Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.

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Roles of epidermal growth factor and Na+/H+ exchanger-1 in esophageal epithelial defense against acid-induced injury

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00238.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. G665-G673 ◽

Cited By ~ 19

Author(s):

Yasuhiro Fujiwara ◽

Kazuhide Higuchi ◽

Takashi Takashima ◽

Masaki Hamaguchi ◽

Tsuyoshi Hayakawa ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Epithelial Cells ◽

Acid Reflux ◽

Dependent Manner ◽

Egf Receptors ◽

Epithelial Proliferation ◽

Epidermal Growth ◽

Esophageal Epithelial Cells ◽

Chronic Esophagitis

Epidermal growth factor (EGF) is predominantly secreted by salivary glands and activates Na+/H+ exchanger-1 (NHE-1), which regulates intracellular pH (pHi). We investigated the roles of EGF and NHE-1 in esophageal epithelial defense against acid using human esophageal epithelial cell lines and a rat chronic esophagitis model. Esophageal epithelial cells were incubated with acidified medium in the absence or presence of EGF. Cell viability and changes in pHi were measured. Chronic acid reflux esophagitis was induced in rats with and without sialoadenectomy. Esophageal lesion index, epithelial proliferation, and expression of EGF receptors and NHE-1 were examined. EGF protected esophageal epithelial cells against acid in a dose-dependent manner, and the cytoprotective effect of EGF was completely blocked by treatment with NHE-1 inhibitors. Tyrosine kinase, calmodulin, and PKC inhibitors significantly inhibited cytoprotection by EGF, whereas MEK, phosphatidylinositol 3-kinase, and PKA inhibitors had no effect. EGF significantly increased pHi recovery after NH4Cl pulse acidification, and this increase in pHi recovery was significantly blocked by inhibitors of calmodulin and PKC. Sialoadenectomy led to an increase in the severity of chronic esophagitis but affected neither epithelial proliferation nor expression of EGF receptors. Expression of NHE-1 mRNA was increased in esophagitis and upregulated in rats with sialoadenectomy. The increasing severity of esophagitis in rats with sialoadenectomy was prevented by exogenous administration of EGF. In conclusion, EGF protects esophageal epithelial cells against acid through NHE activation via Ca2+/calmodulin and the PKC pathway. Deficiency in endogenous EGF is associated with increased severity of esophagitis. EGF and NHE-1 play crucial roles in esophageal epithelial defense against acid.

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Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling

The Journal of Cell Biology ◽

10.1083/jcb.200707022 ◽

2008 ◽

Vol 181 (3) ◽

pp. 537-549 ◽

Cited By ~ 69

Author(s):

Naoto Kakinuma ◽

Badal Chandra Roy ◽

Yun Zhu ◽

Yong Wang ◽

Ryoiti Kiyama

Keyword(s):

Cell Migration ◽

Growth Factors ◽

Growth Factor ◽

Akt Signaling ◽

Stress Fibers ◽

Nih3t3 Cells ◽

Rhoa Activity ◽

Epidermal Growth ◽

Phosphoinositide 3 Kinase ◽

Actin Stress Fibers

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3–binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K–Akt signaling.

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Characterization of Morphological and Cytoskeletal Changes in Trophoblast Cells Induced by Insulin-Like Growth Factor-I

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2002-020550 ◽

2002 ◽

Vol 87 (12) ◽

pp. 5751-5759 ◽

Cited By ~ 18

Author(s):

Maryam Kabir-Salmani ◽

Shigetatsu Shiokawa ◽

Yoshihiro Akimoto ◽

Habib Hasan-Nejad ◽

Keiji Sakai ◽

...

Keyword(s):

