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Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.

Khalfaoui T, Groulx JF, Sabra G, GuezGuez A, Basora N, Vermette P, Beaulieu JF - PLoS ONE (2013)

Bottom Line: Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine.Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments.Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

ABSTRACT
Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

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Reduction of 37/67LR expression inhibits cell proliferation.(A) Proliferation was evaluated in HIEC cells 24 and 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20) by BrdU incorporation. Transfected cells were seeded on plastic and stained using an anti-BrdU antibody (mean ± SEM, n=3, ** p< 0.005; *** p < 0.001). (B) The specificity of the inhibitory effect of a reduction of 37/67LR on BrdU incorporation was confirmed on HIEC treated during 48h with siCtrl (50µM), siLR3 and siLR4 (both at 20 and 50 µM) (mean ± SEM, n=3, ** p < 0.001). (C) Representative iCys laser scanning cytometric images from a single experiment exhibiting changes in the progression of cell cycle in HIEC cells 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20). G1, S, and G2/M in each micrograph represent the percentage of cells present in normal phases of the cell cycle whereas Sub-G1 represents the percentage of cells that have undergone apoptosis. (D) Histogram illustrating the percentage (%) of cells distributed in each of Sub-G1, G1, S and G2+M cell-cycle phases after 48 h for HIEC control (siCtrl) and knockdown (siLR4-20) obtained by iCys laser scanning cytometry analysis (mean ± SEM, n=3, * p < 0.05).
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pone-0074337-g006: Reduction of 37/67LR expression inhibits cell proliferation.(A) Proliferation was evaluated in HIEC cells 24 and 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20) by BrdU incorporation. Transfected cells were seeded on plastic and stained using an anti-BrdU antibody (mean ± SEM, n=3, ** p< 0.005; *** p < 0.001). (B) The specificity of the inhibitory effect of a reduction of 37/67LR on BrdU incorporation was confirmed on HIEC treated during 48h with siCtrl (50µM), siLR3 and siLR4 (both at 20 and 50 µM) (mean ± SEM, n=3, ** p < 0.001). (C) Representative iCys laser scanning cytometric images from a single experiment exhibiting changes in the progression of cell cycle in HIEC cells 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20). G1, S, and G2/M in each micrograph represent the percentage of cells present in normal phases of the cell cycle whereas Sub-G1 represents the percentage of cells that have undergone apoptosis. (D) Histogram illustrating the percentage (%) of cells distributed in each of Sub-G1, G1, S and G2+M cell-cycle phases after 48 h for HIEC control (siCtrl) and knockdown (siLR4-20) obtained by iCys laser scanning cytometry analysis (mean ± SEM, n=3, * p < 0.05).

Mentions: To determine whether 37/67LR was directly implicated in cell proliferation, we first evaluated proliferation by BrdU incorporation 24 and 48h following control and siLR transfection. As expected from previous studies, BrdU incorporation under these experimental conditions resulted in a ~30% proportion of positive cells under control conditions [46,71] as also observed herein (Figure 6A). A significant reduction of cell proliferation was observed at both 24 and 48h after transfection with the siLR4 used at 20 µM (Figure 6A). To confirm the specificity of the effect, we tested a second siRNA. As shown in Figure 6B, siLR3 used at both 20 and 50 µM had comparable effects on inhibition of BrdU incorporation as siLR4.


Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.

Khalfaoui T, Groulx JF, Sabra G, GuezGuez A, Basora N, Vermette P, Beaulieu JF - PLoS ONE (2013)

Reduction of 37/67LR expression inhibits cell proliferation.(A) Proliferation was evaluated in HIEC cells 24 and 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20) by BrdU incorporation. Transfected cells were seeded on plastic and stained using an anti-BrdU antibody (mean ± SEM, n=3, ** p< 0.005; *** p < 0.001). (B) The specificity of the inhibitory effect of a reduction of 37/67LR on BrdU incorporation was confirmed on HIEC treated during 48h with siCtrl (50µM), siLR3 and siLR4 (both at 20 and 50 µM) (mean ± SEM, n=3, ** p < 0.001). (C) Representative iCys laser scanning cytometric images from a single experiment exhibiting changes in the progression of cell cycle in HIEC cells 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20). G1, S, and G2/M in each micrograph represent the percentage of cells present in normal phases of the cell cycle whereas Sub-G1 represents the percentage of cells that have undergone apoptosis. (D) Histogram illustrating the percentage (%) of cells distributed in each of Sub-G1, G1, S and G2+M cell-cycle phases after 48 h for HIEC control (siCtrl) and knockdown (siLR4-20) obtained by iCys laser scanning cytometry analysis (mean ± SEM, n=3, * p < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750003&req=5

pone-0074337-g006: Reduction of 37/67LR expression inhibits cell proliferation.(A) Proliferation was evaluated in HIEC cells 24 and 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20) by BrdU incorporation. Transfected cells were seeded on plastic and stained using an anti-BrdU antibody (mean ± SEM, n=3, ** p< 0.005; *** p < 0.001). (B) The specificity of the inhibitory effect of a reduction of 37/67LR on BrdU incorporation was confirmed on HIEC treated during 48h with siCtrl (50µM), siLR3 and siLR4 (both at 20 and 50 µM) (mean ± SEM, n=3, ** p < 0.001). (C) Representative iCys laser scanning cytometric images from a single experiment exhibiting changes in the progression of cell cycle in HIEC cells 48h after transfection with siCtrl or siLR4 at 20 µM (siLR4-20). G1, S, and G2/M in each micrograph represent the percentage of cells present in normal phases of the cell cycle whereas Sub-G1 represents the percentage of cells that have undergone apoptosis. (D) Histogram illustrating the percentage (%) of cells distributed in each of Sub-G1, G1, S and G2+M cell-cycle phases after 48 h for HIEC control (siCtrl) and knockdown (siLR4-20) obtained by iCys laser scanning cytometry analysis (mean ± SEM, n=3, * p < 0.05).
Mentions: To determine whether 37/67LR was directly implicated in cell proliferation, we first evaluated proliferation by BrdU incorporation 24 and 48h following control and siLR transfection. As expected from previous studies, BrdU incorporation under these experimental conditions resulted in a ~30% proportion of positive cells under control conditions [46,71] as also observed herein (Figure 6A). A significant reduction of cell proliferation was observed at both 24 and 48h after transfection with the siLR4 used at 20 µM (Figure 6A). To confirm the specificity of the effect, we tested a second siRNA. As shown in Figure 6B, siLR3 used at both 20 and 50 µM had comparable effects on inhibition of BrdU incorporation as siLR4.

Bottom Line: Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine.Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments.Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

ABSTRACT
Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

Show MeSH
Related in: MedlinePlus