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Galectin-1 is implicated in the protein kinase C epsilon/vimentin-controlled trafficking of integrin-beta1 in glioblastoma cells.

Fortin S, Le Mercier M, Camby I, Spiegl-Kreinecker S, Berger W, Lefranc F, Kiss R - Brain Pathol. (2009)

Bottom Line: Galectin-1 depletion does not alter the gene expression level of integrin-beta1.Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCepsilon) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells.Our results argue for the involvement of galectin-1 in the PKCepsilon/vimentin-controlled trafficking of integrin-beta1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Toxicology, Institute of Pharmacy, Univesité Libre de Bruxelles, Brussels.

ABSTRACT
Cell motility and resistance to apoptosis characterize glioblastoma (GBM) growth and malignancy. In our current work we report that galectin-1, a homodimeric adhesion molecule and carbohydrate-binding protein with affinity for beta-galactosides, is linked with cell surface expression of integrin beta1 and the process of integrin trafficking. Using immunofluorescence, depletion of galectin-1 through both stable knockdown and transient-targeted small interfering RNA (siRNA) treatment induces an intracellular accumulation of integrin-beta1 coincident with a diminution of integrin-beta1 at points of cellular adhesion at the cell membrane. Galectin-1 depletion does not alter the gene expression level of integrin-beta1. Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCepsilon) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells. Our results argue for the involvement of galectin-1 in the PKCepsilon/vimentin-controlled trafficking of integrin-beta1. The understanding of molecular mediators such as galectin-1 and the pathways through which they drive the cell invasion so descriptive of GBM is anticipated to reveal potential therapeutic targets that promote glioma malignancy.

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Related in: MedlinePlus

Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin-1-targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε. WB analysis of PKCε and vimentin protein expression levels in U87 (A) or Hs683 (B) cells untreated (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) at days 5, 7 and 9 post-transfection. IF analysis of PKCε localization in U87 (C) or Hs683 (D) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post-transfection. IF analysis of vimentin localization in U87 (E) or Hs683 (F) cells scr or galectin-1 siRNA-transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post-transfection. In all IF figures right columns represent the corresponding bright field images.
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fig05: Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin-1-targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε. WB analysis of PKCε and vimentin protein expression levels in U87 (A) or Hs683 (B) cells untreated (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) at days 5, 7 and 9 post-transfection. IF analysis of PKCε localization in U87 (C) or Hs683 (D) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post-transfection. IF analysis of vimentin localization in U87 (E) or Hs683 (F) cells scr or galectin-1 siRNA-transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post-transfection. In all IF figures right columns represent the corresponding bright field images.

Mentions: To begin to assess biochemically whether any of the genes whose expression was modified upon treatment of siRNA targeting galectin-1 were involved in integrin-β1 cellular localization changes, we inquired if whether, similar to integrin-β1, there were changes in the localization of the trafficking and export-related genes. Using the genes affected from the gene expression microarray study under galectin-1-targeted siRNA treatment as the first potential candidates (Table 1), we first selected the gene PKCε to study for its known role in integrin-β1 trafficking (17). We assessed through WB analysis the expression of PKCε in both U87 and Hs683 cells under conditions of siRNA targeting galectin-1 at days 5, 7 and 9 post-transfection, and found no significant changes in PKCε protein expression levels (Figure 5A,B). We then performed IF studies of PKCε localization in both U87 and Hs683 under conditions of siRNA targeting galectin-1 at day 5 (Hs683) or day 7 (U87) post-transfection. Scramble-transfected cells showed a diffuse staining pattern for PKCε in the cytoplasm, whereas the localization under galectin-1 siRNA conditions shifted towards only a strong perinuclear staining in both U87 (Figure 5C) and Hs683 (Figure 5D) cells.


Galectin-1 is implicated in the protein kinase C epsilon/vimentin-controlled trafficking of integrin-beta1 in glioblastoma cells.

