Figure 1.
Expression of 37/67LR in normal human intestinal cells.
As shown by Western blot (A), the anti-37/67LR antibody predominantly detects a major band at ~37 kDa in normal HIEC cells (lane 1) as well as in the positive control Caco-2 cells (lane 2). In the developing human small intestine (B–D) immunolocalization of 37/67LR revealed predominant expression of the receptor in the intervillous area at 10 weeks of gestation (B) and in the crypts at 14 (C) and 20 weeks (D) coinciding with the corresponding proliferative compartment as defined by the proliferative antigen Ki67 on serial sections (arrows in B', C' and D'). In the adult, 37/67LR (E) was mainly located in the middle part of the crypts (brackets) containing the most actively proliferating cells as revealed by Ki67 (E'). Villus (v) cells as well as the Paneth/stem cell region (*), the latter defined by the expression of the Paneth cell marker PLA2 (* in E'') were both negative for 37/67LR. Immunostaining of the stromal compartment was below the detection level at all stages. Evan blue was used as a histological counterstain (brown/red). Nuclei were stained with DAPI (blue). Scale bars in A-D: 50 µm; E: 100 µm. (F) Summary of observations in the adult small intestine where expression of 37/67LR was consistently found to be absent from the Paneth/stem cell zone (1), maximal in the middle crypt region containing the Ki67-positive transit amplifying cells (2) and decreased toward the terminal differentiation compartment (3) and the base of the villus (4).
Figure 2.
Tissular expression of 37/67LR in the intestine.
(A) Representative semi-quantitative PCR analysis of purified epithelial (e) and stromal (s) fractions showed that the 37/67LR transcript can be found in both tissues. RPLPO served as the normalizer. The purity of each fraction was shown by the detection E-cadherin (E-cad: epithelial) and tenascin-C (TnC: stromal). (B) Western blot analyses confirmed the presence of 37/67LR in both the epithelial and stromal fractions by detecting the 37kD component although at higher amounts in the epithelial fraction relative to β-actin (mean ± SEM, n=3, ***: p < 0.0005).
Figure 3.
Characterization of 37/67LR in human intestinal epithelial crypt (HIEC) cells.
(A) Western blot analysis of subconfluent (SC) and 5 day postconfluent (PC) HIEC cells revealed expression of 37/67LR in proliferative cells and a significant reduction of its expression in quiescent cells relative to β-actin (mean ± SEM, n=3, *: p < 0.02). (B) Cell counts over a 10 day period after seeding confirmed that cell number stabilizes after confluence (c ->). (C) Representative Western blot analysis of 37/67LR in enriched HIEC cytoplasmic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions showing that 37/67LR was essentially found in the cytoplasmic and membrane fractions. Enriched fractions were verified by the detection of p27KIP1 (p27; cytoplasmic fraction F1), the β1 integrin subunit (β1; membrane fraction F2), trimethylated lysine 27 on histone 3 (H3K27me3e; nuclear fraction F3) and keratin 18 (K18; cytoskeletal fraction F4; also detectable in the cytoplasmic F1 and membrane F2 fractions). (D) Indirect immunofluorescence for the detection of 37/67LR in HIEC cells showing mainly perinuclear granular and weak membrane staining. Nuclei were stained with DAPI (blue). Scale bar in D: 50 µm.
Figure 4.
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.
Figure 5.
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)).
Figure 6.
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).
Figure 7.
Decreased expression of 37/67LR results in reduction of cell adhesion to LM-111 YIGSR related peptide.
Controls and HIEC cells exhibiting reduced 37/67LR expression (siLR4-20) were used for 1h adhesion assays on low-fouling carboxymethyl-dextran (CMD) layers bearing the tripeptide Arg–Gly–Asp (RGD) or CDPGYIGSR, a laminin nonapeptide (YIGSR). The CMD surface prevents cell adhesion. HIEC cell adhesion to RGD was maximal and comparable for control and siLR-treated cells whereas a statistically significant reduction of cell adhesion on YIGSR was observed in partially 37/67LR-depleted cells (mean ± SEM, n=3, ** p < 0.01).
Figure 8.
Blocking 37/67LR reduces cell proliferation.
HIEC cells were treated for 1h with neutralizing-blocking anti-37/67LR antibody (MLuC5) or anti-integrin β1 antibody (mAb13) as well as non-immune serum (NI) as negative control before plating. BrdU-positive cells were counted and expressed as percentage of total cells determined by DAPI staining (mean ± SEM, n=3, * p < 0.001; ** p < 0.0001).