cells with an expression vector encoding lbc cDNA results in focus formation, demonstrating its biological activity. These data indicate that the lbc oncogene encodes a novel product implicated in distinct cellular signal transduction functions.

Identification and mutation of primary and secondary proteolytic cleavage sites in murine stem cell factor cDNA yields biologically active, cell-associated protein.

Phenotypic abnormalities of melanocytes, germ cells, and hematopoietic cells of Steel mice demonstrate the critical role of stem cell factor (SCF) in development. Production of SCF in the hematopoietic microenvironment as either a membrane-associated or soluble factor leads to pleiotropic effects on hematopoietic stem and progenitor cells and significant effects on the production of erythroid cells. Although the production of these two forms of SCF is highly regulated, the physiologic role(s) of membrane-associated and soluble SCF remain unclear. We have demonstrated that the generation of soluble murine SCF by murine stromal cells derived from the fetal hematopoietic microenvironment is dependent on two distinct proteolytic cleavage sites. The primary site in exon 6 is preferentially utilized in these cells. The secondary site located in exon 7 is utilized only in the absence of the primary site. Proteolytic processing at this secondary site appears to be species-specific, since the human protein sequence diverges at this site, and protein expressed from the human cDNA encoding this site in murine stromal cells remains largely membrane-associated. Site-directed mutagenesis of the murine SCF cDNA encoding both proteolytic cleavage sites leads to the generation of membrane-associated and biologically active SCF on murine stromal cells. These results suggest that the regulation of processing of the secondary proteolytic cleavage site could play a critical role in the function of membrane-associated SCF protein.

Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells.

Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression.

Evaluation of two ELISA's for detecting Chlamydia trachomatis from endocervical swabs.

Two enzyme immunoassays (EIAs) detecting Chlamydia trachomatis from endocervical swabs, Syva MicroTrak (MT) and Abbott Chlamydiazyme (CZ), were compared with a tissue culture (TC) standard. Initially, 8% (100 of 1250) of specimens were TC positive, yielding sensitivities of 94% (94 of 100) for MT and 79% (79 of 100) for CZ with identical 98% specificities (1129 of 1150 for MT and 1130 of 1150 for CZ). Discrepant specimens were retested by both EIAs and assayed for elementary bodies (EBs) by a fluorescent antibody test. After discrepancy analysis, 9.5% (118) of 1240 patients were either TC or EB positive, yielding sensitivities of 94.1% for MT (111 of 118) and 79.7% for CZ (94 of 118) with identical specificities of 100% (1122 of 1122). These results indicate that the MT is significantly more sensitive (p less than 0.05, McNemar test) than CZ in detecting C. trachomatis from endocervical swabs.

Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo.

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia.

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.

N-ras and human cancer.

Activated N-ras genes have been detected in a variety of human tumours and tumour derived cell lines. It has been proposed that mutation or amplification of an N-ras gene represents one of the key steps in the development of some human tumours. Here we discuss the various methods by which activated N-ras genes have been detected, the ways in which they have become activated, and their contributions to the transformed phenotype.

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia.

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

Maintenance of granulocyte-monocyte progenitor cells in liquid cultures of human foetal liver.

These studies describe a liquid suspension culture system for normal myeloid cells derived from human foetal liver. A simple one-step fractionation procedure was employed to obtain a cell population capable of expanding into all stages of myeloid differentiation, including committed myeloid progenitor cells (GM-CFC). Cell proliferation in these cultures resulted in the maintenance of early myeloid populations for up to a month. In order to extend myeloid cell maintenance, a specific factor in the form of media conditioned by human endothelial cells (endo C.M.) was used. Addition of endo C.M. to foetal liver cultures resulted in increased myeloid proliferation coupled to extensive myeloid differentiation. Clonally derived foetal liver culture cells proliferated for up to 2 months in the presence of endo C.M. before maturing into macrophages. These results show that endo C.M. exert an extensive proliferative effect on early myeloid cells as well as inducing their differentiation. The large quantity of cells in early stages of myeloid differentiation provided by foetal liver cultures may be useful for biochemical and molecular biology studies of myelopoiesis. In addition, these cultures are a potential source from which to derive normal myeloid lines. The separation of the potent proliferative activity present in endo C.M. may yield an effector which maintains human myeloid cell proliferation.