Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca²⁺ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca²⁺ pathway and whether it regulates gene expression.     

Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells

Aquaporin (AQP) is a family of transmembrane proteins for water transport. Recent studies revealed that AQPs are likely to play a role in tumor progression and invasion. We aimed to examine the potential role of AQP5 in the progression of human breast cancer cells. Expression of AQP5 mRNA and protein was seen in human breast cancer cell line (both MCF7 and MDA-MB-231) by RT-PCR and immunoblotting analysis. Immunoperoxidase labeling of AQP5 was observed at ductal epithelial cells of human breast tissues. In benign tumor, AQP5 labeling was mainly seen at the apical domains of ductal epithelial cells. In contrast, in invasive ductal carcinoma, prominent AQP5 labeling was associated with cancer cells, whereas some ducts were unlabeled and apical polarity of AQP5 in ducts was lost. Cell proliferation (BrdU incorporation assay) and migration of MCF7 cells were significantly attenuated by lentivirus-mediated AQP5-shRNA transduction. Hyperosmotic stress induced by sorbitol treatment (100 mM, 24 h) reduced AQP5 expression in MCF7 cells, which was also associated with a significant reduction in cell proliferation and migration. Taken together, prominent AQP5 expression in breast cancer cells with the loss of polarity of ductal epithelial cells was seen during the progression of breast carcinoma. shRNA- or hyperosmotic stress-induced reduction in AQP5 expression of MCF7 cells was associated with significantly reduced cell proliferation and migration. In conclusion, AQP5 overexpression is likely to play a role in cell growth and metastasis of human breast cancer and could be a novel target for anti-breast cancer treatment.     

Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.     

Cytogenetic characterization of HB2 epithelial cells from the human breast

HB2 is a cell line originated by subcloning of MTSV1-7 mammary luminal epithelial cells isolated from human milk and immortalization via introduction of the gene encoding simian virus 40 (SV40) large T antigen. Despite its wide utilization as non-neoplastic counterpart in assays aimed to elucidating various biochemical and genetical aspects of normal and tumoral breast cells, to our knowledge no literature data have so far appeared concerning the chromosomal characterization of the HB2 cells. Here, we report the cytogenetic characterization of the karyotype of HB2 cells, which puts in evidence the occurrence of changes in chromosomal number and structure and the presence of unidentified chromosomal markers in variable amount. Our results do not detract from the utility of HB2 cells in illustrating fundamental aspects of breast cell biology, but rather interject a note of caution into generalizing results obtained with this cell line to other non-immortalized epithelial cell populations from the human breast. Therefore, this work represents a useful resource for all who want to perform appropriate and focused future studies on this cell line and proposes precise indications for a knowledgeable use of HB2 cells.     

A soluble form of Wnt-1 protein with mitogenic activity on mammary epithelial cells.

The proto-oncogene Wnt-1 plays an essential role in fetal brain development and causes hyperplasia and tumorigenesis when activated ectopically in the mouse mammary gland. When expressed in certain mammary epithelial cell lines, the gene causes morphological transformation and excess cell proliferation at confluence. Like other members of the mammalian Wnt family, Wnt-1 encodes secretory glycoproteins which have been detected in association with the extracellular matrix or cell surface but which have not previously been found in a soluble or biologically active form. We show here that conditioned medium harvested from a mammary cell line expressing Wnt-1 contains soluble Wnt-1 protein and induces mitogenesis and transformation of mammary target cells. By immunodepletion of medium containing epitope-tagged Wnt-1, we show that at least 60% of this activity is specifically dependent on Wnt-1 protein. These results provide the first demonstration that a mammalian Wnt protein can act as a diffusible extracellular signaling factor.     

miR-29b regulates migration of human breast cancer cells

microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by targeting mRNAs, inhibiting the expression of the associated proteins. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNAs in tumor biology has not been established. We evaluated the expression pattern of miRNAs in human breast cancer cells by qPCR, finding out an up-regulated miRNA miR-29b and studying its biological effect by migration assay. We defined a target gene PTEN by bioinformatics approach and western blot. In breast cancer cell line MDA-MB-231 cell, which migrate faster than MCF-7, we observed that miR-29b was highly over-expressed. Inhibition of miR-29b in cultured cells increased the expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, promoting apoptosis, decreasing migration, and decreasing invasion. In contrast, enhanced miR-29b expression by transfection with pre-miR-29b decreased the expression of PTEN and impaired apoptosis, increasing tumor cell migration and invasion. Moreover, PTEN was shown to be a direct target of miR-29b and was also shown to contribute to the miR-29b-mediated effects on cell invasion. Modulation of miR-29b altered the role of PTEN involved in cell migration and invasion. Aberrant expression of miR-29b, which modulates PTEN expression, can contribute to migration, invasion, and anti-apoptosis.     

