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Am J Physiol Renal Physiol 294: F393-F406, 2008. First published November 21, 2007; doi:10.1152/ajprenal.00315.2007
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Genetic inactivation of the laminin {alpha}5 chain receptor Lu/BCAM leads to kidney and intestinal abnormalities in the mouse

Cécile Rahuel,1 Anne Filipe,1,* Léa Ritie,2,* Wassim El Nemer,1 Natacha Patey-Mariaud,3 Dominique Eladari,4 Jean-Pierre Cartron,1 Patricia Simon-Assmann,2 Caroline Le Van Kim,1 and Yves Colin1

1Institut National de la Santé et de la Recherche Médicale, Unité 665, Institut National de la Transfusion Sanguine, and University Paris Diderot, Paris, France; 2Institut National de la Santé et de la Recherche Médicale, Unité 682, University Louis Pasteur, Strasbourg; 3Service de Néphrologie, Hôpital Necker-Enfants Malades; and 4Institut National de la Santé et de la Recherche Médicale, Unité 872, Paris, France

Submitted 10 July 2007 ; accepted in final form 13 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lutheran blood group and basal cell adhesion molecule (Lu/BCAM) has been recognized as a unique receptor for laminin {alpha}5 chain in human red blood cells and as a coreceptor in epithelial, endothelial, and smooth muscle cells. Because limited information is available regarding the function of this adhesion glycoprotein in vivo, we generated Lu/BCAM-null mice and looked for abnormalities in red blood cells as well as in kidney and intestine, two tissues showing alteration in laminin {alpha}5 chain-deficient mice. We first showed that, in contrast to humans, wild-type murine red blood cells failed to express Lu/BCAM. Lu/BCAM-null mice were healthy and developed normally. However, although no alteration of the renal function was evidenced, up to 90% of the glomeruli from mutant kidneys exhibited abnormalities characterized by a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane. Similarly, intestine analysis of mutant mice revealed smooth muscle coat thickening and disorganization. Because glomerular basement membrane and smooth muscle coat express laminin {alpha}5 chain and are in contact with cell types expressing Lu/BCAM in wild-type mice, these results provide evidence that Lu/BCAM, as a laminin receptor, is involved in vivo in the maintenance of normal basement membrane organization in the kidney and intestine.

lutheran blood group and basal cell adhesion molecule; basement membranes; glomeruli; intestine; knockout mice

IN HUMANS, Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens both reside on two glycoprotein (gp) isoforms of the immunoglobulin (Ig) superfamily, Lu and Lu(v13), that differ by the size of their cytoplasmic tail (8, 45, 47). Lu/BCAM represent the unique receptors for laminins 511/521, major components of basement membrane, in normal and sickle (SS) red blood cells (RBCs) (14, 59). In humans, a pathophysiological role of Lu/BCAM in vasoocclusive crisis of sickle cell patients has been suggested by the observation that stimulation of SS RBCs by the physiological stress mediator epinephrine increases Lu/BCAM-mediated adhesion of SS RBCs to laminin {alpha}5 chain via a cAMP and protein kinase A-dependent signaling pathway (20, 23, 62). Lu/BCAM have been also recognized as the first members of the Ig superfamily acting as laminin {alpha}5 chain coreceptors in kidney epithelial cells, smooth muscle cells, and endothelial cell lines (26). Lu/BCAM have a predominant membrane expression in the basal layer of the epithelium, on the surface of a subset of muscle cells, and on the endothelium of blood vessel walls but were also detected in inflammatory epidermis such as acute contact dermatitis, actinic porokeratosis, and Sweet's syndrome (13, 26, 52), and have been shown to be overexpressed in various carcinoma (19). Lu/BCAM bind to the globular domain 3 of laminin {alpha}5 chain (LG3) (27) by their first three Ig-like domains (15). The cytoplasmic domain of Lu/BCAM interacts with the membrane skeleton (44) through direct binding to spectrin, at least in RBCs (31), and contains serine phosphorylation sites, which is consistent with a receptor signaling function. However, although the adhesion properties of Lu/BCAM have been clearly demonstrated in vitro, limited information is available regarding the biological function of these gps in vivo.

In mouse, Lu/BCAM is expressed from the Lu gene as a unique isoform (44, 46) in various tissues, including heart, lung, kidney, intestine, and skeletal muscle (40). Moreover, localization of Lu/BCAM at the basal surface of many epithelial cells and on muscle cells adjacent to basement membranes suggests that Lu/BCAM could function as a receptor for {alpha}5 chain-containing laminins in mice (26). Supporting this hypothesis, it has been shown that Lu/BCAM expression is reduced dramatically in various tissues from mouse embryos lacking laminin {alpha}5 chain, although it is increased significantly in the heart of transgenic mice overexpressing laminin {alpha}5 chain (6, 40).

Mice lacking laminin {alpha}5 chain die during embryogenesis with various developmental defects, including kidney and intestine developmental abnormalities (6, 36, 38, 42). Hence, laminin {alpha}5 chain mutant embryos exhibit a defective glomerulogenesis associated with an abnormal glomerular basement membrane (GBM) and absence of vascularized glomeruli, an abnormal intestinal smooth muscle development with excessive folding of intestinal loops, and a defective differentiation process of smooth muscle cell. Finally, absence of laminin {alpha}5 chain in lung induces impaired late lung development with abnormal alveolar epithelial cell maturation, perturbed vasculogenesis, and alveolization. In the same way, mice lacking integrin {alpha}3β1, an adhesion molecule shown to act as laminin {alpha}5 chain receptor, present a partially similar phenotype than laminin {alpha}5 chain mice in kidney and lung (30). Together with in vitro studies showing that integrin {alpha}3β1 and Lu/BCAM mediate adhesion of mesangial cells to GBM laminin {alpha}5 chain (29), these results suggest a potential cooperation in vivo between laminin {alpha}5 chain receptors, Lu/BCAM gp and integrin {alpha}3β1, during mouse organogenesis.

