Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.     

Rac is involved in early TCR signaling

The GTPase Rac controls signaling pathways often related to actin polymerization in various cell types. In T lymphocytes, Rac is activated by Vav, a major component of the multiprotein transduction complex associated to the TCR. Although profound signaling defects have been observed in Vav-deficient mice, a role of Rac in the corresponding early TCR signaling has not been tested directly. This question was investigated in Jurkat T cells transfected with either a dominant-negative (RacN17) or a constitutively active (RacV12) form of Rac. In T cells expressing either RacN17 or RacV12, the anti-CD3-induced Ca2+ response and production of inositol-1,4,5-trisphosphate were inhibited. The basal level of phosphatidylinositol-4,5-bisphosphate was not significantly diminished by Rac mutants. The major inhibitory effect of Rac mutants on Ca2+ signaling is exerted on the activity of phospholipase C-gamma and, before that, on the phosphorylation of ZAP-70 and of the linker molecule for activation of T cells, LAT. An anti-CD3-induced increase in actin polymerization was observed in control cells but not in cells transfected with a Rac mutant. In addition, latrunculin, which binds to monomeric actin, simultaneously inhibited basal and CD3-induced actin polymerization and Ca2+ signaling. These findings suggest a link between the effects exerted by Rac mutants on cortical actin polymerization and on TCR signaling. Rac cycling between its GTP- and GDP-bound states is necessary for this signaling. Alterations observed in early TCR-dependent signals suggest that Rac contributes to the assembly of the TCR-associated multiprotein transduction complex.     

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.     

N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.     

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

Scalable producing embryoid bodies by rotary cell culture system and constructing engineered cardiac tissue with ES-derived cardiomyocytes in vitro

Embryonic stem (ES) cells are of significant interest either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. ES cells aggregation is important for embryoid bodies (EBs) formation and the subsequent generation of ES cell derivatives. This study was conducted to describe scalable production of EBs by the rotary cell culture system (RCCS, STLV type) and estimate the feasibility of constructing engineered cardiac tissue (ECT). In comparison with suspension culture in a Petri dish, the efficiency of the dynamic process was analyzed with respect to the yield of EB formation and their cardiomyocyte differentiation. Cardiomyocyte differentiation was evaluated by immunohistochemical analysis. After the elementary enrichment by gradient percoll, ES cell-derived cardiomyocytes were applied to construct ECT. Cell gross morphology, spatial distribution, and ultrastructure were evaluated by using histological analysis, confocal laser scanning microscopy, and transmission electron microscopy. Results showed that EB efficiencies in STLV were nearly 1.5-2.0 times higher than that of liquid suspension cultures, and cardiomyocyte differentiation of EBs progressed in a normal course after the dynamic cultivation in STLV. Additionally, the differentiated cultures could be enriched elementarily by gradient percoll. Once cast into the collagen strand, cells grew well and became more matured in Petri dishes. Synchronous contraction of the cell cluster was observed on the surface of the ECT, and cell connection was also established. It was the first report to have beating ES-derived cardiomyocytes on a 3-D collagen scaffold, which might provide a promising model for physiological and pharmacological studies and tissue replacement therapy.     

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.     

Abl tyrosine kinase promotes dorsal ruffles but restrains lamellipodia extension during cell spreading on fibronectin