Fortin S, Le Mercier M, Camby I, Spiegl-Kreinecker S, Berger W, Lefranc F, Kiss R - Brain Pathol. (2009)

Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin-1-targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε. WB analysis of PKCε and vimentin protein expression levels in U87 (A) or Hs683 (B) cells untreated (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) at days 5, 7 and 9 post-transfection. IF analysis of PKCε localization in U87 (C) or Hs683 (D) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post-transfection. IF analysis of vimentin localization in U87 (E) or Hs683 (F) cells scr or galectin-1 siRNA-transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post-transfection. In all IF figures right columns represent the corresponding bright field images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805865&req=5

fig05: Western blot (WB) and immunofluorescence (IF) analysis of vimentin and protein kinase C epsilon (PKCε) protein expression levels; galectin-1-targeted siRNA increases only vimentin protein levels in Hs683, induces increased perinulear amassment of both PKCε and vimentin, and diminishes the diffuse cytoplasmic staining of PKCε. WB analysis of PKCε and vimentin protein expression levels in U87 (A) or Hs683 (B) cells untreated (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) at days 5, 7 and 9 post-transfection. IF analysis of PKCε localization in U87 (C) or Hs683 (D) scr or siGal1 at either day 5 (Hs683) or day 7 (U87) post-transfection. IF analysis of vimentin localization in U87 (E) or Hs683 (F) cells scr or galectin-1 siRNA-transfected (siGal1) at either day 5 (Hs683) or day 7 (U87) post-transfection. In all IF figures right columns represent the corresponding bright field images.
Mentions: To begin to assess biochemically whether any of the genes whose expression was modified upon treatment of siRNA targeting galectin-1 were involved in integrin-β1 cellular localization changes, we inquired if whether, similar to integrin-β1, there were changes in the localization of the trafficking and export-related genes. Using the genes affected from the gene expression microarray study under galectin-1-targeted siRNA treatment as the first potential candidates (Table 1), we first selected the gene PKCε to study for its known role in integrin-β1 trafficking (17). We assessed through WB analysis the expression of PKCε in both U87 and Hs683 cells under conditions of siRNA targeting galectin-1 at days 5, 7 and 9 post-transfection, and found no significant changes in PKCε protein expression levels (Figure 5A,B). We then performed IF studies of PKCε localization in both U87 and Hs683 under conditions of siRNA targeting galectin-1 at day 5 (Hs683) or day 7 (U87) post-transfection. Scramble-transfected cells showed a diffuse staining pattern for PKCε in the cytoplasm, whereas the localization under galectin-1 siRNA conditions shifted towards only a strong perinuclear staining in both U87 (Figure 5C) and Hs683 (Figure 5D) cells.

Bottom Line: Galectin-1 depletion does not alter the gene expression level of integrin-beta1.Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCepsilon) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells.Our results argue for the involvement of galectin-1 in the PKCepsilon/vimentin-controlled trafficking of integrin-beta1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Toxicology, Institute of Pharmacy, Univesité Libre de Bruxelles, Brussels.

ABSTRACT
Cell motility and resistance to apoptosis characterize glioblastoma (GBM) growth and malignancy. In our current work we report that galectin-1, a homodimeric adhesion molecule and carbohydrate-binding protein with affinity for beta-galactosides, is linked with cell surface expression of integrin beta1 and the process of integrin trafficking. Using immunofluorescence, depletion of galectin-1 through both stable knockdown and transient-targeted small interfering RNA (siRNA) treatment induces an intracellular accumulation of integrin-beta1 coincident with a diminution of integrin-beta1 at points of cellular adhesion at the cell membrane. Galectin-1 depletion does not alter the gene expression level of integrin-beta1. Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCepsilon) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells. Our results argue for the involvement of galectin-1 in the PKCepsilon/vimentin-controlled trafficking of integrin-beta1. The understanding of molecular mediators such as galectin-1 and the pathways through which they drive the cell invasion so descriptive of GBM is anticipated to reveal potential therapeutic targets that promote glioma malignancy.

Show MeSH
Related in: MedlinePlus