The G-protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian cancer cells by blocking tubulin polymerization

The G-protein-coupled estrogen receptor 1 (GPER) has recently been reported to mediate the non-genomic action of estrogen in different types of cells and tissues. G-1 (1-[4-(6-bromobenzo[1,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed as a potent and selective agonist for GPER. G-1 has been shown to induce the expression of genes and activate pathways that facilitate cancer cell proliferation by activating GPER. Here we demonstrate that G-1 has an anticancer potential with a mechanism similar to vinca alkaloids, the commonly used chemotherapy drugs. We found that G-1 blocks tubulin polymerization and thereby interrupts microtubule assembly in ovarian cancer cells leading to the arrest of cell cycle in the prophase of mitosis and the suppression of ovarian cancer cell proliferation. G-1 treatment also induces apoptosis of ovarian cancer cells. The ability of G-1 to target microtubules to suppress ovarian cancer cell proliferation makes it a promising candidate drug for treatment of ovarian cancer.     

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background

Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).

Methodology/Principal Findings

The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10⁻⁸ to 10⁻⁵ M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.

Conclusion

GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.     

Oestrogen regulates SOX9 bioavailability by rapidly activating ERK1/2 and stabilising microtubules in a human testis-derived cell line

Nuclear SOX9 is essential for Sertoli cell differentiation and the development of a testis. Exposure of Sertoli cells to exogenous oestrogen causes cytoplasmic retention of SOX9, inhibiting testis development and promoting ovarian development. The cytoplasmic localisation of SOX9 requires a stabilised microtubule network and a key MAPK complex, ERK1/2, is responsive to oestrogen and known to affect the microtubule network. We hypothesised that oestrogen could stabilise microtubules through the activation of ERK1/2 to promote the cytoplasmic retention of SOX9. Treatment of human testis-derived NT2/D1 cells for 30 min with oestrogen rapidly activated ERK1/2, stabilised the microtubule network and increased cytoplasmic localisation of SOX9. The effects of oestrogen on SOX9 and tubulin were blocked by the ERK1/2 inhibitor U0126, demonstrating that ERK1/2 mediates the stabilisation of microtubules and cytoplasmic retention of SOX9 by oestrogen. Together, these data revealed a previously unknown mechanism for oestrogen in impacting MAPK signalling to block SOX9 bioavailability and the differentiation of Sertoli cells.     

Effects of microtubule modulators on HIV-1 infection of transformed and resting CD4 T cells

Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin,P-selectin,ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish

Estrogen inhibition of oocyte maturation (OM) and the role of GPER (formerly known as GPR30) were investigated in zebrafish. Estradiol-17β (E2) and G-1, a GPER-selective agonist, bound to zebrafish oocyte membranes suggesting the presence of GPER which was confirmed by immunocytochemistry using a specific GPER antibody. Incubation of follicle-enclosed oocytes with an aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian follicle cell layers significantly increased spontaneous OM which was partially reversed by co-treatment with either 100 nM E2 or G-1. Incubation of denuded oocytes with the GPER antibody blocked the inhibitory effects of estrogens on OM, whereas microinjection of estrogen receptor alpha (ERα) antisense oligonucleotides into the oocytes was ineffective. The results suggest that endogenous estrogens produced by the follicle cells inhibit or delay spontaneous maturation of zebrafish oocytes and that this estrogen action is mediated through GPER. Treatment with E2 and G-1 also attenuated the stimulatory effect of the teleost maturation-inducing steroid, 17,20β-dihyroxy-4-pregnen-3-one (DHP), on OM. Moreover, E2 and G-1 down-regulated the expression of membrane progestin receptor alpha (mPRα), the intermediary in DHP induction of OM. Conversely DHP treatment caused a > 50% decline in GPER mRNA levels. The results suggest that estrogens and GPER are critical components of the endocrine system controlling the onset of OM in zebrafish. A model is proposed for the dual control of the onset of oocyte maturation in teleosts by estrogens and progestins acting through GPER and mPRα, respectively, at different stages of oocyte development.     

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells.Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.     