5-Hydroxytryptamine as a potent migration enhancer of human aortic endothelial cells

The purpose of the present study was to investigate whether 5-hydroxytryptamine (5-HT, serotonin) affects migration of vascular endothelial cells. 5-HT significantly enhanced migration of human aortic endothelial cells (HAECs), and this enhancement was completely inhibited by GR 55562, a 5-HT1 receptor antagonist, and fluoxetine, a 5-HT transporter inhibitor, but was not affected by ketanserin, a 5-HT2 receptor antagonist. 5-HT stimulation increased RhoA and ERK activity of HAECs, and inhibitors of RhoA (Y-27632 and H-1152) and inhibitors of MEK (U0126 and PD98059) abolished the 5-HT-induced increase in migration velocity. Inhibition of Rho kinase by Y-27632 blocked stress fiber formation and rear release of HAECs. Thus, 5-HT has a potent enhancing action on migration of HAECs through activating the RhoA and ERK pathways following 5-HT1 receptor stimulation.     

Regulation of CEACAM1 transcription in human breast epithelial cells

Background  

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).     

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells

Summary Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca²⁺ medium (1.05 mM). To clarify the mechanism by which Ca²⁺ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca²⁺ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca²⁺ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca²⁺ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca²⁺-grown cells did not display a network of tubulin fibers, whereas high Ca²⁺-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca²⁺ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatement of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca²⁺ medium had no observable effect on cell morphology. These results indicate that high levels of Ca²⁺ either favor tubulin polymerization or stabilize the polymerized state. This research was supported by NCI grant CA-38921 from the National Cancer Institute, Bethesda, MD, and by an institutional grant from the United Foundation of Greater Detroit.     

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.     

