The galectins are a family of [beta]-galactoside binding proteins with features typical of cytoplasmic resident proteins, such as an acylated N-terminus and lack of a signal sequence for translocation into the endoplasmic reticulum (Barondes et al., 1994; Hughes, 1994, 1997). Nevertheless, the galectins are secreted by nonclassical and as yet poorly defined pathways and become localized at cell surfaces and in the extracellular matrix through binding to appropriately glycosylated ligands (Cooper and Barondes, 1990; Lindstedt et al., 1993; Sato et al., 1993; Mehul and Hughes, 1997).
Galectin-3 is expressed widely in epithelial tissues and cell lines such as Madin-Darby canine kidney MDCK cells. As a preliminary to studying the role of galectin-3 in kidney development in vivo (Winyard et al., 1997), we used (Bao and Hughes, 1995) a well-characterized culture model of renal epithelial morphogenesis (McAteer et al., 1987; Wang et al., 1990a,b). When MDCK cells are seeded at low density within 3D-collagen gels, the cells grow clonally and form small aggregates that in time sort out into polarized cysts with a central lumen and a basal surface in contact with the surrounding collagen gel as well as a laminin-rich basement membrane deposited by the cells (Caplan et al., 1987). Treatment of the cyst cultures with hepatocyte growth factor (HGF/scatter factor) induces the cysts to elongate by localized cell division into tubule- like structures that eventually fuse and form a large syncytium (Montesano et al., 1991a,b). Immunocytochemical studies showed galectin-3 to be excluded from the lumenal/apical surface of the cysts and in the elongating tubules whereas the lateral and basal surfaces of the cyst bodies were heavily stained (Bao and Hughes, 1995). Since high galectin-3 expression was associated with sites of adhesions in the developing MDCK epithelium, we suggested that the lectin may synergism with or activate cell-cell adhesions at lateral surfaces and cell-matrix adhesions at basal surfaces to maintain cyst polarity. In support of this idea, treatment of cyst cultures of MDCK cells with galectin-3-blocking antibodies was found to speed up the growth of the cysts over control cultures and addition of high concentrations of recombinant galectin-3 retarded cyst growth.
In order to test these earlier suggestions we took advantage of a ricin-resistant MDCK mutant (Meiss et al., 1982; Brandli et al., 1998). This cell-line is defective in translocation of UDP-galactose into the Golgi apparatus. As a consequence, the N- and O-linked glycans of nascent glycoproteins cannot be terminated with galactose residues, a defect expressed pleotropically on all cellular glycoconjugates normally containing galactose residues including any receptors for galectin-3. A [beta]-galactoside structure is a necessary, but not sufficient, requirement for high-affinity binding to galectin-3 (Sato and Hughes, 1992; Henrick et al., 1998). We find that the ricin-resistant MDCK cell line produces and secretes wild-type levels of galectin-3 but forms cysts in collagen cultures that grow very quickly and adopt very irregular shapes. These results provide further evidence that a block in binding of galectin-3 to certain carbohydrate receptors at the cell-surface and within the extracellular matrix is correlated with abnormal MDCK cyst formation, consistent with the proposal that galectin-3 appears to be a negative regulator of epithelial cyst growth.
Cyst growth of wild-type and ricin-resistant MDCK cells within collagen gels
In preliminary experiments, wild-type and the ricin-resistant MDCK lines were found to grow at similar rates in routine monolayer culture, ~19-21 h doubling time, in agreement with previous findings (Meiss et al., 1982; Brandli et al., 1988), and to express similar amounts of galectin-3 by Western blotting of equivalent samples of cell lysates (results not shown).