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca²⁺-ATPase (PMCA) is essential for removal of cytoplasmic Ca²⁺ and for shaping the time courses of Ca²⁺-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca²⁺ extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30''s C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other''s function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca²⁺ signaling and GPER/GPR30-mediated activities.     

Activation of G protein coupled estrogen receptor prevents chemotherapy-induced intestinal mucositis by inhibiting the DNA damage in crypt cell in an extracellular signal-regulated kinase 1- and 2- dependent manner

Chemotherapy-induced intestinal mucositis (CIM) is a common adverse reaction to antineoplastic treatment with few appropriate, specific interventions. We aimed to identify the role of the G protein coupled estrogen receptor (GPER) in CIM and its mechanism. Adult male C57BL/6 mice were intraperitoneally injected with 5-fluorouracil to establish the CIM model. The selective GPER agonist G-1 significantly inhibited weight loss and histological damage in CIM mice and restored mucosal barrier dysfunction, including improving the expression of ZO-1, increasing the number of goblet cells, and decreasing mucosal permeability. Moreover, G-1 treatment did not alter the antitumor effect of 5-fluorouracil. In the CIM model, G-1 therapy reduced the expression of proapoptotic protein and cyclin D1 and cyclin B1, reversed the changes in the number of TUNEL⁺ cells, Ki67⁺ and bromodeoxyuridine⁺ cells in crypts. The selective GPER antagonist G15 eliminated all of the above effects caused by G-1 on CIM, and application of G15 alone increased the severity of CIM. GPER was predominantly expressed in ileal crypts, and G-1 inhibited the DNA damage induced by 5-fluorouracil in vivo and vitro, as confirmed by the decrease in the number of γH2AX⁺ cells in the crypts and the comet assay results. Referring to the data from GEO dataset we verified GPER activation restored ERK1/2 activity in CIM and 5-fluorouracil-treated IEC-6 cells. Once the effects of G-1 on ERK1/2 activity were abolished with the ERK1/2 inhibitor PD0325901, the effects of G-1 on DNA damage both in vivo and in vitro were eliminated. Correspondingly, all of the manifestations of G-1 protection against CIM were inhibited by PD0325901, such as body weight and histological changes, the mucosal barrier, the apoptosis and proliferation of crypt cells. In conclusion, GPER activation prevents CIM by inhibiting crypt cell DNA damage in an ERK1/2-dependent manner, suggesting GPER might be a target preventing CIM.Subject terms: Pharmacology, Gastrointestinal diseases     

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.     

Cold exposure reveals two populations of microtubules in pulmonary endothelia

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

Vascular disrupting agent drug classes differ in effects on the cytoskeleton

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 μM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.     

Role of the cytoskeleton during injury-induced cell migration in corneal endothelium

The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming reestablished. When injured corneas are placed in media containing 5 x 10(-7) M cytochalasin B, endothelial cell migration occurs; but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10(-8) M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 micrograms/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.     

M20, the small subunit of PP1M,binds to microtubules

Myosinlight chain phosphatase (PP1M) is composed of three subunits, i.e.,M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosinbinding subunit, the function of M20 is unknown. In the present study,we found that M20 binds to microtubules. The binding activity wasrevealed by cosedimentation of M20 with microtubules and binding oftubulin to M20 affinity resin. Green fluorescent protein (GFP)-taggedM20 (M20-GFP) was expressed in chicken primary smooth muscle cells andCOS-7 cells and was used as a probe for studying the associationbetween M20 and microtubules in living cells. M20-GFP was localized onfilamentous structures in both cell types. Colocalization analysisrevealed that M20-GFP colocalized with tubulin. Treatment withnocodazole, but not cytochalasin B, abolished the filamentous structureof M20-GFP. These results indicate that M20-GFP associates withmicrotubules in cells. Microinjection of rhodamine-tubulin into theM20-expressing cells revealed that incorporation of rhodamine-tubulininto microtubules was significantly facilitated bymicrotubule-associated M20. Consistent with this result, M20 enhancedthe rate of tubulin polymerization in vitro and produced elongatedmicrotubules. These results suggest that M20 has a microtubule bindingactivity and plays a role in regulating microtubule dynamics.

     

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.