Therefore, to gain insight into Lu/BCAM function, we generated mice with targeted disruption of the Lu gene and examined whether they displayed hallmarks of disturbed morphogenesis and differentiation of the kidney and intestine, two organs shown to highly express Lu/BCAM (6, 40). Lu/BCAM-null mice were viable and fertile. We showed that the loss of Lu/BCAM causes structural alterations in kidney (thickening of the GBM) and intestine (enlargement of the smooth muscle layers and abnormal organization of the cells).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gene targeting, breeding, and genotyping of mice. The mouse Lu gene was disrupted by insertional mutagenesis as previously described by Zheng et al. (63). An insertional vector was isolated from the mouse 129S5/SvEvbrd genomic 3'hprt library [a generous gift of Dr. A. Bradley, (63)] using a 300-bp probe from exon 8 of the mouse Lu gene. The targeting insertional vector contained a 7.3-kb genomic fragment of the murine Lu gene starting from intron 1 and finishing in intron 8, as determined by restriction mapping, PCR analysis, and sequencing. A gap of 170 bp was created in the Lu sequence between unique HpaI and SfiI sites to stimulate the single crossover event between the vector and the chromosomal DNA (Fig. 1A). The HpaI/SfiI linearized vector was electroporated into 129/Ola embryonic stem cells (genOway, Lyon, France). Embryonic stem cell culture, electroporation (800 volt, 0.3 µF, 80.106 cells for 100 µg of linearized targeting vector), and puromycin selection of transfected embryonic stem cell clones were performed at genOway, according to standard procedures. As the gap was repaired during the targeted recombination, the HpaI/ SfiI region of the Lu gene was used as a probe for Southern blot screening of embryonic stem cell recombinants after SacI digestion. In targeted clones, a 18-kb SacI fragment was revealed in addition to the wild-type (WT) 5.4-kb fragment (Fig. 1B).


Figure 1
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  		Fig. 1. Targeted disruption of the mouse lutheran blood group (Lu) gene and genotyping strategy. A: targeting insertional vector is shown between the wild-type (WT) and the recombinant Lu gene isolated from the 3'hprt mouse genomic library. The 3'hprt library backbone contains the puromycin resistance gene (Puro), 3'hprt, and the K14Agouti transgene. The gapped insertional vector is linearized at the SfiI and HpaI sites and targeted to the Lu locus. During the targeting event, the SfiI/HpaI gap is repaired, resulting in 2 duplicated regions separated by the vector backbone. B: strategy for mice genotyping by Southern blotting with a probe located in the gap region. After SacI digestion, a 5.4-kb fragment in WT allele and a 18-kb fragment in the mutated allele were detected. After NdeI digestion, a 4.7-kb fragment was present in WT and heterozygous mice, which disappeared in homozygous (–/–) mice. C: Southern blot analysis after SacI or NdeI digestion of genomic DNA extracted from the tail of WT (+/+), heterozygous (+/–), and homozygous (–/–) mice. D: mice genotyping by PCR-restriction fragment length polymorphism. A NdeI site (noted NdeI* in B) present in C57B/6 allele and absent in 129/Ola lineage was used. The PCR generated a 581-bp fragment encompassing the polymorphic site. After Nde I digestion, the 581-bp PCR product was cleaved into two fragments of 302 and 279 bp (comigrating on 2% agarose) in C57BL/6 genotypes (WT), whereas only the uncleaved 581-bp fragment was observed with 129/Ola lineage (KO). All 581-, 302-, and 279-bp fragments were detected in the heterozygous mice.

 
Chimeric mice were generated at the Institut Clinique de la Souris (ICS, Strasbourg, France) after injection of one embryonic stem cell recombinant clone into C57BL/6 blastocysts. Male chimeras were mated to C57BL/6 females, and genotyping of progeny by Southern blot analysis after SacI digestion was performed as described above. Homozygous Lu–/– [knockout (KO)] and WT mice on a hybrid 129/Ola-C57BL/6 background were obtained by heterozygous mating. Targeting was confirmed by a Southern blot analysis of SacI and NdeI fragments from tail biopsy DNA (Fig. 1C). After lineage establishment, genotyping was performed by PCR using an restriction fragment length polymorphism (RFLP) between C57BL/6 and 129/Ola mice: an Nde I site (Fig. 1B) was present in the Lu gene from C57BL/6 and absent in the 129/Ola lineage. Oligonucleotides used for PCR-RFLP were as follows: sense 5'-GGCCACCTCAAGTGTCCGTGTGTTT-3' and antisense 5'-TTTGAGGTCATTCTGGGCTGGCTAT-3'. The PCR generated a 581-bp fragment encompassing the polymorphic site. After Nde I digestion, the 581-bp PCR product was cleaved into two fragments of 279 and 302 bp (comigrating on agarose 2%) in C57BL/6 genotypes (WT), whereas only the uncleaved 581-bp fragment was observed with 129 lineage (KO). All 581-, 302-, and 279-bp fragments were detected in the heterozygous mice (Fig. 1D).

Antibodies. The rabbit polyclonal anti-murine (m) Lu antibody 455 was raised against the NH2-terminal extracellular domain of the murine Lu gp. A cDNA fragment encoding amino acids 21 to 540 was cloned into the pIgplus vector (Ingenius; R & D Systems) and used for transient transfection of COS-7 cells. The secreted mLu protein was recovered from a Protein A-Sepharose purification column as previously described (15) and used to immunize New Zealand rabbits (EFS, Nantes, France). Specificity of the 455 anti-mLu polyclonal antibody (pAb) was determined by Western blot analysis of Triton X-100 extracts prepared either from WT human erythroleukemic K562 cells (34) or from K562 cells expressing the recombinant mLu gp (46). The 455 anti-mLu pAb detected a 85-kDa band in transfected but not in WT K562 cells (see Fig. 2A). The rabbit polyclonal anti-human (h) Lu antibody 602 directed against amino acids 579 to 595 of the COOH-terminal part of the human protein has been described previously (20). As expected from sequence conservation between the human and murine Lu gp cytoplasmic tail (44, 46), the 602 pAb cross reacted with the recombinant mLu gp expressed in K562 cells (see Fig. 2A). Primary antibodies to basement membrane molecules and to integrin subunits used for immunofluorescence studies are listed in Table 1. To label enterocytes and enteroendocrine cells in the intestine, affinity-purified rabbit polyclonal antibodies anti-villin [generous gift of Dr. Robine, Institut Curie, Paris, France (50)] and anti-chromogranin A (DiaSorin) were used. To identify muscle cells, polyclonal smooth muscle desmin antibodies [kindly provided by Prof. Gabbiani, Université de Genève, Suisse (4)] and monoclonal anti-{alpha}-smooth muscle actin (Sigma, St. Louis, MO) were used. Mouse monoclonal anti-β-tubulin isotype III antibody (clone SDL.3D10; Sigma) was used for detection of myenteric plexuses. To identify the cell types in glomerular structures, rat monoclonal antibody (mAb) MEC 13.3 to platelet endothelial cell adhesion molecule (PECAM) (BD Biosciences), mouse mAb GID4 against synaptopodin (PROGEN, Heidelberg, Germany), and mouse mAb clone D33 to desmin (DAKO) were used for labeling endothelial cells, podocytes, and mesangial cells, respectively.