Extracellular Matrix ◽

Rgd Peptide ◽

Stress Fibers ◽

Immunofluorescent Staining ◽

Dependent Manner ◽

Trophoblast Cells ◽

Α Subunit ◽

Igf I ◽

Lamellipodia Formation ◽

Actin Stress Fibers

Abstract IGF-I and IGF-II were appeared to play major roles in the adhesive and migratory events that are considered to be crucial in the implantation process. The purpose of this study was to determine the effects of IGF-I on trophoblast adhesion to extracellular matrix. Trophoblast cells obtained from early gestation at artificial abortion were incubated with the indicated doses of IGF-I at the indicated times. Trophoblast cells were treated with IGF-I in the presence or absence of RGD peptide and an antibody against α-subunit of IGF-I receptor (αIR3). Morphometric and morphological changes were studied using light and electron microscopy. Furthermore, vinculin, actin stress fibers, phosphorylated focal adhesion kinase (FAK), phosphotyrosine, and paxillin were immunolocalized in trophoblast cells after IGF-I treatment in the presence or absence of αIR3. Immunoprecipitation and anti-phosphotyrosine immunoblotting were carried out to detect the phosphorylated FAK and phosphorylated paxillin contents of the IGF-I-treated and untreated trophoblast cells. The results showed that IGF-I promoted trophoblast adhesion to fibronectin substrate in a time- and dose-dependent manner, and addition of RGD peptide and αIR3 monoclonal antibody abolished the effects of IGF-I in these cells. Morphological studies exhibited an increase in the lamellipodia formation upon IGF-I treatment, and confocal images of immunofluorescent staining revealed localization of phosphorylated FAK, paxillin, and vinculin at focal adhesions as well as redistribution of actin microfilaments and formation of actin stress fibers inside the cell. Western blotting, using antiphosphotyrosine demonstrated proteins with molecular masses of 125 kDa (FAK) and 68 kDa (paxillin) present in the IGF-I-treated cells, which were lacking in the control groups. In conclusion, these findings suggest that IGF-I can stimulate lamellipodia formation and promote adhesion of trophoblast cells to extracellular matrix by activating their adhesion molecules that must be activated within the implantation window.

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Small Guanosine Triphospatase RhoA and Rho-Associated Kinase as Regulators of Trophoblast Migration

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2002-020376 ◽

2002 ◽

Vol 87 (12) ◽

pp. 5808-5816 ◽

Cited By ~ 29

Author(s):

Shigetatsu Shiokawa ◽

Mitsutoshi Iwashita ◽

Yoshihiro Akimoto ◽

Shinya Nagamatsu ◽

Ken Sakai ◽

...

Keyword(s):

Cell Adhesion ◽

Stress Fibers ◽

Extravillous Trophoblast ◽

Dependent Manner ◽

Focal Contact ◽

Trophoblast Cells ◽

Human Trophoblast ◽

Trophoblast Migration ◽

Human Trophoblast Cells ◽

Actin Stress Fibers

Abstract The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility. Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility. In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells. The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA. In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast. Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum. Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells. The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632. Cultured human cytotrophoblast cells from 7–9 wk of gestation migrated into a fibronectin-coated membrane. Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner. In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers. Suppression of RhoA-ROCK reduces trophoblast migration. These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration.

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Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

The Journal of Cell Biology ◽

10.1083/jcb.200505018 ◽

2005 ◽

Vol 171 (2) ◽

pp. 209-215 ◽

Cited By ~ 227

Author(s):

Masaaki Yoshigi ◽

Laura M. Hoffman ◽

Christopher C. Jensen ◽

H. Joseph Yost ◽

Mary C. Beckerle

Keyword(s):

Shear Stress ◽

Mechanical Stress ◽

Actin Filaments ◽

Focal Adhesions ◽

Cyclic Stretch ◽

Stress Fibers ◽

Focal Adhesion Proteins ◽

Cytoskeletal Responses ◽

Actin Stress Fibers

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

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Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1964 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1964-1971 ◽

Cited By ~ 52

Author(s):

B L Hempstead ◽

R B Birge ◽

J E Fajardo ◽

R Glassman ◽

D Mahadeo ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Fusion Protein ◽

Pc12 Cells ◽

Dependent Manner ◽

Egf Receptors ◽

A431 Cells ◽

Nerve Growth ◽

Epidermal Growth

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

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Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

10.1101/2021.06.05.447194 ◽

2021 ◽

Author(s):

Alexander Kirschner ◽

Ana N Strat ◽

John Yablonski ◽

Tyler Bague ◽

Haiyan Li ◽

...

