A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development

Coordinated cell migration is essential in many fundamental biological processes including embryonic development, organogenesis, wound healing and the immune response. During organogenesis, groups of cells are directed to specific locations within the embryo. Here we show that the zebrafish miles apart (mil) mutation specifically affects the migration of the heart precursors to the midline. We found that mutant cells transplanted into a wild-type embryo migrate normally and that wild-type cells in a mutant embryo fail to migrate, suggesting that mil may be involved in generating an environment permissive for migration. We isolated mil by positional cloning and show that it encodes a member of the lysosphingolipid G-protein-coupled receptor family. We also show that sphingosine-1-phosphate is a ligand for Mil, and that it activates several downstream signalling events that are not activated by the mutant alleles. These data reveal a new role for lysosphingolipids in regulating cell migration during vertebrate development and provide the first molecular clues into the fusion of the bilateral heart primordia during organogenesis of the heart.     

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.     

Regulation of cancer cell migration and bone metastasis by RANKL

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.     

Cloning and characterization of a gene that regulates cell adhesion.

Molecules of the cadherin and integrin families involved in cell-cell and cell-matrix adhesion have been implicated in epithelial differentiation, carcinogenesis and metastasis. Having observed that a colon cancer cell line bound avidly to collagen type I, inducing integrin-triggered glandular differentiation, we investigated the regulation of integrin function in these cells. We modified a mammalian expression cloning system that used monoclonal antibody selection to clone cell surface molecules. Using attachment to collagen type I to select for adhesive phenotype, we isolated a complementary DNA clone that increases cell adhesion to components of the extracellular matrix. The corresponding gene (cell adhesion regulator, CAR) is located on the long arm of chromosome 16 (16q) and encodes a protein of 142 amino acids, which has an N-terminal myristoylation motif and a consensus tyrosine-kinase phosphorylation site at the C terminus. Removal of this tyrosine residue abolishes enhancement of cell-matrix adhesion. This gene may encode an adhesion signal transduction molecule that functions in the suppression of tumour invasion.     

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.     

MAPK regulates cell cycle progression in pig oocytes and fertilized eggs

Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogenactivated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK₂ was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphorylation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while phosphorylation resumed at 16 h. These results suggest that MAPK may play an important regulatory role in MI-MII transition, pronucleus formation and the initiation of the first mitosis in pig eggs.     

Oxysterols direct immune cell migration via EBI2

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.     

A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division.

Ror kinases are a family of orphan receptors with tyrosine kinase activity that are related to muscle specific kinase (MuSK), a receptor tyrosine kinase that assembles acetylcholine receptors at the neuromuscular junction. Although the functions of Ror kinases are unknown, similarities between Ror and MuSK kinases have led to speculation that Ror kinases regulate synaptic development. Here we show that the Caenorhabditis elegans gene cam-1 encodes a member of the Ror kinase family that guides migrating cells and orients the polarity of asymmetric cell divisions and axon outgrowth. We find that tyrosine kinase activity is required for some of the functions of CAM-1, but not for its role in cell migration. CAM-1 is expressed in cells that require its function, and acts cell autonomously in migrating neurons. Overexpression and loss of cam-1 function result in reciprocal cell-migration phenotypes, indicating that levels of CAM-1 influence the final positions of migrating cells. Our results raise the possibility that Ror kinases regulate cell motility and asymmetric cell division in organisms as diverse as nematodes and mammals.     

WAVE2 is required for directed cell migration and cardiovascular development

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.     

Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. We studied the Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth. Deletion of the Lkb1 (also called Stk11) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors.     

c-Myc regulates mammalian body size by controlling cell number but not cell size.

Overexpression of the proto-oncogene c-myc has been implicated in the genesis of diverse human tumours. c-Myc seems to regulate diverse biological processes, but its role in tumorigenesis and normal physiology remains enigmatic. Here we report the generation of an allelic series of mice in which c-myc expression is incrementally reduced to zero. Fibroblasts from these mice show reduced proliferation and after complete loss of c-Myc function they exit the cell cycle. We show that Myc activity is not needed for cellular growth but does determine the percentage of activated T cells that re-enter the cell cycle. In vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a result of hypotrophy. We find that c-myc substitutes for c-myc in fibroblasts, indicating they have similar biological activities. This suggests there may be fundamental differences in the mechanisms by which mammals and insects control body size. We propose that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size.     

Cytokine-induced pseudopodial protrusion is coupled to tumour cell migration

Pseudopodia protrusion is a prominent feature of actively motile cells in vitro and invading tumour cells in vivo; however, the function and regulation of pseudopodia are poorly understood. Tumour autocrine motility factor (AMF) represents a new class of cytokines which are secreted by tumour cells and embryonic cells and induce random motility in the producer cells or in heterologous cells with appropriate receptors. Here we report that a major effect of this factor is to induce the extension of cell pseudopodia before cell translocation. Using a new method to quantify and isolate pseudopodia, we find that human breast carcinoma cell AMF (at concentrations of 1 nM or below) stimulates random pseudopodia formation in a dose-dependent and time-dependent manner. Anti-AMF antibodies inhibit pseudopodia protrusion and cell motility, showing the importance of pseudopodia formation during locomotion. AMF-stimulated motility and pseudopodia formation occur on a wide variety of adhesive substrata which suggests that certain intrinsic motility events are independent of the attachment mechanism. Induced pseudopodia show a prominent axial actin network in the electron microscope. The number of laminin receptor and fibronectin RGD recognition sites is increased by a factor of 20 in the induced pseudopodia when compared to the average distribution in unstimulated cells. Exploratory pseudopodia regulated by cell-derived motility factors contain receptors for matrix proteins and could serve as 'senseorgans' essential to the process of cell locomotion.     

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.     

Myofibrillogenesis regulator-1 promotes cell adhesion and migration in human hepatoma cells

Myofibrillogenesis regulator-1 (MR-1) is a gene overexpressed usually in many human cancers. However, the effects of MR-1 on cell proliferation, adhesion, migration and genome-wide gene regulation are still unclear. In this study, a human hepatoma cell line that highly overexpresses MR-1, BEL-7402/MR-1 cells was established. While the high expression of MR-1 did not promote cell proliferation, it significantly increased cell spreading, adhesion and migration compared with control cells. A total of 147 genes were regulated by MR-1 expression, 46 genes were down-regulated and 101 genes were up-regulated by MR-1 overexpression. Many of these genes were related to cell adhesion, cytoskeletal regulation, MAPK signaling, and cell cycle related pathways. Western blot analysis further confirmed the regulation of pathways associated with migration by MR-1. These results suggest that MR-1 is involved in the regulation of cancer cell adhesion, migration and related gene expression.     

Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption

Aquaporin-1 (AQP1) is a water channel protein expressed widely in vascular endothelia, where it increases cell membrane water permeability. The role of AQP1 in endothelial cell function is unknown. Here we show remarkably impaired tumour growth in AQP1-null mice after subcutaneous or intracranial tumour cell implantation, with reduced tumour vascularity and extensive necrosis. A new mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type and from AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of non-endothelial cells with AQP1 or with a structurally different water-selective transporter (AQP4) accelerated cell migration and wound healing in vitro. Motile AQP1-expressing cells had prominent membrane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. Our findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumour spread and organ regeneration.     

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.