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ABSTRACT: Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum-localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca(2+) entry across the plasma membrane and the Ca(2+) release-activated Ca(2+) channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca(2+) entry.
Science Signaling 03/2013; 6(267):ra18. · 7.50 Impact Factor
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ABSTRACT: Vascular endothelial (VE) cadherin, the major adherens junction adhesion molecule in endothelial cells, interacts with p120-catenin and β-catenin through its cytoplasmic tail. However, the specific functional contributions of the catenins to the establishment of strong adhesion are not fully understood. Here, we utilize bioengineering approaches to identify the roles of cadherin-catenin interactions in promoting strong cellular adhesion and the ability of the cells to spread on an adhesive surface. Our results demonstrate that the domain of VE-cadherin which binds to β-catenin is required for the establishment of strong steady-state adhesion strength. Surprisingly, p120 binding to the cadherin tail had no impact on the strength of adhesion when the available adhesive area was limited. Rather, the binding of VE-cadherin to p120 regulates adhesive contact area in a Rac1-dependent manner. These findings reveal that p120 and β-catenin have distinct but complementary roles in strengthening cadherin-mediated adhesion.
Molecular biology of the cell 01/2013; · 5.98 Impact Factor
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[show abstract]
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ABSTRACT: p120-catenin (p120) binds to the cytoplasmic tails of classical cadherins and inhibits cadherin endocytosis. Although p120 regulation of cadherin internalization is thought to be important for adhesive junction dynamics, the mechanism by which p120 modulates cadherin endocytosis is unknown. In this paper, we identify a dual-function motif in classical cadherins consisting of three highly conserved acidic residues that alternately serve as a p120-binding interface and an endocytic signal. Mutation of this motif resulted in a cadherin variant that was both p120 uncoupled and resistant to endocytosis. In endothelial cells, in which dynamic changes in adhesion are important components of angiogenesis and inflammation, a vascular endothelial cadherin (VE-cadherin) mutant defective in endocytosis assembled normally into cell-cell junctions but potently suppressed cell migration in response to vascular endothelial growth factor. These results reveal the mechanistic basis by which p120 stabilizes cadherins and demonstrate that VE-cadherin endocytosis is crucial for endothelial cell migration in response to an angiogenic growth factor.
The Journal of Cell Biology 10/2012; 199(2):365-80. · 10.26 Impact Factor
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Amy M Spinelli,
José C González-Cobos,
Xuexin Zhang,
Rajender K Motiani,
Sarah Rowan,
Wei Zhang,
Joshua Garrett, Peter A Vincent,
Khalid Matrougui,
Harold A Singer,
Mohamed Trebak
[show abstract]
[hide abstract]
ABSTRACT: Airway smooth muscle cell (ASMC) remodeling contributes to the structural changes in the airways that are central to the clinical manifestations of asthma. Ca(2+) signals play an important role in ASMC remodeling through control of ASMC migration and hypertrophy/proliferation. Upregulation of STIM1 and Orai1 proteins, the molecular components of the store-operated Ca(2+) entry (SOCE) pathway, has recently emerged as an important mediator of vascular remodeling. However, the potential upregulation of STIM1 and Orai1 in asthmatic airways remains unknown. An important smooth muscle migratory agonist with major contributions to ASMC remodeling is the platelet-derived growth factor (PDGF). Nevertheless, the Ca(2+) entry route activated by PDGF in ASMC remains elusive. Here, we show that STIM1 and Orai1 protein levels are greatly upregulated in ASMC isolated from ovalbumin-challenged asthmatic mice, compared to control mice. Furthermore, we show that PDGF activates a Ca(2+) entry pathway in rat primary ASMC that is pharmacologically reminiscent of SOCE. Molecular knockdown of STIM1 and Orai1 proteins inhibited PDGF-activated Ca(2+) entry in these cells. Whole-cell patch clamp recordings revealed the activation of Ca(2+) release-activated Ca(2+) (CRAC) current by PDGF in ASMC. These CRAC currents were abrogated upon either STIM1 or Orai1 knockdown. We show that either STIM1 or Orai1 knockdown significantly inhibited ASMC proliferation and chemotactic migration in response to PDGF. These results implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation and migration and suggest the potential use of STIM1 and Orai1 as targets for ASMC remodeling during asthma.