Figure 2
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  		Fig. 2. Western blot analysis of Lu/basal cell adhesion molecule (BCAM) expression. A: anti-murine (m) Lu 455 antibody specificity was analyzed by Western blots: murine Lu/BCAM was revealed in the mouse kidney epithelial cell line (mIMCD3) and in the mLu-transfected human erythroleukemic cell line K562 (K562mLu) but not in the mouse erythroleukemic cell line MEL (NI MEL), even after dimethyl sulfoxide-induced terminal differentiation (I MEL), nor in normal human and mouse red blood cells (hRBC and mRBC, respectively) or sickle red blood cells (SAD mRBC) from mice. The difference in size (4 kDa) of Lu/BCAM between red blood cell (RBC) and non-RBCs presumably represents differential glycosylation of the protein. The anti-hLu 602 antibody, which cross-reacts with mLu, shows the same pattern as the anti-mLu 455 antibody, except that hRBC were positive for the human Lu expression. B: in WT mice (+/+), anti-mLu 455 antibody recognizes two bands in kidney (K) and intestine (I) but not in RBCs. The lower band (*) is not recognized by the anti-hLu 602 antibody and might therefore represent a degraded form of Lu/CAM protein lacking part of the COOH-terminal tail. No signal was detected in knockout (KO; –/–) mice. The same results were obtained with anti-hLu 602, which recognizes the intracytoplasmic part of the human Lu/BCAM protein. Bands noted represent nonspecific hybridization in intestine.

 

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  		Table 1. Specificities and sources of antibodies used for detection of basement membrane molecules and integrin subunits

 
Immunoblot analysis. Human erythroleukemic cells (K562, clone CCL-243) and murine inner medullary collecting duct cells (IMCD3, clone CRL-2123) were obtained from the American Type Culture Collection. Mouse leukemic lymphoblastic cells (MEL, clone 707) were obtained from the European Collection of Cell Cultures. Ghosts were prepared as described (12) from normal RBCs, sickle RBCs from SAD mice [a generous gift from Yves Beuzard, Hôpital St. Louis, Paris (56, 57)], and spherocytic RBCs from sph/sph mice (5). Mice kidneys and intestines were homogenized in tetraethylammonium buffer (10 mM triethanolamine, 250 mM sucrose) containing protease inhibitors [complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany)]. Homogenate protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Samples were then solubilized in Laemmli buffer and boiled 5 min before loading. Protein (50 µg) was separated by SDS-PAGE (8%) under nonreducing conditions and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Blots were probed with the anti-mLu 455 antiserum (1:10,000) or the anti-hLu 602 antiserum (1:5,000). Detection was done with a ECL+ chemiluminescence kit (Amersham Biosciences, Buckinghamshire, UK).

Physiological parameter measurements and metabolic cage experiments. All animals were treated in compliance with French and European Union animal welfare policies. Animal protocols were approved by the regional ethics committee (Strasbourg, France). All experiments were performed on age- and sex-matched littermates. Basic plasma chemistry and hematology were performed, noninvasive cardiovascular exploration was monitored, and general parameters, like physical appearance, body weight, and body mass index, were controlled. For urine chemistry and urinary protein excretion analysis, mice were put individually in metabolic cages for 9 days; after 5 days of habituation, 24-h urine was collected every day during four consecutive days. Because there were important standard deviations in urine parameter values between mice, a mean between the 4 days collection was calculated for each parameter. The following parameters were measured in urine every day: 24-h urine volume, sodium, potassium, chloride, calcium, urea, phosphorus, total proteins, glucose, and osmolarity. Urine osmolarity was analyzed by using a Fiske 110 osmometer. Creatinine clearance and fractional sodium reabsorption were calculated. Urine albumin dosage was performed by enzyme-linked immunosorbent assay. Qualitative analysis of urinary protein was performed by SDS-PAGE. Intestinal transit was determined by assessing the appearance in stools of an artificial colored alimentation. After a 36-h fasting, the mice had access to powdered chow mixed with a green food dye, and the time at which the first colored stools appeared was noted.

Conventional histology and transmission electron microscopy. Six males and females of WT and Lu–/– mice were killed at 4 and 6 mo. The entire intestines (from duodenum to colon) and kidneys were removed and processed immediately. For light microscopy examination, tissues were fixed in 4% buffered formaldehyde or Bouin solution overnight, embedded in paraffin, and sectioned at 10 and 5 µm for segments of intestine (jejunum, colon) and kidney, respectively. Sections were stained with hematoxylin-eosin or with periodic acid-Schiff (PAS) for light microscopy.

For transmission electron microscopy (facilities of the IGBMC, Strasbourg, France), intestinal and kidney specimens were fixed for 2 h at 4°C in 0.2 M cacodylate-buffered 2% glutaraldehyde, pH 7.4, postfixed for 30 min at 4°C in cacodylate-buffered 1% osmium tetroxide, pH 7.4, dehydrated, and embedded in araldite or mounted on aluminum stubs coated with palladium gold. Semithin 0.5-µm sections were stained with toluidine blue for histological observations. Ultrathin sections were stained with uranyl acetate and lead citrate for kidney and colon before ultrastructural observation using an electron microscope (Morgagni 268D-Philips), and images were recorded into the Soft Imaging System.

Immunohistochemical analysis. For indirect immunofluorescence staining, kidneys were cut in half in a midsaggital plane and cryoprotected by incubation overnight in 30% sucrose in PBS, immersed in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura), frozen in isopentane cooled in liquid nitrogen, and then stored at –80°C until used. Intestines were directly embedded in OCT and frozen with the same procedure. Kidney sections (2 µm) were placed on superfrost/plus slides (Kindler, Freiburg, Germany), washed two times in PBS, blocked in 10% goat serum-1% BSA in PBS 30 min at room temperature, and then incubated with primary antibodies diluted in 1% PBS-BSA for 1 h at room temperature in a moist chamber. After unbound antibodies were rinsed off with PBS, Alexa 488- or Alexa 568-conjugated secondary antibodies (goat anti-rabbit, -rat, or -mouse; Molecular Probes) were applied in a similar fashion. Transverse intestinal sections (7 µm) were incubated with primary antibodies overnight at 4°C in a moist chamber. Bound antibodies were visualized using fluorescein isothiocyanate-conjugated antibody [goat anti-rat (Jackson Laboratories); sheep anti-mouse (Bio-Rad), and goat anti-rabbit (Nordic)]. After being mounted in a glycerol-PBS-phenylenediamine solution, kidney and intestine slides were analyzed using an epifluorescence microscope AX 60 (Olympus Optical) or a confocal microscope C1 Nikon. Control sections were processed as above with omission of the primary antibodies.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Targeting of the Lu gene prevents renal and intestinal Lu/BCAM protein expression. The mouse Lu gene was disrupted by insertional targeting as described in MATERIALS AND METHODS and shown in Fig. 1, using an original method previously described by Zheng et al. (63). Mice heterozygous for the targeted Lu gene (Lu–/+) were bred, and genotype analysis of the progeny (n = 129) showed a transmission of the targeted allele with an expected Mendelian ratio (+/+26%, +/–46%, and –/–27%). Western blot analysis revealed that the 81-kDa band detected in kidney and intestinal WT protein samples by antibodies directed against the extracellular (pAb 455) and intracellular (pAb 602) domains of Lu/BCAM was undetectable in Lu–/– samples (Fig. 2B). Immunohistochemistry experiments with the 455 pAb revealed expression of Lu/BCAM in kidney and intestine (small intestine and colon) sections from WT mice, whereas no staining was detected in KO mice (Fig. 3). All of these results demonstrated that the insertional gene targeting event was effective in preventing expression of Lu/BCAM in two murine tissues previously reported to express this gp (6, 40).