Cdk5: A regulator of epithelial cell adhesion and migration

Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.Key words: Cdk5, Src, Rho, stress fibers, epithelial cells, cell adhesion, cell migrationAnchoring of epithelial cells to their basement membrane is essential to maintain their morphology, normal physiological function and survival. Cells attach to extracellular matrix components by means of membrane-spanning integrins, which cluster and link to the actin cytoskeleton via components of focal adhesions. At focal adhesions, actin is bundled into stress fibers, multi-protein cellular contractile machines that strengthen attachment and provide traction during migration.¹ Stress fiber contraction is generated by myosin II, a hexamer containing one pair of each non-muscle heavy chains (NMHCs), essential light chains, and myosin regulatory light chains (MRLC). Myosin motor activity is regulated by phosphorylation of MRLC at Thr18/Ser19 and is required to generate tension on actin filaments and to maintain stress fibers.¹ Although a number of kinases have been identified which phosphorylate MRLC at Thr18/Ser19, the principal kinases in most cells are myosin light chain kinase (MLCK)² and Rho-kinase (ROCK),³ a downstream effector of the small GTPas, RhoA.Rho family small GTPases play a central role in regulating many aspects of cytoskeletal organization and contraction.⁴ These GTPases are subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1,^5,6 and negative regulation by GTPase-activating proteins (GAPs), such as p190RhoGAP.⁷ As cells spread, the Rho-family GTPase, Cdc42, is activated at the cell periphery, leading to the formation of numerous filapodia. Focal adhesion formation is first seen at the tips of these filapodia as focal adhesion proteins such as talin and focal adhesion kinase (FAK) bind to the intracellular domains of localized integrins.⁸ Src is recruited to activated FAK at the nascent focal adhesion and generates binding sites for additional focal adhesion proteins by phosphorylating FAK and paxillin.⁹ Src activity is essential for the further maturation of the focal adhesion and for activating the Rho-family GTPase, Rac, leading to Arp2/3-dependent actin polymerization, formation of a lamellipodium and extension of the cell boundary. Simultaneously, Src inhibits RhoA by phosphorylating and activating its upstream inhibitor, p190RhoGAP. As the focal adhesion matures, Src is deactivated, allowing the Rho activation necessary for mDia-dependent actin polymerization,¹⁰ myosin-dependent cytoskeletal contraction⁵ and tight adhesion to the extracellular matrix. Since new focal adhesions continually form at the distal boundary of the spreading cell, the most mature and highly contracted stress fibers are localized at the center of the cell.Cell adhesion is an essential component of cell migration: if adhesion is too weak, cells can not generate the traction necessary for migration; if it is too strong, they are unable to overcome the forces that anchor them in place. Thus, the relationship between adhesion force and migration rate is a bell-shaped curve.¹¹ Migration rate increases as adhesive strength increases until an optimum value is reached. Thereafter, increases in adhesive strength decrease migration rate. Since the strength of adhesion depends on extracellular matrix composition as well as the types of integrin expressed in the cell, a decrease in adhesive strength may result in either faster or slower cell migration.Several lines of evidence indicate that the proline-directed serine/threonine kinase cyclin dependent kinase 5 (Cdk5) plays an integral role in regulating cell adhesion and/or migration in epithelial cells.^12–17 Cdk5 is an atypical member of cyclin dependent kinase family, which is activated by the non-cyclin proteins, p35 or p39.¹⁸ Cdk5 is most abundant in neuronal cells where it also regulates migration and cytoskeletal dynamics.¹⁹ In neurons, Cdk5 exerts its effects on migration at least in part by phosphorylating FAK,¹⁹ and the LIS1 associated protein, NDEL1.²⁰ In contrast, recent findings have revealed two novel pathways involved in Cdk5-dependent regulation of migration in epithelial cells.^16,17One of these newly discovered mechanisms links Cdk5 activation to control of stress fiber contraction.¹⁶ We have found that Cdk5 and its activator, p35, co-localize with phosphorylated myosin regulatory light chain (MRLC) on centrally located stress fibers in spreading cells.¹⁶ Moreover, Cdk5 is strongly activated in spreading cells as central stress fiber contraction becomes pronounced.²¹ Since contraction of these central stress fibers is primarily responsible for tight attachment between the cell and the extracellular matrix,⁵ the above findings suggested that Cdk5 might regulate cell adhesion by regulating MRLC phosphorylation. To test this possibility we inhibited Cdk5 activity by several independent means and found that MRLC phosphorylation was likewise inhibited. In addition, we found that inhibiting Cdk5 either prevented the formation of central stress fibers or led to their dissolution. The concave cell boundaries characteristic of contracting cells were also lost.¹⁶ Since MRLC lacks a favorable site for phosphorylation by Cdk5, we asked whether Cdk5 might affect the upstream signaling pathways that regulate MRLC phosphorylation. Experiments with specific pathway inhibitors indicated that the MRLC phosphorylation involved in stress fiber contraction in lens epithelial cells was regulated largely by Rho-kinase (ROCK). Inhibiting Cdk5 activity not only significantly reduced ROCK activity, but also blocked activation of its upstream regulator, Rho. To explore the mechanism behind the Cdk5-dependent regulation of Rho, we turned our attention to p190RhoGAP, which appears to play a major role in regulating Rho-dependent stress fiber contraction.⁷ This RhoGAP must be phosphorylated by Src to be active; as a result, Rho activity is low in the early stages of cell spreading, when Src activity is high. At later times, Src activity falls, p190RhoGAP activity is lost, and Rho-GTP is formed, enabling Rho-dependent myosin phosphorylation and stress fiber contraction.^9,10 We have found that inhibiting Cdk5 activity during this later stage of cell spreading increases Src activity and Src-dependent phosphorylation of its substrate, p190RhoGAP. This in turn leads to decreased Rho activity accompanied by loss of Rho-dependent myosin phosphorylation, dissolution of central stress fibers, and loss of cell contraction (Fig. 1). Moreover, inhibiting Src protects cells from the loss of Rho activation and dissolution of central stress fibers produced by inhibiting Cdk5.