Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.     

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.     

Stem cell factor induces ERM proteins phosphorylation through PI3K activation to mediate melanocyte proliferation and migration

Stem cell factor (SCF) activates a variety of signals associated with stimulation of proliferation, differentiation, migration, and survival in melanocytes. However, the molecular mechanisms by which SCF and its receptor Kit activates these signaling pathways simultaneously and independently are still poorly defined. Here, we examined whether SCF induces ezrin/radixin/moesin (ERM) proteins phosphorylation as a downstream target of PI3K in melanocytes. ERM proteins are cross-linkers between the plasma membrane and the actin cytoskeleton and are activated by phosphorylation of a C-terminal threonine residue. Our results demonstrated that SCF-induced ERM proteins phosphorylation on threonine residue and Rac1 activation in cultured normal human melanocytes through the activation of PI3K. The functional role of phosphorylated-ERM proteins was examined using melanocytes infected with adenovirus carrying a dominant negative mutant (Ala-558, TA) or wild type of moesin. In the TA moesin-overexpressing melanocytes, SCF-induced cell proliferation and migration were inhibited. Thus, our results indicate that phosphorylation of ERM proteins plays an important role in the regulation of SCF-induced melanocyte proliferation and migration.     

Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway

The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT(1)R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension.     

Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors.     

Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis

Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis "decision." Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.     

Distinct functions of erythropoietin and stem cell factor are linked to activation of mTOR kinase signaling pathway in human erythroid progenitors

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis. The concerted effects of these two growth factors are responsible for guiding initial commitment, expansion and differentiation of progenitors. The aim of the study was to identify signaling elements pertinent to translational control and elucidate whether both cytokines can contribute to protein translation providing some functional redundancy as seen with respect to apoptosis. The current study focused on non-apoptotic functions of SCF mediated through mTOR/p70S6 leading to protein translation and cell proliferation. We utilized a human primary erythroid progenitors and erythroblasts that are responsive to EPO and SCF to investigate the activation of mTOR/p70S6 kinases and their downstream effectors, the pathway primarily responsible for protein translation. We showed that mTOR, p70S6 kinases and their downstream signaling elements 4EBP1 and S6 ribosomal protein are all activated by SCF but not by EPO in primary erythroid progenitors. We also found that SCF is the sole contributor to activation of the protein translational machinery and activation of mTOR/p70S6 pathway is confined to the proliferative phase of erythroid differentiation program. Altogether these results demonstrate that unlike the survival function which is supported by both EPO and SCF protein translation essential for proliferation is governed by only SCF.     

Specificity of Rho insert-mediated activation of phospholipase D1

Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.     

Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress

The development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte that is now beginning to be characterized. Little is known about the mechanisms underlying the beginning of the oocyte growth and the acquisition of the competence to resume meiosis by the growing oocyte. In the present study, we devised a multistep culture system for mouse oocytes obtained from 15.5- to 16.5-days postcoitum embryos (mean diameter +/- SEM, 9.7 +/- 1.3 microm), allowing three stages of the oocyte growth to be identified: (i) an early stage in which the oocyte growth is induced by direct stimulation of a soluble growth factor, namely stem cell factor (SCF), independent of the formation of gap junctions with granulosa cells; (ii) a second phase in which the oocyte growth depends on the combined action of SCF and contacts with granulosa cells; and (iii) a third phase of granulosa cell-dependent, SCF-independent growth. At each stage, key events of oocyte development and differentiation, such as the c-kit reexpression, the early zona pellucida assembly, and the beginning of follicologenesis, were observed to occur independently by the presence of SCF. At the end of the in vitro growing phases, lasting 18-20 days, oocytes reached a size (50 +/- 2.5 microm) and a chromatin differentiation (stage I-II) equivalent to those of 9- to 10-day-old preantral oocytes and were unable to complete the growth phase. About 50% of the in vitro-grown oocytes were induced to resume meiosis by okadaic acid (OA) treatment. However, a significant fraction of them (48%) showed inability to maintain the chromosome condensation in M-phase. When in vitro-grown oocytes were treated with UO126, a specific MEK inhibitor that prevents activation of mitogen-activated protein kinases (ERK-1 and ERK-2), for 1 h before, during, and following OA treatment, only 22% of oocytes underwent germinal vesicle breakdown after 24 h from the OA treatment. These studies demonstrate that SCF alone can induce the onset of the oocyte growth. This is, however, not sufficient to fully activate the mechanisms governing the acquisition of the meiotic competence previously described as a 15-day oocyte-autonomous clock starting at the onset of growth. The inability of oocytes to progress into the last stages of growth and the lack of synchrony between nuclear and cytoplasm maturation showed by a subset of them resemble the characteristics of oocytes from connexin-37- and -43-deficient mice and indicate the preantral/antral transition point as a critical stage of oocyte development requiring the coordinated differentiation of the oocyte with granulosa cells and the maintenance of adequate communication between these two cell types to assure the correct oocyte meiotic maturation.     

Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila

The final stage of spermatid terminal differentiation involves the removal of their bulk cytoplasm in a process known as spermatid individualization. Here we show that apoptotic proteins play an essential role during spermatid individualization in Drosophila melanogaster. Several aspects of sperm terminal differentiation, including the activation of caspases, are reminiscent of apoptosis. Notably, caspase inhibitors prevent the removal of bulk cytoplasm in spermatids and block sperm maturation in vivo, causing male sterility. We further identified loss-of-function mutations in one of the two Drosophila cyt-c genes, cyt-c-d, which block caspase activation and subsequent spermatid terminal differentiation. Finally, a giant ubiquitin-conjugating enzyme, dBruce, is required to protect the sperm nucleus against hypercondensation and degeneration. These observations suggest that an apoptosis-like mechanism is required for spermatid differentiation in Drosophila.     

The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4

The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.     

Down-regulation of Myc is essential for terminal erythroid maturation

Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.