The nonreceptor Abl tyrosine kinase stimulates F-actin microspikes and membrane ruffles in response to adhesion and growth factor signals. We show here that induced dimerization of Abl-FKBP, but not the kinase-defective AblKD-FKBP, inhibits cell spreading on fibronectin. Conversely, knockdown of cellular Abl by shRNA stimulates cell spreading. The Abl kinase inhibitor, imatinib, also stimulates cell spreading and its effect is overridden by the imatinib-resistant AblT315I. Expression of Abl but not AbkKD in Abl/Arg-deficient cells again inhibits spreading. Furthermore, Abl inhibits spreading of cells that express the activated Rac, RacV12, correlating with RacV12 localization to dorsal membrane protrusions. Ectopic expression of CrkII, a Rac activator that is inactivated by Abl-mediated tyrosine phosphorylation, antagonizes Abl-mediated dorsal membrane localization of RacV12. Ectopic expression of a dynamin-2 mutant, previously shown to induce Rac-GTP localization to the dorsal membrane, abolishes the stimulatory effect of imatinib on cell spreading. These results suggest that Abl tyrosine kinase, through CrkII phosphorylation and in collaboration with dynamin-2 can regulate the partitioning of Rac-GTP to favor dorsal ruffles during cell spreading. The Abl-dependent dorsal membrane localization of activated Rac explains its positive role in ruffling and negative role in cell spreading and migration.     

Differentiation of nonbeating embryonic stem cells into beating cardiomyocytes is dependent on downregulation of PKC beta and zeta in concert with upregulation of PKC epsilon

Cardiomyocyte differentiation overall has been analyzed in vivo and in vitro at the molecular level by homologous recombination, gene mutation studies, and by transgenics; however, the roles of many signal transduction mechanisms that drive this differentiation process are still not fully understood. One set of signal transduction components that has been studied in detail in mature, differentiated cardiomyocytes is the PKC isotype superfamily. However, while the function of each isotype is slowly being uncovered in adult cardiomyocytes, limited information persists concerning their function in the differentiation process of cardiomyocytes. To begin analyzing the function of specific PKC isotypes in the differentiation process, we employed an established model for differentiating ES cells into cardiomyocyte-positive embryoid bodies (EBs) in vitro. RT-PCR, Western analyses, and confocal microscopy all showed that the expression of specific PKC isotypes was significantly changed as ES cells differentiated into cardiomyocytes. More importantly, by using antagonists specific for each isotype we found that this change was a final step in the differentiation process. PKC beta and zeta downregulation served to promote differentiation (beating), while upregulation of PKC epsilon appeared to amplify differentiation (beating). Finally, melding classical tools (i.e., ionic exchange glass beads) with recently developed methods for differentiating ES cells creates a possible novel technique for investigating differentiation of ES cells into cardiomyocytes as well as other cell types.     

Activation of protein kinase D3 by signaling through Rac and the alpha subunits of the heterotrimeric G proteins G12 and G13

PKD is the founding member of a novel protein kinase family that also includes PKD2 and PKD3. PKD has been the focus of most studies up to date, but little is known about the mechanisms that mediate PKD3 activation. Here, we show that addition of aluminum fluoride to COS-7 cells cotransfected with PKD3 and Galpha13 or Galpha12 induced PKD3 activation, which was associated with a transient plasma membrane translocation of cytosolic PKD3. Treatment with Clostridium difficile toxin B blocked PKD3 activation induced by either bombesin or by aluminum fluoride-stimulated Galpha12/13 but did not affect Galphaq-induced PKD3 activation. Furthermore, PKD3 immunoprecipitated from cells cotransfected with a constitutively active Rac (RacV12) exhibited a marked increase in PKD3 basal catalytic activity. In contrast, cotransfection with active Rho (RhoQ63L), Cdc42 (Cdc42Q61L), or Ras (RasV12) did not promote PKD3 activation. Expression of either COOH-terminal dominant-negative fragment of Galpha13 or dominant negative Rac (Rac N17) attenuated bombesin-induced PKD3 activation. Treatment with protein kinase C (PKC) inhibitors prevented the increase in PKD3 activity induced by RacV12 and aluminum fluoride-stimulated Galpha12/13. The catalytic activation of PKD3 in response to RacV12, alpha12/13 signaling or bombesin correlated with Ser-731/Ser-735 phosphorylation in the activation loop of this enzyme. Our results indicate that Galpha12/13 and Rac are important components in the signal transduction pathways that mediate bombesin receptor-induced PKD3 activation.     

Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.