¹⁶ Since the effects of Cdk5 on Rho-dependent cytoskeletal contraction appear to be mediated almost entirely through Cdk5-dependent regulation of Src, it will be particularly important to determine how Cdk5 limits Src activity.Open in a separate windowFigure 1Cdk5 inhibition reduces contraction of preformed stress fibers. (A) Cells were spread on fibronectin for 60 min to adhere, allowing them to form focal adhesion and stress fibers (pre-incubation) and then further incubated for 2 h in absence (control) or presence of Cdk5 inhibitor (olomoucine) and stained with phalloidin. The cells without olomoucine (control) had concave boundaries and well-formed stress fibers. Olomoucine treated cells showed loss of central stress fibers and failure to contract. Scale bar = 20 µ. (B) experimental conditions were same as shown in (A). Cdk5 inhibitor, olomoucine, was added after 1 h of spreading (indicated as t = 0) and cells were incubated for an additional 2h in absence or presence of olomoucine. Cell lysates were immunoblotted with antibodies for pMRLC (upper) and MRLC (middle). Tubulin was used as a loading control (lower). Lane 1: untreated (at 0 h); Lane 2: untreated (at 2 h); Lane 3: Cdk5 inhibitor (olomoucine) treated. (C) results of three independent experiments of the type shown in (B) were quantified by densitometry and normalized to determine the relative levels of pMRLC at each time. Statistical analysis demonstrated a significant (p < 0.05) decrease in pMRLC level in olomoucine treated cells compared to untreated cells.The central stress fibers regulated by Cdk5 play a central role in anchoring cells to the substratum, and their loss when Cdk5 is inhibited will reduce adhesion. As discussed above, reduction in cell adhesion may either increase or decrease the rate of cell migration, depending on the cell type and extracellular matrix composition. In lens and corneal epithelial cells, the reduction in adhesion produced by Cdk5 inhibition promotes cell migration.^13,15,16,22 Moreover, regulation of Rho/Rho-kinase signaling by Cdk5 seems to be a major factor in determining the migration rate, since inhibitors of Cdk5 and Rho-kinase increased lens epithelial cell migration rate equivalently and inhibiting both produced no additional effect.¹⁶Interestingly, an independent line of investigation has shown that this is not the only mechanism underlying Cdk5-dependent regulation of cell adhesion and migration. Cdk5 also localizes at focal contacts at the cell periphery and phosphorylates the focal adhesion protein talin.¹⁷ The talin phosphorylation site has been identified as S425, near the FERM domain in the talin head region. Upon focal adhesion disassembly, this region is separated from the talin rod domain by calpain-dependent cleavage.²³ Phosphorylation at S425 by Cdk5 blocks ubiquitylation and degradation of the talin head by inhibiting interaction with the E3 ligase, Smurf1. This leads, ultimately, to greater stability of lamellipodia and newly formed focal adhesions, thus strengthening adhesion to the substrate.¹⁷ Although the exact molecular events involved in this stabilization are not yet clear, it has been suggested that the talin head may “prime” integrins to bind full length talin.²⁴ One possible scenario describing how this might occur is shown in Figure 2. By permitting the isolated head region to escape degradation following calpain cleavage, Cdk5-dependent phosphorylation may stablize a pool of talin head domains to bind focal contacts within the lamellipodium. It is known that the isolated talin head region can bind and activate integrins during cell protrusion.²⁵ The resulting integrin activation would be expected to stabilize the lamellipodium by strengthening integrin-dependent adhesion. Since the head domain lacks sites for actin binding, which are located in the talin rod domain,²⁶ the bound head domain would have to be replaced by full length talin to enable focal adhesion attachment to the cytoskeleton.²⁵ The head domain might promote this replacement by recruiting the PIP-kinase needed to generate PI(4,5) P₂,²³ which facilitates binding of full length talin to integrin by exposing the auto-inhibited integrin binding sites.²⁷ The binding of full length talin and the resulting link between the integrins and the actin cytoskeleton would then further strengthen adhesion.²⁵ This model predicts that full length talin would bind poorly in the absence of Cdk5 activity, due to degradation of the talin head and the resulting limited availability of PI(4,5)P₂, and thus provides a possible explanation for the observed rapid turnover of peripheral focal adhesions.¹⁷ Clearly, other models may be proposed to explain the increase in adhesion produced by talin head phosphorylation, and deciding among them will be an active area for future investigation. Nonetheless, it is now certain that talin is a key substrate for Cdk5 at focal adhesions.Open in a separate windowFigure 2Mechanism of Cdk5-dependent regulation of cell adhesion and migration. Binding of p35 to Cdk5 forms the active Cdk5/p35 kinase, which regulates cell adhesion and migration in two distinct ways. Cdk5-dependent phosphorylation of the talin head domain at Ser425 prevents its ubiquitylation and degradation, allowing it to persist following calpain cleavage. The phosphorylated talin head may then bind to integrin at peripheral sites and recruit PIP-K, which converts PI(4)P to PI(4,5)P₂. PI(4,5)P₂ may promote replacement of the talin head by full length talin. Full length talin recruits other focal adhesion proteins to form the mature focal adhesion. The talin tail provides the site for the actin binding and polymerization. Polymerized actin is subsequently bundled into stress fibers. Cdk5/p35 also regulates the Rho-dependent myosin phosphorylation necessary for stress fiber stability and cytoskeletal contraction by limiting Src activity. This in turn decreases Src-dependent phosphorylation of p190RhoGAP, favoring Rho-GTP formation, Rho-dependent stress fiber polymerization, stabilization and contraction. Both pathways modulate cell migration by increasing adhesive strength.In summary, the presently available data indicate that Cdk5 has at least two distinct functions in cell adhesion (Fig. 2). On the one hand, it stabilizes peripheral focal adhesions and promotes their attachment to the cytoskeleton by phosphorylating the talin head. On the other hand, once the actin cytoskeleton has been organized into stress fibers, Cdk5 enhances the Rho activation essential for stability and contraction of central stress fibers by limiting Src activity. The discovery that Cdk5 is involved in two separate events required for efficient migration, suggests that it may coordinate multiple signaling pathways. The known involvement of Cdk5 and its activator, p35, in regulating microtubule stability suggests yet another mechanism by which Cdk5 activity may regulate cytoskeletal function. Microtubules are closely associated with stress fibers²⁸ and their depolymerization has been shown to release the Rho activating protein, GEF-H1, leading to Rho activation and Rho-dependent myosin contraction.⁶ Since cell adhesion and migration play an important role in the progression of many pathological conditions, Cdk5, its substrates and its downstream effectors involved in cell adhesion may provide novel targets for therapeutic intervention.^15,29     