Figure 3
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  		Fig. 3. Immunolocalization of Lu/BCAM in mouse kidney, small intestine, and colon. Frozen sections from WT and Lu/BCAM-null (KO) mice were stained with the 455 anti-Lu polyclonal antibody (pAb). In kidney sections, low-power magnification shows Lu/BCAM expression in glomeruli (g), distal tubules (dt), and blood vessels (bv) of WT mice. Cross section staining of small intestines and colon from WT show that Lu/BCAM is present in muscular layers (ml), in the serosal layer (arrow), in the muscularis mucosae (mm), in blood vessels (bv), and at the base of epithelial cells (arrowheads) along the crypt-villus axis in the WT small intestine or along the colonic gland. Insets in small intestine panels point to enlargement of the muscular layers. No staining was detected in kidney, small intestine, or colon or in of Lu/BCAM-null mice. Scale bars: kidney, 50 µm; small intestine and colon, 100 µm; insets, 25 µm.

 
Lu/BCAM is not expressed on murine RBCs. Because expression and function of Lu/BCAM is being extensively investigated in human RBCs (16), we wished to check if Lu–/– mice could also constitute an animal model of Lu/BCAM deficiency in the RBC lineage. Surprisingly, the 455 and 602 anti-Lu pAbs failed to detect a protein band in RBC ghosts prepared either from WT or from Lu–/– mice (Fig. 2B). Whereas murine Lu/BCAM was revealed in the mouse kidney epithelial cell line mIMCD3 and in the mLu-transfected human erythroleukemic cell line K562, Lu/BCAM could not be detected in the mouse erythroleukemic cell line MEL, even after dimethyl sulfoxide (DMSO)-induced terminal differentiation, nor in SS RBC from SAD mice (Fig. 2A). Murine SS RBCs were analyzed because human SS RBCs express a higher amount of Lu/BCAM compared with control RBCs (14, 58). Lu/BCAM was not detected by Western blot analysis of RBC ghost membranes from sph/sph hereditary spherocytosis mice, whose reticulocytes comprise up to 95% of the circulating hemoglobinized cells (5 and data not shown). In agreement with the Western blot analysis, flow cytometric analysis indicated that the 455 pAb was positive in a test with mIMCD3 cells but not with WT, SAD, and sph/sph mouse RBCs nor with uninduced and DMSO-induced MEL cells (data not shown). Furthermore, RT-PCR experiments indicated that the Lu/BCAM cDNA could be amplified from the mIMCD3 RNA preparation but not from whole blood RNAs (data not shown). These results demonstrated that, in mouse, and in contrast to humans, Lu/BCAM is not expressed in mature RBCs nor in reticulocytes.

Immunolocalization of Lu/BCAM in the mouse kidney and intestine. Lu/BCAM was localized in frozen sections of WT mouse kidney and intestinal tissues at the adult stage by indirect immunofluorescence using the 455 anti-mLu pAb. The specificity of this antibody was assessed by its reactivity with Lu/BCAM-transfected but not with untransfected cell lines (see above) and confirmed by its reactivity with WT but not with KO tissue samples. In kidney, glomeruli, distal tubules, collecting duct, and blood vessels were strongly labeled by the 455 pAb (Fig. 3), in agreement with previous analysis performed with a different anti-Lu pAb (40). Within glomeruli, colabeling experiments with cell type-specific antibodies and Lu/BCAM were analyzed by focusing on each cellular marker to adjust the confocal microscope on optimal labeling. Expression pattern of Lu/BCAM protein was analyzed on the same horizontal section. These experiments revealed a colocalization of Lu/BCAM with PECAM [endothelial cell marker (60)] and synaptopodin [podocyte marker (41)] and to a lesser extent with desmin [mesangial cell marker (43)] at what appears to be the junction with GBM (Fig. 4A). These results indicated that, within the glomeruli, Lu/BCAM is expressed not only in podocytes and mesangial cells, as previously suggested (29, 40), but also in endothelial cells. Furthermore, immunohistochemical analysis of WT glomeruli revealed a colocalization of Lu/BCAM with {alpha}5 and β2 laminin chains but not with laminin β1 chain (Fig. 4B). Because laminins 521 ({alpha}5β2{gamma}1) and 511 ({alpha}5β1{gamma}1) are components of the GBM and of the mesangial matrix (MM), respectively, these results together with those presented in Fig. 4A indicated that, in all three cell types, Lu/BCAM is expressed at cell contact with GBM.


Figure 4
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  		Fig. 4. Identification of cell type expressing Lu/BCAM in WT glomeruli. A: confocal microscopy analysis of double staining with anti-Lu 455 pAb (green) and monoclonal antibody (mAb) to platelet endothelial cell adhesion molecule (PECAM), synaptopodin, or desmin (red) to label endothelial cells, podocytes, and mesangial cells, respectively. The colocalization of Lu/BCAM with a cell specific marker yields a yellow color on podocytes and endothelial cells and to a lesser extent on mesangial cells. B: immunostaining of Lu/BCAM with laminin {alpha}5, β2, and β1 chains in glomeruli of WT mice. Lu/BCAM colocalized with {alpha}5 chain in GBM (yellow in merge). Lu/BCAM colocalized completely with the laminin β2 chain in the GBM (yellow in merge) but did not colocalize with the β1 chain in the mesangial matrix. Scales bar: 30 µm.

 
The intestine is characterized by a morphological and functional proximo-distal regionalization; the small intestine (duodenum, jejunum, and ileum) is formed of villi composed of differentiated cells (enterocytes, mucus cells, endocrine, and Paneth cells), whereas the distal part (the colon) is formed of deep glands in which colonocytes and mucus cells are present. Overall, Lu/BCAM was detected in different areas across the intestinal wall: the outer smooth muscle layers, at the base of the epithelium, and in the endothelium of blood vessel walls (Fig. 3). Concerning the small intestinal muscle region, a strong reaction with the 455 pAb was noted in the external serosa (or mesothelium) and in the region delineating the individual smooth muscle cells in both internal and external muscular layers (Fig. 3). In the colon, the muscularis mucosae underlying the colonic glands, the muscular layers were also stained, whereas the external serosa was not labeled. The staining found in the epithelium, basally concentrated in the polarized epithelial cells, presented a gradient of expression from crypt base to villous tip in the small intestine and along the colonic glands (Fig. 3).

