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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 without affecting endogenous protein translation.(A) Representative western blot analysis of 37/67LR (37kDa), human fibronectin (HFN), insulin like growth factor binding protein 7 (IGFBP7) and β-actin in HIEC cells 48h after transfection with siCtrl and siLR4 at various concentrations (0-50 µM). (B) Relative 37/67LR, HFN and IGFPB7 expression were found to be stable in the presence of increasing concentrations of siCtrl. With siLR4, 50% knockdown of 37/67LR expression was achieved at 20 µM, a concentration that had no effect on HFN or IGFBP7 expression in contrast to higher concentrations such as 40 and 50 µM which resulted in more than 80% knockdown of 37/67LR expression and inhibition of protein synthesis as observed for HFN and IGFBP7. Relative amounts of 37/67LR, HFN and IGFBP7 were determined by optical densitometry and expressed relative to β-actin (mean ± SEM, n=3 separate experiments, * p < 0.05 relative to 0 µM)).
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pone-0074337-g005: Reduction of 37/67LR expression without affecting endogenous protein translation.(A) Representative western blot analysis of 37/67LR (37kDa), human fibronectin (HFN), insulin like growth factor binding protein 7 (IGFBP7) and β-actin in HIEC cells 48h after transfection with siCtrl and siLR4 at various concentrations (0-50 µM). (B) Relative 37/67LR, HFN and IGFPB7 expression were found to be stable in the presence of increasing concentrations of siCtrl. With siLR4, 50% knockdown of 37/67LR expression was achieved at 20 µM, a concentration that had no effect on HFN or IGFBP7 expression in contrast to higher concentrations such as 40 and 50 µM which resulted in more than 80% knockdown of 37/67LR expression and inhibition of protein synthesis as observed for HFN and IGFBP7. Relative amounts of 37/67LR, HFN and IGFBP7 were determined by optical densitometry and expressed relative to β-actin (mean ± SEM, n=3 separate experiments, * p < 0.05 relative to 0 µM)).

Mentions: Because 37/67LR is thought to be required for protein translation [35], we performed a 0-50 µM dose–response analysis of siLR4 efficiency in order to evaluate 37/67LR expression relative to other control gene products such as fibronectin, IGFBP7 and TIMP3 which are constitutively produced by HIEC cells [47,71,74] and used to monitor translation. Control siRNA (siCtrl) dose–response showed that expression of 37/67LR, fibronectin, IGFBP7 and TIMP3 was not altered at any tested concentration (Figure 5A, left panel; Figure 5B, upper panel; TIMP3 data: 1.0 at 0 µM vs 1.05 ± 0.15 at 20 µM). In contrast, siLR4 showed maximal apparent efficiency at 30 µM and higher for both 37/67LR and fibronectin and 40 and 50 µM for IGFBP7 (Figure 5A, right panel). However at 20 µM, a ~50% knockdown of the expression of 37/67LR was observed with siLR4 while neither fibronectin, IGFBP-7 nor TIMP3 expression was significantly affected (Figure 5B, lower panel; TIMP3: 1.0 at 0 µM vs 1.22 ± 0.14 at 20 µM). These results indicated that siLR4 at a concentration of 20µM (siLR4-20) can significantly reduce 37/67LR expression without altering general translation. We then used these conditions to investigate the role of 37/67LR in normal HIEC cells.


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 without affecting endogenous protein translation.(A) Representative western blot analysis of 37/67LR (37kDa), human fibronectin (HFN), insulin like growth factor binding protein 7 (IGFBP7) and β-actin in HIEC cells 48h after transfection with siCtrl and siLR4 at various concentrations (0-50 µM). (B) Relative 37/67LR, HFN and IGFPB7 expression were found to be stable in the presence of increasing concentrations of siCtrl. With siLR4, 50% knockdown of 37/67LR expression was achieved at 20 µM, a concentration that had no effect on HFN or IGFBP7 expression in contrast to higher concentrations such as 40 and 50 µM which resulted in more than 80% knockdown of 37/67LR expression and inhibition of protein synthesis as observed for HFN and IGFBP7. Relative amounts of 37/67LR, HFN and IGFBP7 were determined by optical densitometry and expressed relative to β-actin (mean ± SEM, n=3 separate experiments, * p < 0.05 relative to 0 µM)).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074337-g005: Reduction of 37/67LR expression without affecting endogenous protein translation.(A) Representative western blot analysis of 37/67LR (37kDa), human fibronectin (HFN), insulin like growth factor binding protein 7 (IGFBP7) and β-actin in HIEC cells 48h after transfection with siCtrl and siLR4 at various concentrations (0-50 µM). (B) Relative 37/67LR, HFN and IGFPB7 expression were found to be stable in the presence of increasing concentrations of siCtrl. With siLR4, 50% knockdown of 37/67LR expression was achieved at 20 µM, a concentration that had no effect on HFN or IGFBP7 expression in contrast to higher concentrations such as 40 and 50 µM which resulted in more than 80% knockdown of 37/67LR expression and inhibition of protein synthesis as observed for HFN and IGFBP7. Relative amounts of 37/67LR, HFN and IGFBP7 were determined by optical densitometry and expressed relative to β-actin (mean ± SEM, n=3 separate experiments, * p < 0.05 relative to 0 µM)).
Mentions: Because 37/67LR is thought to be required for protein translation [35], we performed a 0-50 µM dose–response analysis of siLR4 efficiency in order to evaluate 37/67LR expression relative to other control gene products such as fibronectin, IGFBP7 and TIMP3 which are constitutively produced by HIEC cells [47,71,74] and used to monitor translation. Control siRNA (siCtrl) dose–response showed that expression of 37/67LR, fibronectin, IGFBP7 and TIMP3 was not altered at any tested concentration (Figure 5A, left panel; Figure 5B, upper panel; TIMP3 data: 1.0 at 0 µM vs 1.05 ± 0.15 at 20 µM). In contrast, siLR4 showed maximal apparent efficiency at 30 µM and higher for both 37/67LR and fibronectin and 40 and 50 µM for IGFBP7 (Figure 5A, right panel). However at 20 µM, a ~50% knockdown of the expression of 37/67LR was observed with siLR4 while neither fibronectin, IGFBP-7 nor TIMP3 expression was significantly affected (Figure 5B, lower panel; TIMP3: 1.0 at 0 µM vs 1.22 ± 0.14 at 20 µM). These results indicated that siLR4 at a concentration of 20µM (siLR4-20) can significantly reduce 37/67LR expression without altering general translation. We then used these conditions to investigate the role of 37/67LR in normal HIEC cells.

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