Pflügers Archiv - European Journal of Physiology 09/2012; 464(5):481-92. · 4.46 Impact Factor
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Amy M. Spinelli,
José C. González-Cobos,
Xuexin Zhang,
Rajender K. Motiani,
Sarah Rowan,
Wei Zhang,
Joshua Garrett, Peter A. Vincent,
Khalid Matrougui,
Harold A. Singer,
Mohamed Trebak
[show abstract]
[hide abstract]
ABSTRACT: Airway smooth muscle cell (ASMC) remodeling
contributes to the structural changes in the airways that are
central to the clinical manifestations of asthma. Ca2+ signals
play an important role in ASMC remodeling through control
of ASMC migration and hypertrophy/proliferation. Upregulation
of STIM1 and Orai1 proteins, the molecular components
of the store-operated Ca2+ entry (SOCE) pathway, has recently
emerged as an important mediator of vascular remodeling.
However, the potential upregulation of STIM1 and Orai1 in
asthmatic airways remains unknown. An important smooth
muscle migratory agonist with major contributions to ASMC
remodeling is the platelet-derived growth factor (PDGF).
Nevertheless, the Ca2+ entry route activated by PDGF in
ASMCremains elusive. Here, we show that STIM1 and Orai1
protein levels are greatly upregulated in ASMC isolated from
ovalbumin-challenged asthmatic mice, compared to control
mice. Furthermore, we show that PDGF activates a Ca2+ entry
pathway in rat primary ASMC that is pharmacologically
reminiscent of SOCE. Molecular knockdown of STIM1 and
Orai1 proteins inhibited PDGF-activated Ca2+ entry in these
cells. Whole-cell patch clamp recordings revealed the activation
of Ca2+ release-activated Ca2+ (CRAC) current by PDGF
in ASMC. These CRAC currents were abrogated upon either
STIM1 or Orai1 knockdown. We show that either STIM1 or
Orai1 knockdown significantly inhibited ASMC proliferation
and chemotactic migration in response to PDGF. These results
implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation
and migration and suggest the potential use of STIM1
and Orai1 as targets for ASMC remodeling during asthma.
Pflügers Archiv - European Journal of Physiology 09/2012; · 4.46 Impact Factor
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Amy M. Spinelli,
José C. González-Cobos,
Xuexin Zhang,
Rajender K. Motiani,
Sarah Rowan,
Wei Zhang,
Joshua Garrett, Peter A. Vincent,
Khalid Matrougui,
Harold A. Singer,
Mohamed Trebak
[show abstract]
[hide abstract]
ABSTRACT: Airway smooth muscle cell (ASMC) remodeling
contributes to the structural changes in the airways that are
central to the clinical manifestations of asthma. Ca2+ signals
play an important role in ASMC remodeling through control
of ASMC migration and hypertrophy/proliferation. Upregulation
of STIM1 and Orai1 proteins, the molecular components
of the store-operated Ca2+ entry (SOCE) pathway, has recently
emerged as an important mediator of vascular remodeling.
However, the potential upregulation of STIM1 and Orai1 in
asthmatic airways remains unknown. An important smooth
muscle migratory agonist with major contributions to ASMC
remodeling is the platelet-derived growth factor (PDGF).
Nevertheless, the Ca2+ entry route activated by PDGF in
ASMCremains elusive. Here, we show that STIM1 and Orai1
protein levels are greatly upregulated in ASMC isolated from
ovalbumin-challenged asthmatic mice, compared to control
mice. Furthermore, we show that PDGF activates a Ca2+ entry
pathway in rat primary ASMC that is pharmacologically
reminiscent of SOCE. Molecular knockdown of STIM1 and
Orai1 proteins inhibited PDGF-activated Ca2+ entry in these
cells. Whole-cell patch clamp recordings revealed the activation
of Ca2+ release-activated Ca2+ (CRAC) current by PDGF
in ASMC. These CRAC currents were abrogated upon either
STIM1 or Orai1 knockdown. We show that either STIM1 or
Orai1 knockdown significantly inhibited ASMC proliferation
and chemotactic migration in response to PDGF. These results
implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation
and migration and suggest the potential use of STIM1
and Orai1 as targets for ASMC remodeling during asthma.