MDCK wild-type (WT) and ricin-resistant (RIC)cells seeded at a similar low density within a three-dimensional hydrated collagen type 1 gel grew clonally and formed spherical cysts in which a single epithelial layer surrounded a clearly defined central lumen (Figure
Figure 1. Growth of MDCK wild-type (WT) and ricin-resistant (RIC) cells in three-dimensional collagen gels. (A) Typical view of cysts formed by RIC cells after 1 week in culture. Note the large size distribution of polarized cysts with a central lumen (Lu). (B) and (C), Cysts appearing after prolonged 4 weeks culture of RIC cells showing the appearance of sprouting (open arrow head) and tubular (solid arrows) structures. (D) and (E), Cysts formed by WT cells after 1 week (D) and 4 weeks (E) in culture. Polarized cysts of various sizes are seen. (F) WT cells after growth in presence of hepatocyte growth factor HGF. Note persistence of some cysts with a central lumen (Lu) and many cysts with sprouting (open arrowhead) and tubular (solid arrows) outgrowths. Scale bars, 100 µm.
Figure 2. Rates of expansion of wild-type (WT) and ricin-resistant (RIC) MDCK cysts in collagen culture. The mean and range of maximum diameters of cysts were measured as described in Materials and methods and are expressed as a function of time in culture. Polarity of wild-type and ricin-resistant MDCK cysts
We considered the possibility that the cysts formed by MDCK RIC cells were less well polarized than MDCK WT cysts. MDCK cysts are fluid-filled (Valentich et al., 1979; McAteer et al., 1987; Mangoo Karim et al., 1989) and intralumenal pressure alone could possibly affect the turgidity and rate of expansion of incompletely polarized cysts. However, we found that the overall polarity of cysts formed by MDCK RIC cells was indistinguishable from that formed by MDCK WT cells.
Laminin, which is produced endogenously by MDCK cells and incorporated into a basement membrane (Caplan et al., 1987), was found to be located exclusively at the basal surface of MDCK RIC (Figure
Figure 3. Polarity of wild-type (WT, panels A-F) and ricin-resistant (RIC, panels a-f) MDCK cysts growing in collagen culture. Cysts were stained in situ with biotinylated plant lectins DBA, PNA before and after treatment with neuraminidase, LEA, and ConA or with anti-galectin-3 and anti-laminin antibodies. Cell nuclei were counterstained with propidium iodide. In (A/a) the lumen (Lu) and the apical surface (white broken line) are indicated. Apical and basal surfaces are also shown by the arrowheads and arrows, respectively. Scale bars, 20 µm.
Figure 4. Polarity of MDCK WT (A-D) and RIC (a-d) cells growing as sealed monolayers on Transwell filters. (A/a) through (C/c) are vertical z-axis views. (D/d) are single horizontal x-y sections at the level of the tight junctions at lateral membrane domains. Apical and basal surfaces are indicated by the thin and thick arrows, respectively. Cells were stained with DBA, or with antibodies against laminin, the apical marker gp135 or the tight-junction ZO 1 protein. See Figure 3 for further explanation. Scale bars, 10 µm. Galectin-3 expression in MDCK cysts
When we reexamined the distribution of galectin-3 in MDCK WT cysts (Figure
Thus, it appears that during MDCK WT cyst growth galectin-3 is secreted into the pericellular space at basolateral domains and is retained there by binding to galactosylated receptors including basement membrane laminin. In RIC cysts the fate of galectin-3 after secretion is unknown but we assume that, since the endogenous binding proteins are absent, lectin secreted basolaterally diffuses away through the surrounding collagen type I matrix. Galectin-3 secretion from MDCK cell monolayers grown on collagen
In contrast to the strong basolateral secretion of galectin-3 from MDCK WT cysts observed here and previously (Bao and Hughes, 1995), galectin-3 is secreted only at low levels and predominantly from the apical surface of MDCK WT cells grown to confluency as monolayers on permeable filters (Lindstedt et al., 1993; Sato et al., 1993). There is increasing evidence that membrane proteins may change their surface location in the same epithelial cell, in some cases due to signals deriving from components in the extracellular matrix (Al-Awqati, 1996; Wilson, 1997).We considered the possibility that the differences between the polarity of secretion of galectin-3 from MDCK cysts compared to monolayers might simply be due to a general change in polarity related to the different environment at the basal surface of the polarized cells. In the MDCK cysts the basal surface contacts an hydrated collagen type 1 matrix, whereas cells in monolayer culture are in contact with an inert polycarbonate substratum that is conditioned only by the basal secretion of an endogenous pericellular matrix, rich in epithelial matrix components such as collagen type 1V and laminin, but lacking collagen type 1.