Keyword(s):

Optic Nerve ◽

Optic Nerve Head ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Stress Fibers ◽

Mechanosensitive Channel ◽

Dependent Manner ◽

Cytoskeletal Remodeling ◽

Channel Inhibition ◽

Actin Stress Fibers

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining for GFAP. Levels of fibronectin deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (Iba1) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP and fibronectin fluorescence intensity, which were decreased with GsMTx4 treatment. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

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Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway

Biochemical Journal ◽

10.1042/bj2980655 ◽

1994 ◽

Vol 298 (3) ◽

pp. 655-660 ◽

Cited By ~ 24

Author(s):

T R Pettitt ◽

M Zaqqa ◽

M J Wakelam

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phospholipase C ◽

Phospholipase D ◽

Kinase Inhibitor ◽

Inositol Phosphates ◽

Egf Receptors ◽

3T3 Fibroblasts ◽

Epidermal Growth ◽

Hydrolysis Of

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

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Presence of epidermal growth factor receptors on human thyroid membranes

Acta Endocrinologica ◽

10.1530/acta.0.1140396 ◽

1987 ◽

Vol 114 (3) ◽

pp. 396-401 ◽

Cited By ~ 11

Author(s):

Kikuo Kasai ◽

Masaki Hiraiwa ◽

Yoshinobu Suzuki ◽

Tatsushi Emoto ◽

Nobuyuki Banba ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Human Thyroid ◽

Tissue Homogenate ◽

Dependent Manner ◽

Egf Receptors ◽

Thyroid Cells ◽

High Affinity Receptor ◽

Epidermal Growth ◽

Affinity Receptor

Abstract. The nature of epidermal growth factor (EGF) receptors in normal and pathological thyroid membranes was studied on a crude membrane fraction (10 000 × g pellet) of tissue homogenate. Optimal binding of 125I-EGF to human thyroid membranes was obtained at 25°C with 1-h incubation at pH 7.4. The study revealed the presence of both high and low affinity receptors in normal and pathological thyroid membranes. The association constants of high affinity receptor (3.2 × 10−9 mol/l) and of low affinity receptor (1.8 × 10−8 mol/l) observed in normal thyroid membranes were similar to those of thyroid membranes from neoplastic as well as thyrotoxic thyroid tissues. [3H]thymidine incorporation into DNA of cultured human thyroid cells was stimulated by EGF in a dose-dependent manner. A half-maximal stimulation was found around 1 × 10−10 mol/l. These results suggest that EGF may have a possible role in the regulation of thyroid growth and function in physiological and pathological situations.

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Gelsolin-actin interaction and actin polymerization in human neutrophils.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.1983 ◽

1990 ◽

Vol 110 (6) ◽

pp. 1983-1991 ◽

Cited By ~ 38

Author(s):

T Howard ◽

C Chaponnier ◽

H Yin ◽

T Stossel

Keyword(s):

Human Blood ◽

Actin Filaments ◽

Actin Polymerization ◽

Regulatory Proteins ◽

Human Neutrophils ◽

Blood Neutrophils ◽

Acute Increase ◽

Actin Regulatory Proteins ◽

In Cells

The fraction of polymerized actin in human blood neutrophils increases after exposure to formyl-methionyl-leucyl-phenylalanine (fmlp), is maximal 10 s after peptide addition, and decreases after 300 s. Most of the gelsolin (85 +/- 11%) in resting ficoll-hypaque (FH)-purified neutrophils is in an EGTA resistant, 1:1 gelsolin-actin complex, and, within 5 s after 10(-7) M fmlp activation, the amount of gelsolin complexed with actin decreases to 42 +/- 12%. Reversal of gelsolin binding to actin occurs concurrently with an increase in F-actin content, and the appearance of barbed-end nucleating activity. The rate of dissociation of EGTA resistant, 1:1 gelsolin-actin complexes is more rapid in cells exposed to 10(-7) M fmlp than in cells exposed to 10(-9) M fmlp, and the extent of dissociation 10 s after activation depends upon the fmlp concentration. Furthermore, 300 s after fmlp activation when F-actin content is decreasing, gelsolin reassociates with actin as evidenced by an increase in the amount of EGTA resistant, 1:1 gelsolin-actin complex. Since fmlp induces barbed end actin polymerization in neutrophils and since in vitro the gelsolin-actin complex caps the barbed ends of actin filaments and blocks their growth, the data suggests that in FH neutrophils fmlp-induced actin polymerization could be initiated by the reversal of gelsolin binding to actin and the uncapping of actin filaments or nuclei. The data shows that formation and dissociation of gelsolin-actin complexes, together with the effects of other actin regulatory proteins, are important steps in the regulation of actin polymerization in neutrophils. Finally, finding increased amounts of gelsolin-actin complex in basal FH cells and dissociation of the complex in fmlp-activated cells suggests a mechanism by which fmlp can cause actin polymerization without an acute increase in cytosolic Ca++.

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