Pflügers Archiv - European Journal of Physiology 09/2012; · 4.46 Impact Factor
-
Amy M. Spinelli,
José C. González-Cobos,
Xuexin Zhang,
Rajender K. Motiani,
Sarah Rowan,
Wei Zhang,
Joshua Garrett, Peter A. Vincent,
Khalid Matrougui,
Harold A. Singer,
Mohamed Trebak
[show abstract]
[hide abstract]
ABSTRACT: Airway smooth muscle cell (ASMC) remodeling
contributes to the structural changes in the airways that are
central to the clinical manifestations of asthma. Ca2+ signals
play an important role in ASMC remodeling through control
of ASMC migration and hypertrophy/proliferation. Upregulation
of STIM1 and Orai1 proteins, the molecular components
of the store-operated Ca2+ entry (SOCE) pathway, has recently
emerged as an important mediator of vascular remodeling.
However, the potential upregulation of STIM1 and Orai1 in
asthmatic airways remains unknown. An important smooth
muscle migratory agonist with major contributions to ASMC
remodeling is the platelet-derived growth factor (PDGF).
Nevertheless, the Ca2+ entry route activated by PDGF in
ASMCremains elusive. Here, we show that STIM1 and Orai1
protein levels are greatly upregulated in ASMC isolated from
ovalbumin-challenged asthmatic mice, compared to control
mice. Furthermore, we show that PDGF activates a Ca2+ entry
pathway in rat primary ASMC that is pharmacologically
reminiscent of SOCE. Molecular knockdown of STIM1 and
Orai1 proteins inhibited PDGF-activated Ca2+ entry in these
cells. Whole-cell patch clamp recordings revealed the activation
of Ca2+ release-activated Ca2+ (CRAC) current by PDGF
in ASMC. These CRAC currents were abrogated upon either
STIM1 or Orai1 knockdown. We show that either STIM1 or
Orai1 knockdown significantly inhibited ASMC proliferation
and chemotactic migration in response to PDGF. These results
implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation
and migration and suggest the potential use of STIM1
and Orai1 as targets for ASMC remodeling during asthma.
The FASEB Journal 09/2012; · 5.71 Impact Factor
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Wei Zhang,
Katharine E Halligan,
Xuexin Zhang,
Jonathan M Bisaillon,
José C Gonzalez-Cobos,
Rajender K Motiani,
Guoqing Hu, Peter A Vincent,
Jiliang Zhou,
Margarida Barroso,
Harold A Singer,
Khalid Matrougui,
Mohamed Trebak
[show abstract]
[hide abstract]
ABSTRACT: The molecular correlate of the calcium release-activated calcium current (I(CRAC)), the channel protein Orai1, is upregulated in proliferative vascular smooth muscle cells (VSMC). However, the role of Orai1 in vascular disease remains largely unknown.
The goal of this study was to determine the role of Orai1 in neointima formation after balloon injury of rat carotid arteries and its potential upregulation in a mouse model of VSMC remodeling.
Lentiviral particles encoding short-hairpin RNA (shRNA) targeting either Orai1 (shOrai1) or STIM1 (shSTIM1) caused knockdown of their respective target mRNA and proteins and abrogated store-operated calcium entry and I(CRAC) in VSMC; control shRNA was targeted to luciferase (shLuciferase). Balloon injury of rat carotid arteries upregulated protein expression of Orai1, STIM1, and calcium-calmodulin kinase IIdelta2 (CamKIIδ2); increased proliferation assessed by Ki67 and PCNA and decreased protein expression of myosin heavy chain in medial and neointimal VSMC. Incubation of the injured vessel with shOrai1 prevented Orai1, STIM1, and CamKIIδ2 upregulation in the media and neointima; inhibited cell proliferation and markedly reduced neointima formation 14 days post injury; similar results were obtained with shSTIM1. VSMC Orai1 and STIM1 knockdown inhibited nuclear factor for activated T-cell (NFAT) nuclear translocation and activity. Furthermore, Orai1 and STIM1 were upregulated in mice carotid arteries subjected to ligation.