However, immunocytochemical studies showed that monolayer MDCK WT cells retained the same overall polarity as when these cells were grown in cyst culture, as indicated by the distinct distributions of the basal components laminin (Figure
Figure 5. Galectin-3 secretion from MDCK WT and RIC cells in monolayer cultures. WT (A) and RIC (B) cells grown on Transwell filters coated with collagen types I/III were pulsed with 35S-methionine for 4 h and chased for up to 20 h as indicated by addition of fresh medium to the upper and lower compartments of the Transwell unit. Some cultures were pretreated with tunicamycin (5 µg/ml) before labeling. Galectin-3 was identified in the apical (a) and basal (b) medium and in cell lysates (c) by immunoprecipitation, SDS-PAGE and radioautography. The migration of galectin-3 and of protein standards is indicated. (C) Confluent monolayers of WT cells grown on uncoated Transwell filters or filters coated as indicated were labeled with 35S-methionine for 24 h. Apical and basal medium fractions were analyzed as in (A) and (B). Filter coating: None, naked filter; Lam, laminin; Mat, Matrigel; Col, collagen type I.
In pulse-chase experiments of MDCK WT cells grown on collagen types 1/III-coated permeable filters, a small part (about 10-15%) of total galectin-3 was secreted and this was predominantly from the apical domain (Figure
The results reported here support and extend our previous suggestion (Bao and Hughes, 1995) for a role of galectin-3 and cognate galactose-containing receptors in epithelial growth and polarity. It seems that ligation by galectin-3 of surface carbohydrate receptors is important for normal MDCK cyst formation and growth, and for orderly elongation and branching morphogenesis induced by HGF acting through it's receptor, the c-met proto-oncogene product. The very similar polarity of the wild-type and ricin-resistant cysts, as determined by marker analysis (Figure
How then can the glycosylation defect, and the failure of mutant cysts to retain galectin-3 in the pericellular space, be correlated with aberrant growth of the mutant cysts? Clearly, galectin-3 retention at basolateral membranes is not an obligatory factor in the maintenance of cyst polarity. However, a transient role in some early events in establishment of polarity cannot be excluded. It is known that clonal growth of MDCK cysts in collagen gels occurs by localized cell divisions within the epithelium and formation of new junctional complexes at the contiguous surfaces of daughter cells (McAteer et al., 1987). Formation of occluding tight junctions recloses the epithelium, segregating again the apical and basolateral membranes of the newly divided cells, and their polarity is stabilized by formation of desmosomal and adherens junctions at the apposing lateral domains and re-establishment of cell-matrix interactions at the basal surfaces. Many studies show that cell-cell and cell-substratum contacts are crucial in the establishment of the epithelial axis and the development of the central lumen (Wang et al., 1990a,b; Wollner et al., 1992), and these processes must be highly regulated to maintain continuity and shape of the expanding cysts. Polarity is well-preserved even in branching MDCK cysts (Montesano et al., 1991a,b), including the MDCK RIC cysts (Figure
The present results, in agreement with earlier data (Bao and Hughes, 1995), suggest strongly that a major part of cytosolic galectin-3 is exported from MDCK cells when these are grown as cysts within collagen type 1 gels. Unfortunately, the cyst culture conditions do not lend themselves easily to biochemical analysis. Although any galectin-3 exported from the basolateral surface of cysts can be readily recovered in the bathing medium, since it diffuses through the hydrated collagen, the cysts are less easily accessible and all attempts to access the lumenal contents in sufficient amounts have been unsuccessful. Therefore, we cannot accurately quantitate the proportion of total lectin that becomes externalized. Our impression, from confocal immunofluorescence studies (Figure
The increased export of galectin-3 in cyst culture must be taken into account when considering the apparent reversal of polarity in lectin secretion from cysts compared to monolayer cultures. It may be that the low level of galectin-3 secretion in the latter conditions, most of which is apically directed, represents a nonregulated pathway that still exists in the cysts, but at too low a level to detect by the methods used here. In this model the basolateral pathway is activated in cyst culture and dominates the extracellular patterning of galectin-3 expression, as we have found. Perhaps secretion is upregulated in order to satisfy a specific functional requirement at basolateral sites as described above. In contrast, in order to establish a polarized monolayer culture, the cells need to be grown into a confluent and quiescent state over several days, epithelial polarization is already accomplished and static and the basolateral secretory pathway is not required.