Phenotypic and macroscopic organ analysis of Lu/BCAM-null mice. Lu/BCAM-null mice appeared healthy and developed normally. Fertility analysis (WT females crossed with Lu–/– males and Lu–/– females crossed with WT males) indicated that Lu/BCAM-null mice were fertile and produced normal-sized litters of Lu+/– mice. When analyzed at 6 mo of age, the body weight of KO mice was higher than that of their WT littermates [KO vs. WT males (gr): 36 ± 2.6 vs. 29 ± 5.9, n = 7, P < 0.03; KO vs. WT females: 25.7 ± 2.6 vs. 23.3 ± 1.5, n = 8, P < 0.05]. In addition, the body mass index of KO males was higher than that of the WT males (0.34 vs. 0.28, P < 0.03, n = 7). No differences were observed between KO and control mice for other general parameters (body shape, head shape, whiskers, teeth, ear, eye, limbs, digits, tail, coat, skin, swelling and edema, gait, motricity, rectal temperature, bone mineral density) nor for blood common chemistry and hematology (plasma urea, creatinine, sodium, potassium, phosphorus, chloride, calcium, magnesium, bicarbonates, total proteins, and osmolarity; white blood cells, RBCs, and platelets count; hemoglobin and hematocrit) (data not shown).

Apart from a significant difference in kidney weight between KO and WT mice (see below), none of the organs examined at the macroscopic levels (lung, heart, prostate, liver, small intestine, colon, ovary, oviducts, testis, and prostate) exhibited abnormalities in KO mice compared with controls.

Renal abnormalities in Lu/BCAM-null mice. A significant difference of kidney weight between Lu/BCAM-null and WT mice was observed, with the mean kidney weight of male and female Lu/BCAM-null mice being higher than that of their respective controls at 4 mo of age [KO vs. WT males (gr): 0.27 ± 0.03 vs. 0.21 ± 0.03, n = 7, P < 0.001; KO vs. WT females: 0.16 ± 0.01 vs. 0.14 ± 0.02, n = 7, P < 0.02] and at 6 mo of age [KO vs. WT males (gr): 0.26 ± 0.04 vs. 0.21 ± 0.03, n = 7, P < 0.01; KO vs. WT females: 0.16 ± 0.02 vs. 0.13 ± 0.01, n = 7, P < 0.02]. However, these differences did not remain significant when values were normalized to the body weight.

Renal abnormalities in Lu/BCAM-null mice compared with the WT exclusively concerned the glomerulus. Serial sectioning of kidneys (6 WT and 5 KO males as well as 6 WT and 6 KO females of 4 mo, 4 WT and 8 KO males, and 9 WT and 7 KO females of 6 mo) followed by hematoxylin and eosin staining revealed that, regardless of their position within the cortex, a twofold higher number of Lu/BCAM-null glomeruli from males (~80–90%) than from females (~40–50%) exhibited a significant reduction in the number of visible capillary lumens (Fig. 5A). PAS staining confirmed this observation and revealed, in addition, an increase of the PAS-positive surface in the glomeruli of male mutants (15 KO), but not of female mutants (13 KO), compared with the WT mice (15 males and 13 females) at both 4 and 6 mo (data not shown).


Figure 5
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  		Fig. 5. Histological analysis of glomeruli from WT and Lu/BCAM-null mice (6 mo old). A: hematoxylin-eosin staining shows a significant reduction of the visible capillary lumens (arrow) in Lu-null mice compared with WT mice. Scale bar: 30 µm. B: electron micrograph analysis of glomerulus. In mutant mice, a significant thickening of the glomerular basement membrane (GBM) was observed with extensive protusions toward the capsular space (big arrow). Scale bar: 0.05 µm. C: electron micrograph analysis of the glomerular filter. Er, RBC; 1, foot process; 2, capillary lumen; 3, podocyte; 4, capsular space; small arrows, fenestrated endothelium; star, electron dense endothelial cytoplasmic material. Scale bar: 0.025 µm.

 
Glomerular abnormalities were characterized in more detail by electron microscopy in 4- and 6-mo-old mice (Fig. 5B). In agreement with light microscopy, electron micrographs revealed abnormalities of ~40–50% and 80–90% of the glomeruli from all female (5 KO vs. 3 WT) and male (5 KO vs. 3 WT) Lu/BCAM-null mice, respectively. The main feature is a considerable thickening of the lamina densa of the GBM with the presence of numerous protusions toward the podocytic side. These formations were absent in age-paired WT mice. No podocyte changes were detected, in particular; foot processes and slit diaphragms were normally present. Except for the presence of electron dense endothelial cytoplasmic materials (Fig. 5C) and for a slight accumulation of MM (data not shown), there was no evidence for major abnormalities of endothelial and mesangial cells.

Because the GBM plays critical roles in glomerular filtration (35), these glomerular abnormalities led us to investigate potential alteration of the renal function in Lu/BCAM-null mice. The study was performed on 24 mice between 23 and 31 wk of age: 6 WT and 6 KO males and 5 WT and 7 KO females. In spite of the abnormally thick GBM in Lu/BCAM-null mice, renal function was not disturbed. No significant differences were observed between Lu/BCAM-null and WT mice when the following parameters were measured: 24-h urine volume, sodium, potassium, chloride, calcium, urea, creatinine, phosphorus, total proteins, glucose, and osmolarity.

Basement membrane protein analysis in Lu/BCAM-null glomeruli. Because Lu/BCAM has been shown to specifically bind the laminin {alpha}5 chain (27, 33), we tested if Lu/BCAM deficiency could modify expression of the {alpha}5 chain-containing laminin 511 (MM) and laminin 521 (GBM), as well as that of integrins {alpha}3β1 and {alpha}6β1 shown to act as laminin {alpha}5 chain coreceptors (26). Figure 6 shows that, compared with control (3 WT males of 6 mo old), staining of Lu/BCAM-null kidneys (3 KO males of 6 mo old) reveals an overall retraction of the glomeruli with all the GBM and MM markers examined. Moreover, laminin {alpha}5, β2, and {gamma}1 antibodies gave a diffuse labeling pattern and revealed a narrowing of visible capillary lumens in Lu/BCAM-null glomeruli. In contrast, laminin β1 as well as the laminin chains {alpha}2 and {alpha}4 exhibited a normal labeling pattern, indicating no abnormalities of the MM in KO mice.


Figure 6
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  		Fig. 6. Basement membrane protein analysis in Lu/BCAM-null glomeruli. Expression of laminins 511 and 521 ({alpha}5, β1, β2, and {gamma}1 chains) in glomeruli in 6-mo-old mutant and WT mice was analyzed by confocal microscopy. Laminin {alpha}2 or {alpha}4 chains and collagen IV{alpha}5 chain or entactin were used as mesangial matrix (MM) and GBM specific markers, respectively.

