Notch signaling plays a key role in cardiac cell differentiation

Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Cardiac conduction cells are specialized cells responsible for orchestrating the rhythmic contractions of the heart. Here, we show that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker, HNK-1, at early stages. Misexpression of constitutively active Notch1 (NIC) in early heart tubes in chick exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in expression of cardiac muscle markers, but an increase in expression of conduction cell markers, HNK-1, and SNAP-25. However, the expression of the conduction marker connexin 40 was inhibited. Loss-of-function study, using a dominant-negative form of Suppressor-of-Hairless, further supports that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.     

Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration

Water influx through aquaporin-1 (AQP-1) has been linked to the ability of different cell types to migrate, and therefore plays an important part in processes like metastasis and angiogenesis. Since the erythroid growth factor erythropoietin (Epo) is now recognized as an angiogenesis promoter, we investigated the participation of AQP-1 as a downstream effector of this cytokine in the migration of endothelial cells. Inhibition of AQP-1 with either mercury ions (Hg²⁺) or a specific siRNA led to an impaired migration of EA.hy926 endothelial cells exposed to Epo (wound-healing assays). Epo also induced the expression of AQP-1 at mRNA and protein levels, an effect which was dependent on the influx of extracellular calcium through L-type calcium channels as well as TRPC3 channels.The relationship between Epo and AQP-1 was further confirmed at shorter exposure times, as the cytokine was unable to trigger calcium influxes in cells where AQP-1 had previously been knocked down. Moreover, Epo promoted changes in the subcellular localization of AQP-1 as well as rearrangements in the actin cytoskeleton, which are consistent with a migratory phenotype. Worthy of note, carbamylated erythropoietin (cEpo), the non-erythropoietic and non-promigratory derivative of Epo, was incapable of AQP-1 modulation.The therapeutical implications of aquaporin targeting in angiogenesis-related diseases highlight the importance of the present results in the context of the relationship between AQP-1 and Epo.     

Phosphoinositide signaling plays a key role in cytokinesis

To perform the vital functions of motility and division, cells must undergo dramatic shifts in cell polarity. Recent evidence suggests that polarized distributions of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, which are clearly important for regulating cell morphology during migration, also play an important role during the final event in cell division, which is cytokinesis. Thus, there is a critical interplay between the membrane phosphoinositides and the cytoskeletal cortex that regulates the complex series of cell shape changes that accompany these two processes.     

An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival

We recently identified neuregulin‐1 (NRG1) as a novel target of Notch1 required in Notch‐dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1‐dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1‐ERBB3‐ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells.

This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.     

Id-1 plays a key role in cell adhesion in neural stem cells through the preservation of RAP1 signaling

Reorganization of the cytoskeleton is necessary for apoptosis, proliferation, migration, development and tissue repair. However, it is well established that mutations or overexpression of key regulators contribute to the phenotype and progression of several pathologies such as cancer. For instance, c-src mutations and the overexpression of FAK have been implicated in the invasive and metastatic process, suggesting that components of the motility system may represent a new class of therapeutic targets. Over the last several years, we and others have established distinct roles for the Ste20-like kinase SLK, encompassing apoptosis, growth, motility and development. Here, we review the SLK field from its initial cloning to the most recent findings from our laboratory. We summarize the various roles of SLK and the biochemical mechanisms that regulate its activity. These various findings reveal very complex functions and pattern of regulation for SLK in development and cancer, making it a potential therapeutic target.     

Phospholipase D1 plays a key role in TNF-alpha signaling

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.     

Nuclear localization signal of ING4 plays a key role in its binding to p53

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.     

Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling

Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.     

Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells

Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a. Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.     