Unfortunately, nothing is known as yet about the intracellular sorting mechanisms for cytoplasmic proteins such as galectin-3. Since the available evidence suggests that galectins may not enter classical secretory pathways (Cooper and Barondes, 1990; Lindstedt et al., 1993; Sato and Hughes, 1993), sorting signals identified for membrane and secretory proteins are unlikely to be used directly (Schelffele et al., 1995; Keller and Simons, 1997). However, some basolateral sorting information resides in the cytoplasmic tails of membrane proteins (Keller and Simons, 1997), and it cannot be excluded that galectins might utilize such information through specific protein-protein interactions with the cytoplasmically exposed signals. Perhaps the apparent block in limited basal secretion of galectin-3 in tunacamycin-treated cells (Figure
How does this work relate to the potential role(s) of galectin-3 in formation of epithelia in the embryonic kidney? Galectin-3 expression is regulated developmentally in the kidney (Foddy et al., 1990) and more recent studies (Winyard et al., 1997) of human kidney are consistent with the idea that galectin-3 may be implicated in both the regulation of normal growth and differentiation of the ureteric epithelia, as well as in the pathogenesis of cystic epithelia. It is particularly notable that, as in the in vitro models, galectin-3 sorting to particular membrane domains of the polarized cells is very variable. Although galectin-3 is found predominantly at lumenal/apical domains of the ureteric bud epithelium as it actively branches into the nephrogenic cortex, the lectin relocates to a more basolateral pattern as the medullary collecting duct lineage matures. In human multicystic dysplastic kidney disease the malformed dysplastic tubules and cysts retain an immature apical expression of galectin-3 (Winyard et al., 1997). Similar variation in the polarity of membrane proteins during epithelial development and in association with disease states has been reported (Al-Awqati, 1996; Wilson, 1997) and more understanding of the mechanisms underlying such plasticity is an important goal for the future.
Materials
Recombinant hamster galectin-3 and specific rabbit polyclonal or rat monoclonal antibodies directed against whole lectin or the N- or C-terminal domains were obtained as described previously (Mehul et al., 1994, 1995). Laminin from EHS tumor and polyclonal anti-laminin antibodies were obtained as described previously (Bao and Hughes, 1995). Mouse monoclonal antibodies against the apical membrane glycoprotein gp135 (Ojakian and Schwimmer, 1988) and tight junction ZO1 protein were obtained from Ian Burdett, NIMR, UK. Matrigel and human Hepatocyte Growth Factor (HGF) were from Universal Biologicals, London, UK. Collagen Type I, biotinylated derivatives of Dolichos biflorus agglutinin (DBA), Arachis hypogaea agglutinin (PNA), Concanavalin A (ConA), Helix pomatia agglutinin (HPA), Lycopersicon esculentum agglutinin (LEA), Ricinus communis agglutinin I, and FITC- and Texas red conjugates of goat anti-rabbit, mouse, or rat IgGs and streptavidin were from Sigma Chemical, Poole, UK.