 
To determine if the abnormal laminin pictures obtained with laminin {alpha}5, β2, and {gamma}1 antibodies account for an alteration of the overall GBM structure, or for specific modifications of some GBM components, we analyzed the expression pattern of other basement membrane components. Type IV collagen {alpha}5 chain and entactin, expressed in GBM (22, 32, 39), revealed the glomerular retraction but did not exhibit a diffuse labeling pattern. No difference of expression for the laminin receptors {alpha}3, {alpha}6, and β1 integrin subunits between Lu/BCAM-null and WT glomeruli were detected (data not shown).

Absence of Lu/BCAM is accompanied by intestinal smooth muscle thickening and disorganization. We examined the jejunum and colon segments of 4- and 6-mo-old control (4 cases of 4-mo-old and 4 cases of 6-mo-old) and mutant (7 cases of 4-mo-old and 5 cases of 6-mo-old) animals (Fig. 7). The global architecture of the small intestine and colon upon histological analysis was similar between WT and Lu/BCAM-null mice: formation of crypt-villus structures and development of the smooth muscle layers were not affected whatever the stage analyzed. The normal program of epithelial differentiation was not compromised in the intestines of mutant animals (Fig. 8). Indeed, no obvious modifications in the apical brush-border membrane of enterocytes were detected, as confirmed by staining of the brush border-associated villin. Morphology and number of mucus cells assessed by PAS-hematoxylin staining was identical in jejunum or colon segments of mutant mice compared with intestinal samples from control animals. In both WT and Lu/BCAM-null mice, enteroendocrine cells, examined by immunostaining for chromogranin A, were seen at typically low numbers in the jejunum. Finally, Paneth cells were identified in the crypt region of small intestinal segments in WT and Lu/BCAM-null mice. However, an increase in the smooth muscle coat thickness [where Lu/BCAM is highly expressed in control intestines (see Fig. 3)] was observed in the 4-mo-old mutant intestines (7 cases out of 9 mutants analyzed vs. 9 controls). This difference was mostly visible at the distal part of the intestine, the colon, leading in some cases to a threefold thickening of the muscle coat. At 6 mo, these differences between WT and KO were not detectable anymore (Fig. 7).


Figure 7
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  		Fig. 7. Thickening of the intestinal muscle coat in 4-mo-old Lu/BCAM null mice. Representative sections of the small intestine (a, b, d, e, g, and i) and colon (c, f, h, and j) from WT (a–c, g, and h) and KO (d–f, i, and j) animals at 4 (a–f) and 6 (g–j) mo stained with hematoxylin-eosin. The thickness of the muscle coat is increased at 4 mo in the KO mice in both regions of the intestine, the jejunum (e vs. b), and the colon (f vs. c) although some irregularity can be noted. The overall organization of the small intestine and colon is not modified as attested by similar numbers, sizes, and organizations of the crypt/villus units (d and i vs. a and g) or glands (j vs. h). ml, Muscular layer; cml, circular muscular layer; lml, longitudinal muscular layer; v, villus; c, crypts. Scales bars: 200 µm in a and d; 25 µm in b, c, e, and f; and 100 µm in g, h, i, and j.

 

Figure 8
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  		Fig. 8. Cytodifferentiation of intestinal epithelial cells is not modified in Lu/BCAM-null mice. Representative sections of the small intestine from WT (a–d) and KO (e–h) adult mice stained for representation of enterocytes (anti-villin antibody; a and e), of goblet cells (periodic acid/Schiff staining; arrows in b and f), of enteroendocrine cells (anti-chromogranin A antibody; arrows in c and g), and of Paneth cells located in the crypt region (ultrathin sections; arrows in d and h point to Paneth granules). e, Epithelium; lp, lamina propria; ml, muscular layers; c, crypt. Scale bar: 25 µm.

 
To examine possible modifications in the muscle compartment and in basement membranes, sections of intestinal segments (jejunum and colon) were processed for electron microscopy on 4-mo-old (2 females and 2 males) and on 6-mo-old (1 female) WT and Lu/BCAM-null mice (Fig. 9). Two major alterations were noted in jejunum and colon from mutant mice, whatever the stage examined, and were essentially obvious at 4 mo: 1) disorganization of myofilaments that are multidirectional but in controls fibers are found parallel to the main axis of the cell (Fig. 9A) and 2) abnormal enlarged spaces between individual smooth muscle cells concomitant with an accumulation of electron-dense material not organized as a well-defined basement membrane (Fig. 9B). Occasionally, abnormal accumulation of organelles were noted, such as clusters of mitochondria in the center of cells and presence of numerous vesicles of pinocytosis under the plasma membrane of smooth muscle cells (data not shown); other abnormal features found in mutant intestinal samples include sporadically damaged mitochondria and nuclei necrosis (data not shown). These alterations led us to investigate potential modification of the bowel function by analyzing the intestinal transit. This transit was not significantly modified in 4-mo-old mutant mice (6 females) compared with controls (6 females). The first colored stools appeared at 170.5 ± 37.3 (SE) min in control animals vs. 179.7 ± 35.4 min for Lu/BCAM-null animals (P > 0.05; n = 6).


Figure 9
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  		Fig. 9. Ultrastructural analysis showing the smooth muscle alterations in Lu/BCAM-null intestines. Ultrathin sections of jejunal (a and c: 4-mo-old animals) and colonic (b and d: 6-mo-old animals) samples from WT (a and b) and KO (c and d) mice. Note the dramatic disorganization of the cytoskeletal elements and of the intercellular space between smooth muscle cells in mutant intestines when compared with controls. Arrows in b and d point to the basement membrane region. Scale bars: 0.5 µm in a and 1 µm in b, c, and d.

 
Basement membrane composition and laminin-binding receptors in Lu/BCAM-null intestines. To determine if deficiency of the Lu/BCAM receptor is associated with changes in the expression pattern of {alpha}5 chain-containing laminins, we examined the intestines of Lu/BCAM-null mice (3 WT and 5 KO of 4 mo old and 2 WT and 2 KO of 6 mo old) by immunofluorescence for laminin {alpha}5, β2, {gamma}1, and {gamma}2 chains. The labeling pattern of the anti-laminin β2, {gamma}1, and {gamma}2 chains (Fig. 10) was not altered in Lu/BCAM-null intestine, neither in the smooth muscle region nor at the epithelial/stromal interface region, areas in which, however, Lu/BCAM was detected in WT intestine sections (see Fig. 3). For laminin {alpha}5 chain, staining disappeared only in the external serosal layer of Lu/BCAM-null intestines (Fig. 10), a region in which Lu/BCAM was highly expressed in control intestines (see Fig. 3). Previously, we found that deletion of {alpha}5 laminin chain in intestine was associated with decreased expression of Lu/BCAM with ectopic deposition of laminin {alpha}2 and {alpha}4 chains (6). Similarly, we here found that laminin {alpha}4 chain was induced in this serosal layer of Lu/BCAM-null intestine (Fig. 10). These modifications were noted in both 4- and 6-mo-old mice in the jejunal part of the intestine where the serosal layer is clearly visible. We also examined the localization of the most-expressed laminin {alpha}5 receptors found in the intestine, the integrin {alpha}3, {alpha}6, β1, and β4 subunits. Similar staining was observed in both epithelial and muscle cells, except for the {alpha}6 subunit that showed a slight increase in the muscle region of 4-mo-old jejunum (data not shown).