Human epididymis protein 4 (HE4) plays a key role in ovarian cancer cell adhesion and motility

Human epididymis protein 4 (HE4) is a novel and specific biomarker for epithelial ovarian cancer (EOC). We previously demonstrated that serum HE4 levels were significantly elevated in the majority of EOC patients but not in subjects with benign disease or healthy controls. However, the precise mechanism of HE4 protein function is unknown. In this study, we generated HE4-overexpressing SKOV3 cells and found that stably transduced cells promoted cell adhesion and migration. Knockdown of HE4 expression was achieved by stable transfection of SKOV3 cells with a construct encoding a short hairpin DNA directed against the HE4 gene. Correspondingly, the proliferation and spreading ability of HE4-expressed cells were inhibited by HE4 suppression. Mechanistically, impaired EGFR and Erk1/2 phosphorylation were observed in cells with HE4 knockdown. The phosphorylation was restored when the knockdown cells were cultured in conditioned medium containing HE4. Moreover, in vivo tumorigenicity showed that HE4 suppression markedly inhibited the growth of tumors. This suggests that expression of HE4 is associated with cancer cell adhesion, migration and tumor growth, which can be related to its effects on the EGFR-MAPK signaling pathway. Our results provide evidence of the cellular and molecular mechanisms that may underlie the motility-promoting role of HE4 in EOC progression. The role of HE4 as a target for gene-based therapy might be considered in future studies.     

