TAGLN expression is deregulated in endometriosis and may be involved in cell invasion, migration, and differentiation

We found an increased expression of the TAGLN gene in endometriotic lesions compared with the eutopic endometrium of the same patients by real-time polymerase chain reaction. It is possible that this deregulation contributes to the development and maintenance of endometriosis by being involved in the pathways of organization of cytoskeletal architecture. (Fertil Steril (R) 2011;96:700-3. (C)2011 by American Society for Reproductive Medicine.)

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

Redox dysregulation in schizophrenia : implication for oligodendrocyte maturation and structural connectivity

Schizophrenia, which results from an interaction between gene and environmental factors, is a psychiatric disorder characterized by reality distortion. The clinical symptoms, which are generally diagnosed in late adolescence or early adulthood, partly derive from altered brain connectivity especially in prefrontal cortex. Disruption of neuronal networks implies oligodendrocyte and myelin abnormalities in schizophrenia pathophysiology. The mechanisms of these impairments are still unclear. Converging evidences indicate a role of redox dysregulation, generated by an imbalance between pro-oxidants and antioxidant defense mechanisms, in the development of schizophrenia pathophysiology. In particular, genetic and biochemical data indicate impaired synthesis of glutathione, the main cellular antioxidant and redox regulator. As oligodendrocyte maturation is dependent on redox state, we evaluated whether abnormal redox control could contribute to oligodendrocyte and myelin impairments in schizophrenia. We found that glutathione in prefrontal cortex of early psychosis patients and control subjects positively correlated with white matter integrity. We then further explored the interplay between glutathione and myelin using a translational approach. Our data showed that in mice with genetically impaired glutathione synthesis, oligodendrocyte late maturation as well as myelination was delayed in the anterior cingulate cortex. Specifically, oligodendrocyte number and myelin levels were lowered at peripubertal age, coincident in time with the peak of myelin- related gene expression during normal brain development. These data suggest that early adolescence is a vulnerable developmental period during which an adequate redox control is required for oligodendrocyte maturation and active myelination process. Consistently, oxidative stress mediated by psychosocial stress also delayed myelination in peripubertal mice. At cellular levels, impaired glutathione synthesis altered oligodendrocyte development at several levels. Using oligodendrocyte progenitor cells cultures, our data showed that glutathione deficiency was associated with (i) cell cycle arrest and a reduction in oligodendrocyte proliferation, and (ii) an impairment in oligodendrocyte maturation. Abnormal oligodendrocyte proliferation was mediated by upregulation of Fyn kinase activity. Consistently, under oxidative stress conditions, we observed abnormal regulation of Fyn kinase in fibroblasts of patients deficient in glutathione synthesis. Together, our data support that a redox dysregulation due to glutathione deficit could underlie myelination impairment in schizophrenia, possibly mediated by dysregulated Fyn pathway. Better characterization of Fyn mechanisms would pave the way towards new drug targets. -- La schizophrénie est une maladie psychiatrique qui se définit par une distorsion de la perception de la réalité. Les symptômes cliniques sont généralement diagnostiqués durant l'adolescence ou au début de l'âge adulte et proviennent de troubles de la connectivité, principalement au niveau du cortex préfrontal. Les dysfonctionnements des réseaux neuronaux impliquent des anomalies au niveau des oligodendrocytes et de la myéline dans la pathophysiologie de la schizophrénie. Les mécanismes responsables des ces altérations restent encore mal compris. Dans le développement de la schizophrénie, des évidences mettent en avant un rôle de la dérégulation rédox, traduit par un déséquilibre entre facteurs pro-oxydants et défenses antioxydantes. Des données génétiques et biochimiques indiquent notamment un défaut de la synthèse du glutathion, le principal antioxydant et rédox régulateur des cellules. Etant donné que la maturation des oligodendrocytes est dépendante de l'état rédox, nous avons regardé si une dérégulation rédox contribue aux anomalies de la myéline dans le cadre de la schizophrénie. Dans le cortex préfrontal des sujets contrôles et des patients en phase précoce de psychose, nous avons montré que le glutathion était positivement associé à l'intégrité de matière blanche. Afin d'explorer plus en détail la relation entre le glutathion et la myéline, nous avons mené une étude translationnelle. Nos résultats ont montré que des souris ayant un déficit de la synthèse du glutathion présentaient un retard dans les processus de maturation des oligodendrocytes et de la myélinisation dans le cortex cingulaire antérieure. Plus précisément, le nombre d'oligodendrocytes et le taux de myéline étaient uniquement diminués durant la période péripubertaire. Cette même période correspond au pic de l'expression des gènes en lien avec la myéline. Ces données soulignent le fait que l'adolescence est une période du développement particulièrement sensible durant laquelle un contrôle adéquat de l'état rédox est nécessaire aux processus de maturation des oligodendrocytes et de myélinisation. Ceci est en accord avec la diminution de myéline observée suite à un stress oxydatif généré par un stress psychosocial. Au niveau cellulaire, un déficit du glutathion affecte le développement des oligodendrocytes à différents stades. En effet, dans des cultures de progéniteurs d'oligodendrocytes, nos résultats montrent qu'une réduction du taux de glutathion était associée à (i) un arrêt du cycle cellulaire ainsi qu'une diminution de la prolifération des oligodendrocytes, et à (ii) des dysfonctionnements de la maturation des oligodendrocytes. Par ailleurs, au niveau moléculaire, les perturbations de la prolifération étaient générées par une augmentation de l'activité de la kinase Fyn. Ceci est en accord avec la dérégulation de Fyn observée dans les fibroblastes de patients ayant une déficience en synthèse du glutathion en condition de stress oxydatif. Les résultats de cette thèse soulignent qu'une dérégulation rédox induite par un déficit en glutathion peut contribuer aux anomalies des oligodendrocytes et de la myéline via le dysfonctionnement des voies de signalisation Fyn. Une recherche plus avancée de l'implication de Fyn dans la maladie pourrait ouvrir la voie à de nouvelles cibles thérapeutiques.

