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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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Knockdown of 37/67LR expression in HIEC cells.(A) Representative Western blot analysis showing the expression level of 37/67LR following a period of 48h after transfection of the 4 predesigned siRNA sequences (siLR1-4) and control si (siCtrl). All sequences induced a significant reduction of 37/67LR, but siLR4 was most efficient. (B) Representative graphs showing relative amounts of 37/67LR mRNA by qPCR and protein by Western blot in control (siCtrl) and knockdown (siLR4) HIEC cells. Relative amounts were obtained from data normalized with RPLPO (mRNA) and β-actin (protein) (mean ± SEM, n=3, **: p<0.0005). (C) Phase contrast micrographs of HIEC cells taken 48h after transfection with siCtrl and siLR4.
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pone-0074337-g004: Knockdown of 37/67LR expression in HIEC cells.(A) Representative Western blot analysis showing the expression level of 37/67LR following a period of 48h after transfection of the 4 predesigned siRNA sequences (siLR1-4) and control si (siCtrl). All sequences induced a significant reduction of 37/67LR, but siLR4 was most efficient. (B) Representative graphs showing relative amounts of 37/67LR mRNA by qPCR and protein by Western blot in control (siCtrl) and knockdown (siLR4) HIEC cells. Relative amounts were obtained from data normalized with RPLPO (mRNA) and β-actin (protein) (mean ± SEM, n=3, **: p<0.0005). (C) Phase contrast micrographs of HIEC cells taken 48h after transfection with siCtrl and siLR4.

Mentions: As a first step to investigate the function of 37/67LR in HIEC cells, we transiently knocked down its expression by transfection with siRNA targeting the 37/67LR sequence. Analysis of 37/67LR expression after 48 hours in the presence of 40 µM of one of 4 predesigned siRNA sequences (siLR1,2,3 and 4) and siCtrl was performed by Western blot (Fig. 4A). While all 4 sequences tested showed some inhibition of expression relative to control, siLR4 showed more efficiency and reproducibility. Using siLR4, 37/67LR expression was found to be reduced by more than 5 times at both the transcript and protein levels (Figure 4B) while the appearance and viability of the cells appeared to be altered under these conditions (Figure 4C).


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)

Knockdown of 37/67LR expression in HIEC cells.(A) Representative Western blot analysis showing the expression level of 37/67LR following a period of 48h after transfection of the 4 predesigned siRNA sequences (siLR1-4) and control si (siCtrl). All sequences induced a significant reduction of 37/67LR, but siLR4 was most efficient. (B) Representative graphs showing relative amounts of 37/67LR mRNA by qPCR and protein by Western blot in control (siCtrl) and knockdown (siLR4) HIEC cells. Relative amounts were obtained from data normalized with RPLPO (mRNA) and β-actin (protein) (mean ± SEM, n=3, **: p<0.0005). (C) Phase contrast micrographs of HIEC cells taken 48h after transfection with siCtrl and siLR4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750003&req=5

pone-0074337-g004: Knockdown of 37/67LR expression in HIEC cells.(A) Representative Western blot analysis showing the expression level of 37/67LR following a period of 48h after transfection of the 4 predesigned siRNA sequences (siLR1-4) and control si (siCtrl). All sequences induced a significant reduction of 37/67LR, but siLR4 was most efficient. (B) Representative graphs showing relative amounts of 37/67LR mRNA by qPCR and protein by Western blot in control (siCtrl) and knockdown (siLR4) HIEC cells. Relative amounts were obtained from data normalized with RPLPO (mRNA) and β-actin (protein) (mean ± SEM, n=3, **: p<0.0005). (C) Phase contrast micrographs of HIEC cells taken 48h after transfection with siCtrl and siLR4.
Mentions: As a first step to investigate the function of 37/67LR in HIEC cells, we transiently knocked down its expression by transfection with siRNA targeting the 37/67LR sequence. Analysis of 37/67LR expression after 48 hours in the presence of 40 µM of one of 4 predesigned siRNA sequences (siLR1,2,3 and 4) and siCtrl was performed by Western blot (Fig. 4A). While all 4 sequences tested showed some inhibition of expression relative to control, siLR4 showed more efficiency and reproducibility. Using siLR4, 37/67LR expression was found to be reduced by more than 5 times at both the transcript and protein levels (Figure 4B) while the appearance and viability of the cells appeared to be altered under these conditions (Figure 4C).

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