Cell culture
MDCK cells type II (Richardson et al., 1981) were obtained from Porton Down, Salisbury, UK. Ricin-resistant MDCK type 11 cells (Meiss et al., 1982; Brandli et al., 1988) were kindly provided by Kai Simons, EMBL, Germany. Cells were cultured at 37°C routinely in a 1:1 mixture of Dulbecco's Modified Eagles Medium (DMEM) and Nutrient Mixture Ham's F12 (Gibco BRL, Paisley, UK) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). For cyst culture cells trypsinized from monolayer culture were resuspended in ice-cold 2.4 mg/ml collagen type I solution at 1 × 105 cells/ml and dispensed into multi-well plates (1 ml/well). After gelation at 37°C for 45 min the cells were grown in DMEM containing 10% FBS for up to 2 months exactly as described previously (Bao and Hughes, 1995). Some cultures were grown throughout in DMEM, 10% FBS containing human HGF at 20 ng/ml. Growth rates of the cysts were measured by estimations of the maximum diameter of randomly selected spherical cysts as described before (Bao and Hughes, 1995). Data from 10 fields in duplicate wells were averaged and standard deviations obtained. Non-spherical cysts exhibiting at least one cytoplasmic sprouting or tubular process were also counted in each field. For growth of polarized monolayer cultures, cells were seeded at 2-4 × 10 5/ml DMEM, 10% FBS into 12 mm diameter 0.4-µm-pore size polycarbonate Transwell or Transwell-Col (collagen types I/III-coated) filter units (Costar, High Wycombe, UK) and cultured at 37°C in DMEM, 10% FBS (0.5 ml and 1.5 ml in the upper and lower chambers, respectively). In some cases naked Transwell filters were coated by incubation with laminin or Matrigel solutions (10 µg/ml) for 1 h. The coated filters were washed briefly with complete medium before addition of the cells. Hydrated collagen-coated filters were produced by addition of a collagen type I solution (2.5 mg/ml, 0.5 ml/filter unit) and cells were added to the top of the fully gelled collagen layer. Electrical resistance across polarized monolayers was measured routinely (Brandli et al., 1988), and only monolayers showing maximal resistivity similar to the reported values (Richardson et al., 1981; Brandli et al., 1988), usually attained after 4-5 days of culture, were used.
Immunocytochemistry
The methods are detailed in Bao and Hughes (1995) and Sato et al. (1993). Briefly, MDCK cell cultures in collagen blocks or on Transwell filters were excised from culture wells using a scalpel, washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde-PBS for 15 min. The fixed gel blocks were diced and the gel fragments or the filters were used for immunocytochemistry after treatment with 0.5% Triton X-100 in PBS for 15 min. Incubations with primary antibodies, diluted appropriately in 1% BSA in PBS, were for 2 h at room temperature followed by extensive washing in PBS and staining with an appropriate dilution of a chromophore-conjugated secondary antibody as before. For staining with lectins, fixed cells were incubated for 1-2 h at room temperature with each biotinylated lectin at 50 µg/ml in PBS containing 0.2% gelatin, followed by washing in PBS and incubation with chromophore-conjugated streptavidin reagent at 50 µg/ml in PBS-gelatin as before. Cells were usually counterstained with 5 mg/ml propidium iodide in PBS at room temperature for 10 min. Confocal microscopy was then performed using either a Bio-Rad MRC 600 or a Leica TCS NT laser scanning confocal microscope LSCM system and associated data processing software as described previously (Bao and Hughes, 1995). Stained samples were examined in vertical z-axis by serial optical sectioning at 1-2 µm intervals. The images shown here were all derived with similar PMT attenuation.
Cell labeling and immunoprecipitation
For pulse-chase experiments on cells growing on permeable filters of Transwell units, the cells were starved at 37°C for 30min. in methionine-free DMEM, 10% FBS before labeling with 35S-methionine (O.5 mCi/filter) in this same medium usually for 4 h. The cells were then washed 2-3 times on the filters with complete DMEM, 10% FBS containing 1000-fold excess methionine and chased for various times. For immunoprecipitation, the medium in the upper (apical) and lower (basal) compartments were harvested and the cells on the filter were solubilized on ice in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.4% SDS, 0.2% Triton X-100 in presence of a cocktail of protease inhibitors (CompleteTM, Boehringer, Mannheim, Germany). Medium fractions and cell lysates were incubated overnight at 4°C with a particular antibody and immune complexes were collected on Protein A-Sepharose (Pharmacia BioTech). The beads were washed several times with 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1% SDS, 0.5% Triton X-100, and protease inhibitors and twice with 50 mM Tris-HCl pH 7.5 before boiling in sample buffer for SDS-PAGE. Separation by SDS-PAGE and fluorography were as described previously (Mehul et al., 1994). Amounts of labeled protein were determined using a scanning densitometer.
1To whom correspondence should be addressed