Orai1 is upregulated in VSMC during vascular injury and is required for NFAT activity, VSMC proliferation, and neointima formation following balloon injury of rat carotids. Orai1 provides a novel target for control of VSMC remodeling during vascular injury or disease.
Circulation Research 08/2011; 109(5):534-42. · 9.49 Impact Factor
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ABSTRACT: The association of p120-catenin (p120) with the juxtamembrane domain (JMD) of vascular endothelial (VE)-cadherin is required to maintain VE-cadherin levels and transendothelial resistance (TEER) of endothelial cell monolayers. To distinguish whether decreased TEER was due to a loss of p120 and not to the decrease in VE-cadherin, we established a system in which p120 was depleted by short hairpin RNA delivered by lentivirus and VE-cadherin was restored via expression of VE-cadherin fused to green fluorescent protein (GFP). Loss of p120 resulted in decreased TEER, which was associated with decreased expression of VE-cadherin, β-catenin, plakoglobin, and α-catenin. Decreased TEER was rescued by restoration of p120 but not by the expression of VE-cadherin-GFP, despite localization of VE-cadherin-GFP at cell-cell borders. Expression of VE-cadherin-GFP restored levels of β-catenin and α-catenin but not plakoglobin, indicating that p120 may be important for recruitment of plakoglobin to the VE-cadherin complex. To evaluate the role of p120 interaction with Rho GTPase in regulating endothelial permeability, we expressed a recombinant form of p120, lacking the NH(2) terminus and containing alanine substitutions, that eliminates binding of Rho to p120. Expression of this isoform restored expression of the adherens junction complex and rescued permeability as measured by TEER. These results demonstrate that p120 is required for maintaining VE-cadherin expression and TEER independently of its NH(2) terminus and its role in regulating Rho.
AJP Heart and Circulatory Physiology 10/2010; 300(1):H36-48. · 3.71 Impact Factor
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[hide abstract]
ABSTRACT: Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.
Journal of Biological Chemistry 05/2010; 285(28):21303-12. · 4.77 Impact Factor
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ABSTRACT: P120 catenin (p120ctn) belongs to the family of Armadillo repeat-containing proteins, which are believed to have dual functions of cell-cell adhesion and transcriptional regulation. In vascular endothelium, p120ctn is mostly recognized for its cell-cell adhesion function through its ability to regulate VE-cadherin. The current study investigated whether p120ctn in endothelial cells also has the capability to signal transcription events. Examination of several endothelial cell types indicated that Kaiso, a p120ctn-binding transcription factor, was abundantly expressed, with a predominant localization to the perinuclear region. Immunoprecipitation of endothelial cell lysates with a p120ctn antibody resulted in p120ctn-Kaiso complex formation, confirming the interactions of the two proteins. Transfection of the KBS (Kaiso-binding sequence) luciferase reporter plasmid into endothelial cells resulted in a 40% lower reporter activity compared to the mutant Kaiso-insensitive construct or empty vector pGL3, indicating that the suppressed reporter activity was attributed to endogenous Kaiso. The knock-down of p120ctn increased the KBS reporter activity 2-fold over control, but had no effects on the mutant KBS reporter activity. Furthermore, p120ctn knock-down also reduced Kaiso expression, suggesting that p120ctn functioned to stabilize Kaiso. Overall, the findings provide evidence that in endothelial cells, p120ctn has a transcription repression function through regulation of Kaiso, possibly as a cofactor with the transcription factor.
Microvascular Research 04/2010; 80(2):233-9. · 2.83 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: p120-catenin (p120) is an armadillo family protein that binds to the cytoplasmic domain of classical cadherins and prevents cadherin endocytosis. The role of p120 in vascular development is unknown.
The purpose of this study is to examine the role of p120 in mammalian vascular development by generating a conditionally mutant mouse lacking endothelial p120 and determining the effects of the knockout on vasculogenesis, angiogenic remodeling, and the regulation of endothelial cadherin levels.