Figure 10
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  		Fig. 10. Immunostaining of constitutive chains of laminin in the small intestines. Cryosections of intestines from WT (A) and KO (B) mice were immunostained with antibodies against laminin {alpha}5, β2, {gamma}1, {gamma}2, and {alpha}4 chains. Laminin {alpha}5 chain expression disappeared only in the serosal layer of Lu/BCAM-null intestines, whereas laminin {alpha}4 chain was induced. e, Epithelium; lp, lamina propria; ml, muscular layers; arrows point to the serosal layer. Scales bars: 100 µm for {alpha}5, {gamma}1, and {gamma}2 laminin chains and 25 µm for β2 and {alpha}4 chains.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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RBC Lu/BCAM gps were originally identified as carriers of human blood group Lutheran (9, 10), and recent in vitro studies pointed out their potential role in the exaggerated adhesion of human SS RBCs to endothelium (for review, see Refs. 16 and 26). Furthermore, expression of Lu/BCAM in the later stages of RBC maturation (7, 55), together with the presence of the {alpha}5 chain-containing laminins 511/521 in human and mouse bone marrow (21) suggested that Lu/BCAM-laminin interaction may be involved in the movement of maturing RBCs from the subendothelium of bone marrow sinusoids to the peripheral blood. Therefore, our aim to generate Lu/BCAM-null mice was originally to establish an animal model to evaluate in vivo and ex vivo the relative importance of Lu/BCAM-mediated RBC adhesion properties in normal and pathological situations. The human and mouse Lu genes and proteins are highly conserved, and experiments carried out with transfected cells indicated that adhesive properties of Lu/BCAM to laminin are conserved between humans and mice (45, 47), but the presence of Lu/BCAM in murine RBCs has not been demonstrated. Surprisingly, we showed here that Lu/BCAM is not expressed in murine mature RBCs and reticulocytes. However, the observation that reticulocytes from mice with hereditary spherocytosis are able to bind human laminin 511/521 under flow conditions (61) raised the question of the presence and of the nature of the laminin receptor on murine RBCs, at least in pathological conditions.

Apart from RBCs, Lu/BCAM is expressed in many human fetal and adult tissues (45, 47) and is overexpressed in various carcinomas (19). Similarly, the murine LU gene is also widely expressed among tissues, and, during mouse embryonic development, the Lu transcript was detected as early as 7 days of gestation (44, 46). The presence of large amounts of Lu/BCAM transcript in fetal tissues might suggest a role in development for this gp. Moreover, it has been shown previously that Lu/BCAM specifically binds the laminin {alpha}5 chain (33), which plays a critical role in mouse organogenesis and development (38). Lu/BCAM expression along the cell surface is correlated with the presence of laminin {alpha}5 chain in adjacent basement membrane and is modulated by this interaction (6, 36–38, 40, 54). Therefore, to gain insight into the potential role of Lu/BCAM in physiological processes such as morphogenesis, organogenesis, and differentiation, we analyzed Lu/BCAM-null kidney and intestine, two tissues that express Lu/BCAM and in which the crucial role of the laminin {alpha}5 chain has been previously demonstrated (6, 29, 38).

The Lu/BCAM-null mice are viable, appear healthy, and develop normally. Thus the inactivation of the Lu gene did not reproduce the defective glomerulogenesis and intestinal smooth muscle development that had been observed for the targeted disruption of the laminin {alpha}5 chain (6, 38). However, we found that basement membranes in kidney glomeruli and intestinal smooth muscle layers were disturbed in the absence of Lu/BCAM.

We have shown here that, in glomeruli, Lu/BCAM localizes at cell contact with GBM in podocytes and mesangial and endothelial cells. Accordingly, Lu/BCAM was shown to colocalize with laminin 521 ({alpha}5β2{gamma}1) but not with laminin 511 ({alpha}5β1{gamma}1). Consistent with this expression of Lu/BCAM on both sides of the GBM, histological analysis revealed that Lu/BCAM deficiency is associated with an abnormal thickening of the GBM with protusions through the epithelial side. Such GBM architecture is reminiscent of that described in rat and mouse kidneys during normal glomerular maturation. Initially, at the S-shaped and developing capillary loop stages, the GBM develops from the fusion of endothelial and epithelial basement membranes. Next, during glomerular maturation, an additional basement membrane, derived primarily from podocytes, appears as subepithelial outpockets that are eventually inserted in the fused GBM. This addition of newly formed basement membrane corresponds to the developmental transition from type IV collagen {alpha}1, {alpha}2 to {alpha}3, {alpha}4, and {alpha}5 and from laminin 111 and 511 to laminin 521. Later, double basement membrane and outpockets are not seen in normal animals, but it has been suggested that remodeling continues during the process of aging (1, 2). Furthermore, the presence of laminin 521 and type IV collagen {alpha}5 suggests that the defective remodeling of the GBM in Lu/BCAM-null mice might not result from glomerular abnormalities of maturation but rather from deficient splicing of additional basement membrane in the GBM during aging, as was proposed for kidney failure in mice lacking the tetraspanin CD151 (51).

Studies of KO mice deficient for laminin {alpha}5 chains or for {alpha}3β1 integrin, an {alpha}5 chain-containing laminin coreceptor (28), have shown the role of outside-in signaling between adherent cells and laminin in the proper organization of the GBM. Absence of laminin {alpha}5 chain results in disruption of GBM and aberrant behavior of associated cells, apparently because of a lack of interactions between endothelial and epithelial cells and GBM (38). On the other side, absence of {alpha}3β1 integrin located specifically at the foot process-GBM interface results in failure in foot-process development and in an extreme disorganization and fragmentation of the GBM along both the podocytic and the endothelial sides. These observations indicated that {alpha}3β1 integrin is necessary for initiating and maintaining the structural organization of the GBM (30) involving reciprocal dynamic interactions between the GBM and adjacent cells. Similarly, the glomerular abnormalities of Lu/BCAM-null mice described above suggest that Lu/BCAM might represent a major laminin 521 receptor on the podocytes essential for signaling. Although Lu/BCAM and integrin {alpha}3β1 are colocalized in podocytes in WT glomeruli (our present results) and although it has been recently shown that these two laminin 521 receptors share the same binding site on the LG3 domain of laminin {alpha}5 chain (28), we did not observed any modification in the expression of {alpha}3β1 integrin in the glomeruli of Lu/BCAM-null mice compared with WT. Together with the observations that the GBM abnormalities of Lu/BCAM-null glomeruli occurred in the presence of normal podocytes, these results support the hypothesis that, as shown in SS RBCs (20, 23), Lu/BCAM acts in podocytes as a signaling molecule whose absence cannot be compensated by {alpha}3β1 rather than as a membrane protein involved in the maintenance of cellular structures.