A novel role for the Ste20 kinase SLK in adhesion signaling and cell migration

With over 60 members, the Sterile 20 family of kinases has been implicated in numerous biological processes, including growth, survival, apoptosis and cell migration. Recently, we have shown that, in addition to cell death, the Ste20-like kinase SLK is required for efficient cell migration in fibroblasts. We have observed that SLK is involved in cell motility through its effect on actin reorganization and microtubule-induced focal adhesion turnover. Scratch wounding of confluent monolayers results in SLK activation. The induction of SLK kinase activity requires the scaffold FAK and a MAPK-dependent pathway. However, its recruitment to the leading edge of migrating fibroblasts requires the activity of the Src family kinases. Since SLK is microtubule-associated, it may represent one of the signals delivered to focal contacts that induces adhesions turnover. A speculative model is proposed to illustrate the mechanism of SLK activation and recruitment at the leading edge of migrating cells.Key words: cell migration, cell adhesion, SLK, microtubules, adhesion turnoverCell migration is involved in multiple biological processes such as development, tissue regeneration, immune surveillance and tumor metastasis. Numerous studies reported a multitude of cellular and molecular players that take part in the signaling networks that regulate cell migration.^1,2 Recently, we reported the participation of a new member, the Ste20 serine/threonine kinase SLK, in the regulation of cell migration. We have shown that SLK is a novel adhesion disassembly signal that is activated and recruited downstream of the FAK/Src complex following scratch wound-induced migration.³ Furthermore, SLK-dependent signals are required to mediate microtubule-dependent focal adhesion tunrnover.³ These findings provide new insights into the mechanisms of cell migration and adhesion dynamics.Since sterile 20 protein (Ste20p) acts as a MAP4K in yeast, it was suggested that mammalian homologues of Ste20p also function as MAP4K.⁴ Several members of the Ste20 family of kinases have been identified in mammals and implicated in various biological processes such as stress responses, cell death and cytoskeletal reorganization.⁵ We and others previously identified a novel Ste20-related kinase termed SLK, which is a part of a signaling pathway mediating c-Jun terminal kinase 1 (JNK1) activation and apoptosis in cultured fibroblasts.^6–8 In addition, recent reports showed that SLK is involved in C2C12 myoblast differentiation and plays a role in cell cycle progression.^9,10 SLK is ubiquitously expressed, but during embryogenesis it is highly enriched in muscle and neuronal tissues.¹¹ It has been shown that SLK is associated with the microtubule cytoskeleton and we have demonstrated that SLK-induced disassembly of actin stress fibers can be inhibited by dominant negative Rac1.^12–14Recently, SLK was identified as a member of a new signaling pathway that induce vasodilatation in response to angiotensin II type 2 receptor activation.¹⁵ It was reported that SLK negatively regulates RhoA-dependent functions by phosphorylation of RhoA at Ser188.¹⁵ These findings suggest that SLK represents a novel relaxation signal involved in cytoskeletal remodeling and cell migration.We have observed that SLK is recruited to the leading edge of migrating fibroblasts by a mechanism involving c-Src signaling.³ The molecular mechanism regulating SLK recruitment is still unclear but is likely to implicate the association of SLK with another protein. The translocation of SLK could involve a microtubule-dependent mechanism leading to its redistribution to peripheral adhesions, using actin stress fibers as tracks. The Rho GTPases have been shown to be important in the targeting of signaling components, such as c-Src, to specific adhesion sites.^16,17 Whether SLK recruitment to the leading edge requires the Rho GTPases remains to be investigated. The Rho-mDia pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and is responsible for delivering APC/Cdc42 and c-Src to their respective sites of action.¹⁸ One attractive possibility is that mDia facilitates SLK-microtubule translocation in a c-Src dependent manner.Integrin molecules which link the extracellular matrix to the intracellular machinery are key players in initiating polarized cell migration into the wound. We investigated SLK activity in a scratch-induced migration model and have been able to decipher various signaling components regulating SLK activation.³ Using knockdown and dominant negative approaches, we showed that SLK is required for microtubule-dependent focal adhesion turnover and cell migration downstream of the FAK/Src complex.³The molecular mechanisms by which microtubules contribute to cell migration have been intensively studied. Geiger''s group provided the first demonstration that cytoskeletal modulation, such as microtubule disruption, triggers integrin-dependent signaling in the absence of external growth factor stimulation.¹⁹ The authors suggested that the involvement of microtubules in adhesion dependent signaling is related to microtubule interaction with the contractile actin-myosin system.¹⁹ By using a nocodazole washout system, it was shown that FAK and the GTPase dynamin are required for microtubule-induced focal adhesion disassembly.²⁰Adhesion turnover involves a number of adapters and signaling molecules, most of which are engaged in FAK signaling pathways.²¹ FAK stimulates adhesion disassembly through a signaling pathway that includes extracellular signal-regulated kinase (ERK) and myosin light chain kinase (MLCK).²² Our data have shown that SLK is activated downstream of FAK/Src/MAPK signaling, suggesting that SLK may be a new target of this pathway that leads to adhesion disassembly. Furthermore, if RhoA is a bona fide substrate for SLK in fibroblasts, then by phosphorylating and inhibiting RhoA, SLK could tilt the Rho/Rac antagonistic interplay toward relaxation and adhesion disassembly. Downstream targets of FAK and Src kinase activity often regulate the recruitment of adapter and structural protein complexes to adhesions.²² The integration of molecules such as zyxin, α-actinin or paxillin into focal contacts can lead to their stabilization and maturation into focal adhesions.²² Interestingly, depending on their phosphorylation state, these components can promote adhesion destabilization and turnover. Therefore, it is tempting to speculate that activated SLK at the leading edge may phosphorylate key signaling components to induce adhesion turnover.A recent study has shown that the frequency of microtubule catastrophes is higher at focal adhesion sites and this event leads to a local release of microtubule regulatory proteins, such as GEF-H1 and APC.²³ Signaling molecules that are released from the microtubules at adhesions could directly associate with molecular factors concentrated at the adhesion plaques, such as Src, PAK and Arp2/3. Furthermore, it was speculated that microtubule catastrophe could be associated with phosphorylated paxillin-dependent protein complexes.²³ One possibility is that through the microtubule, SLK is delivered to focal contacts or adhesions where it serves as a scaffold for disassembling signals. Alternatively, SLK may be phsophorylating key signaling molecules, which ultimately leads to adhesion destabilization and turnover.Overall, our recent findings suggest that SLK is novel regulator of focal adhesion turnover and cell migration (Fig. 1). The molecular mechanisms regulating SLK activity and SLK-dependent adhesion turnover remain to be uncovered and await the identification of SLK substrates.Open in a separate windowFigure 1Model for SLK activation and recruitment at the leading edge. A proportion of SLK is microtubule-associated, likely through a microtubule-binding protein (X). Following activation of the FAK/c-Src complex, signaling through the MAPK pathway can activate and recruit the microtubule-SLK complex, inducing adhesion turnover by destabilization of the actin network or focal contacts/adhesions through an unknown mechanism. (C) denotes a cargo protein coupling the microtubule to polymerized actin. Nocodazole treatment fails to recruit SLK resulting in stable adhesions.