A conditional Cre/loxP gene deletion strategy was used to ablate p120 expression, using the Tie2 promoter to drive endothelial Cre recombinase expression. Mice lacking endothelial p120 died embryonically beginning at embryonic day 11.5. Major blood vessels appeared normal at embryonic day 9.5. However, both embryonic and extraembryonic vasculature of mutant animals were disorganized and displayed decreased microvascular density by embryonic day 11.5. Importantly, both vascular endothelial cadherin and N-cadherin levels were significantly reduced in vessels lacking p120. This decrease in cadherin expression was accompanied by reduced pericyte recruitment and hemorrhaging. Furthermore, p120-null cultured endothelial cells exhibited proliferation defects that could be rescued by exogenous expression of vascular endothelial cadherin.
These findings reveal a fundamental role for p120 in regulating endothelial cadherin levels during vascular development, as well as microvascular patterning, vessel integrity, and endothelial cell proliferation. Loss of endothelial p120 results in lethality attributable to decreased microvascular density and hemorrhages.
Circulation Research 03/2010; 106(5):941-51. · 9.49 Impact Factor
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[hide abstract]
ABSTRACT: Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.
Journal of Biological Chemistry 03/2010; 285(10):7045-55. · 4.77 Impact Factor
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[hide abstract]
ABSTRACT: p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.
Molecular biology of the cell 03/2009; 20(7):1970-80. · 5.98 Impact Factor
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ABSTRACT: Endothelial cells (ECs) express VE-cadherin and N-cadherin, and recent data suggest that VE-cadherin levels are dependent on N-cadherin expression. While investigating changes in N-cadherin levels during endothelial monolayer maturation, the authors found that VE-cadherin levels are maintained in ECs despite a decrease in N-cadherin, suggesting that VE-cadherin levels may not depend on N-cadherin. Knockdown of N-cadherin did not affect VE-cadherin levels in ECs with low endogenous N-cadherin expression. Surprisingly, however, knockdown of N-cadherin in ECs with high endogenous N-cadherin expression increased VE-cadherin levels, suggesting an inverse relationship between the two. This was further supported by a decrease in VE-cadherin following overexpression of N-cadherin. Experiments in which p120, a catenin that binds N- and VE-cadherin, was knocked down or overexpressed indicate that these two cadherins compete for p120. These data demonstrate that VE-cadherin levels are not directly related to N-cadherin levels but may be inversely related due to competition for p120.
Cell Communication & Adhesion 12/2008; 15(4):333-49. · 1.18 Impact Factor
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04/2008: pages 169 - 195; , ISBN: 9783527622092
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ABSTRACT: Alternative splicing of the fibronectin (FN) gene transcript provides an efficient mechanism for generating functionally appropriate forms of this adhesive glycoprotein in situ. Cellular FNs that include the EIIIA and/or EIIIB FN-III segments are prominently expressed during embryogenesis, wound healing, tumor progression, and inflammation. However, the roles of this domain in altering overall FN protein structure and regulating cellular function remain unclear. We previously reported that two integrins, alpha9beta1 and alpha4beta1, ligate the EIIIA segment ( Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D., and Van De Water, L. (2002) J. Biol. Chem. 277, 14467-14474 ) and that the epitopes for function-blocking monoclonal antibodies lie within the C-C' loop of EIIIA ( Liao, Y. F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884 ). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp(41) and Gly(42) residues within the C-C' loop of EIIIA are necessary for integrin alpha9beta1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C' loop encode sufficient information to completely inhibit alpha9beta1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in alpha9beta1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.
Journal of Biological Chemistry 03/2008; 283(5):2858-70. · 4.77 Impact Factor
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ABSTRACT: Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA. All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interference contrast optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. We conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.
AJP Cell Physiology 11/2007; 293(4):C1309-18. · 3.54 Impact Factor
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ABSTRACT: VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.
Molecular Biology of the Cell 12/2005; 16(11):5141-51. · 4.94 Impact Factor
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ABSTRACT: Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a "substrate-trapping" MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction.
Journal of Biological Chemistry 07/2005; 280(22):20995-1003. · 4.77 Impact Factor