Thy-1-mediated cell-cell contact induces astrocyte migration through the engagement of αVβ3 integrin and syndecan-4.

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.

mTORC2 regulates PGE2-mediated endothelial cell survival and migration.

Prostaglandin E(2) (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE(2)-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE(2)-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE(2)-mediated endothelial cell responses.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration.

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.

mTORC2 regulates PGE(2)-mediated endothelial cell survival and migration

Prostaglandin E-2 (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE2-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE2-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE2-mediated endothelial cell responses.

mTORC2 is an important signaling intermediary in VEGF-mediated endothelial cell proliferation, survival and migration

Objective: The vascular endothelial growth factor (VEGF) is a prominent¦contributor of tumor angiogenesis. VEGF induces endothelial cell migration,¦proliferation and survival, which are critical steps for the development of new¦blood vessels, through the activation of the Mek/Erk and PI3K/Akt signaling¦pathways. Recent findings have demonstrated that mTORC2 regulates Akt and¦Erk in endothelial cells. The role of mTORC2 in VEGF-mediated endothelial¦cell responses has however not been characterized.¦Methods: We used human umbilical vein endothelial cells (HUVEC). The¦effects of VEGF on the Mek/Erk and PI3K/Akt pathway were analyzed by¦Western blot. Inhibition of mTORC2 was achieved using small interfering¦RNAs to rictor. Cell proliferation rate was assessed by BrdU incorporation and¦immunocytofluorescence. Apoptosis rate was determined by ELISA as well as¦propidium iodine staining and FACS analysis. Migration of endothelial cells¦was evaluated using a modified Boyden chamber assay.¦Results:Wefound thatVEGF activatesmTORC2 in endothelial cells. Indeed,¦treatment of endothelial cells with VEGF increases Akt phosphorylation, a¦downstream effector of mTORC2. We have further determined the role¦of mTORC2 in VEGF signaling by knocking down rictor, a component¦of mTORC2. We observed that VEGF failed to activate Akt and Erk in¦endothelial cells transfected with rictor siRNA. To next analyze the functional¦significance of mTORC2 inhibition on VEGF-mediated endothelial cell¦responses we performed proliferation, survival and migration assays. We found¦that VEGF failed to induce endothelial cell proliferation, survival and migration¦in endothelial cell lacking mTORC2 activity.¦Conclusion: These results show that mTORC2 is an important signaling¦intermediary in VEGF-induced endothelial cell responses and thus represents¦an interesting target to block VEGF-induced angiogenesis.

Cell proliferation and migration in the jejunum of suckling rats submitted to progressive fasting

Cell proliferation and migration in the intestinal crypts, and cell migration in the villus are controlled by different mechanisms in adult rats. In the present study, weanling rats and fasting rats were used to quantitatively study the correlation of cell cycle parameters and epithelial cell migration in crypts and intestinal villi. Eighteen-day-old rats received a single injection of tritiated thymidine [3H]TdR (23:00 h); half of the pups were submitted to fasting 5 h earlier. Cell proliferation was determined in radioautographs of jejunal crypts, on the basis of the labeling indices (LI) taken 1, 8, 13 and 19 h after [3H]TdR. The results showed that the labeling index did not differ 1 h or 19 h after [3H]TdR between the fed (38.7% or 48%) and fasting groups (34.6% or 50.4%). The modified method of grain count halving indicated that cell cycle time did not differ between fed (16.5 h) and fasting rats (17.8 h); the growth fraction, however, had lower values in fasting (59%) than in fed rats (77%). Cell migration in the crypt, estimated by the LI obtained for each cell position, did not change with treatment. As for the villi, the cell migration rate was significantly retarded by 3 cell positions (8%). These results suggest that the cell migration in the villi of weanling pups does not depend directly on the cell proliferation and migration in the intestinal crypt, but is directly affected by the absence of food in the lumen

Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.