Although up to 90% of the Lu/BCAM-null glomeruli exhibited abnormalities characterized by important reduction of visible capillary lumens and irregular GBM thickening, no functional renal abnormalities such as proteinuria were found associated with the deficiency of Lu/BCAM in mice. At present, the GBM is considered as the critical barrier to protein filtration. Recent analysis of laminin β2 chain-null mice indicated that changes in GBM permselectivity properties preceded and were independent of observable alterations in podocytes (24). In Lu/BCAM-null mice, it is possible that, as shown for mice lacking entactin-1/nidogen-1 (32), the properties of the glomerular wall were altered, but glomerular and tubular epithelial cells were able to reabsorb the filtered proteins.

In the adult intestine, Lu/BCAM expression was found mostly in two distinct areas: at the base of epithelial cells facing the basement membrane along the crypt-villus axis and more intensively on the surface of smooth muscle fibers. As opposed to the kidney, an organ in which there are obviously some alterations in laminin deposition in Lu/BCAM-deficient mice, no visible changes in several laminin chains occur either at the subepithelial basement membrane or around individual smooth muscle cells. The only striking modification concerns the serosal layer in which the Lu/B-CAM defect was accompanied by absence of laminin {alpha}5 that is clearly compensated by other laminin {alpha} chains, a phenomenon already observed in intestine and kidney of laminin {alpha}5-null mice (6, 36).

The lack of Lu/BCAM in the intestinal smooth muscle cells, which mainly express laminin 521 (3), has a major consequence. Indeed, we found a thickening of the smooth muscle layers accompanied by the perturbation of the individual smooth muscle cells. The most striking features were 1) the impaired organization of the myofilaments, which are multidirectional in Lu/BCAM-null intestines, 2) disorganization of the smooth muscle basement membrane, and 3) presence of enlarged spaces in between smooth muscle cells filled by an accumulation of electron-dense material. Together with the observation that expression of integrins is grossly normal, meaning that probably no compensation had occurred, these results pointed out Lu/BCAM as a major intestinal muscle cell laminin {alpha}5 receptor in vivo. Yet, in contrast to Hirschsprung's disease (megacolon associated with failure of enteric ganglia formation) in which dilated muscle of the ganglionic bowel results in impaired transit (3), this transient hypertrophy observed in Lu/BCAM null mice does not significantly affect the intestinal transit. Similarly, no modifications of enteric plexuses were noted, since immunostaining of neural-specific β-tubulin was similar between control and Lu/BCAM mutant intestines (data not shown).

In contrast to muscle cells, the absence of Lu/B-CAM from epithelial cells, which face the basement membrane region along the crypt-villus axis and mainly contain laminin 511 (3), did not affect epithelial cell differentiation. This result supports previous analysis of laminin {alpha}5 chain null mice indicating that {alpha}3β1 integrin is the major {alpha}5 chain receptor in intestinal epithelial cells, at least at the embryonic stages (6).

The visceral musculature is a rather stable tissue, with turnover of muscle cells being extremely low (18). Hypertrophy of intestinal smooth muscle, linked to an increase in muscle cell size and number, has been described mostly due to pathology of chronic nature (17). Interestingly, modifications in mitochondria and intermediate filaments or myofilaments were also noted (18). As recently illustrated in the case of β1-integrin, shown to regulate the microtubule cytoskeleton (48), one can postulate that Lu/BCAM could affect the organization of the cytoskeleton network. However, preliminary experiments indicate that the observed defect in ultrastructure was not associated with any obvious quantitative changes in {alpha}-smooth muscle actin and desmin, checked by immunostaining and/or immunoblot (data not shown). We have previously shown that Lu/BCAM binds to spectrin in RBCs (31), and we are currently determining whether this interaction also occurs between Lu/BCAM and fodrin, the non-RBC form of spectrin. If so, this will prompt us to investigate whether fodrin is expressed differently in muscle and epithelial cells and whether the lack of Lu/BCAM could result in muscle, but not in epithelial cells, in altered qualitative/quantitative expression of the spectrin-based cytoskeleton.

In conclusion, we have shown in vivo that Lu/BCAM is a laminin 521 receptor involved in the maintenance of normal basement membrane organization, at least in kidney and intestine, and that these abnormalities occurred on normally developed organs. Further experiments will be necessary to characterize the signaling events resulting from Lu/BCAM-laminin 521 interaction. It is noteworthy that, as shown here in mice, there is no evidence that the lack of human Lu/BCAM in the few characterized individuals with true Lutheran-null blood group phenotype can cause any known detrimental clinical conditions under physiological situations (25). However, it is anticipated that the Lu/BCAM-null animal model described here should prove useful to evaluate the potential involvement of Lu/BCAM in kidney and intestinal disorders under pathological or stressing conditions.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Institut National de la Transfusion Sanguine, by the GIS-Maladies Rares, and by a Ligue contre le Cancer (Comité départemental du Haut-Rhin) grant to P. Simon-Assmann.


   ACKNOWLEDGMENTS
 
Drs. M. C. Gubler and L. H. Noël (Hopital Necker-Enfants Malades, Paris, France) are acknowledged for their help in kidney tissue section analysis. L. Klein and C. Arnold [Unité (U) 682 Institut National de la Santé et de la Recherche Médicale (INSERM), Strasbourg, France] and Emmanuel Collec [U665/ Institut National de la Transfusion Sanguine (INTS), Paris, France], N. Messaddeq [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Strasbourg, France], and K. Antal [Institut Clinique de la Souris (ICS), Strasbourg, France] are greatly acknowledged for technical assistance in tissue sample preparation, conventional histology, transmission electron microscopy, and confocal microscopy analysis. We thank Dr. L. Sorokin (Münster, Germany), Dr. M. DiPersio (Albany Medical College, Albany, NY), G. Gabbiani (Université de Genève, Suisse), Dr. S. J. Kennel (Oak Ridge, TN), Dr. S. Robine (Institut Curie, Paris, France), and Dr. T. Sasaki (Shriners Hospital for Children Research Center, Portland, OR) for supplying antibodies. Drs. D. Goossens, Drs. G. Nicolas and M. M. Trinh-Trang-Tan (INSERM U665, INTS) are acknowledged for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Colin, INSERM U665, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris, France (e-mail: colin{at}idf.inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. Filipe and L. Ritie have contributed equally to this work. Back


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 MATERIALS AND METHODS
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 REFERENCES
 

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