CLDN-1 promoted the epithelial to migration and mesenchymal transition (EMT) in human bronchial epithelial cells via Notch pathway

Sleep is a profound regulator of cellular immunity, and the curtailment of sleep in present day lifestyle leads to disruption of neuro-immune–endocrine interactions. No therapeutic remedy is yet known for the amelioration of detrimental effects caused by sleep deprivation (SD). The current study was aimed to elucidate the effects of acute SD on immune function and its modulation by water extract from leaves of Withania somnifera (ASH-WEX). Three groups of animals, i.e. Vehicle-Undisturbed sleep (VUD), Vehicle-Sleep deprived (VSD) and ASH-WEX fed sleep deprived (WSD) rats were tested for their anxiety-like behaviour and further used for the study of inflammatory and apoptotic markers expression in piriform cortex and hippocampus regions of the brain. VSD animals showed high level of anxiety in elevated plus maze test, which was ameliorated in WSD group. The stress induced expression of inflammatory and immune response markers GFAP, TNFα, IL-6, OX-18 and OX-42 in VSD animals was found to be modulated by ASH-WEX. Further, the stress induced apoptosis was suppressed in WSD group as indicated by expression of NF-κB, AP-1, Bcl-xL and Cytochrome c. This study provides scientific validation to the anxiolytic, anti-inflammatory and anti-apoptotic properties of ASH-WEX, which may serve as an effective dietary supplement for management of SD induced stress and associated functional impairments.     

Enhancement of the migration of metastatic human breast cancer cells by phosphatidic acid

Phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (SPP) are naturally occurring phospholipids which induce a variety of effects as extracellular messengers. In this study, we compared the effects of these phospholipid signaling molecules on the migration of invasive and noninvasive breast cancer cell lines, an index of the metastatic potential of these cells. As previously demonstrated, invasive MDA-MB-231 breast cancer cells exhibited increased constitutive (nonstimulated) migration in comparison to poorly invasive MCF-7 cells. Phosphatidic acid employed at nanomolar concentrations markedly potentiated migration of the invasive cells but had no effect on migration of either the noninvasive MCF-7 cells or nonneoplastic human epithelial cells. Lysophosphatidic acid and sphingosine 1-phosphate inhibited both the directed (chemotactic) and random (chemokinetic) migration of MDA-MB-231 cells. Experiments were undertaken to characterize the signaling pathway involved in constitutive and PA-stimulated migration of MDA-MB-231 cells. The tyrosine kinase inhibitors staurosporine and genistein inhibited constitutive and PA-induced migration in a dose-dependent manner, consistent with a role for tyrosine phosphorylation in the migratory response. In addition, the phosphatidylinositol (PI) 3' kinase inhibitors wortmannin and LY294002 strongly inhibited both the constitutive and PA-stimulated migration of the invasive breast cancer cells, indicating that PI-3' kinase plays an important role in the metastatic migration of breast cancer cells. Finally, PA-induced migration of MDA-MB-231 was markedly attenuated by pretreatment of cells with Clostridium difficile Toxin B, pertussis toxin and suramin, implying a role for a Gi receptor-dependent process involving activation of the small GTP-binding protein Rho. Since an enhanced ability to migrate heightens the metastatic potential of cells within solid tumors, our results suggest that the metastatic capabilities of breast cancer cells may be enhanced by a receptor-driven cellular process initiated by phosphatidic acid or related lipid phosphate messengers.     

Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.     

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.