(Received for publication, May 23, 1995)
From the
The human cell line HT-29 provides a model system for studying regulation of proliferation and differentiation in intestinal epithelial cell lineages: (i) HT-29 cells cultured in glucose resemble undifferentiated multipotent transit cells located in the lower half of intestinal crypts; (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt; (iii) nonproliferating, confluent HT-29-inosine cells have features of differentiated enterocytes and goblet cells that overlie small intestinal villi. A cDNA library prepared from HT-29-inosine cells was screened with a series of subtracted cDNA probes to identify proteins that regulate proliferation/differentiation along the crypt-villus axis. A cDNA was recovered that encodes a 202-amino acid protein with four predicted membrane spanning domains and two potential sites for N-linked glycosylation. Levels of this new member of the superfamily of tetraspan membrane proteins (TMPs) increase dramatically as nondividing epithelial cells exit the proliferative compartment of the crypt-villus unit and migrate onto the villus. The protein is also produced in nondividing hepatocytes that have the greatest proliferative potential within liver acini. Three sets of observations indicate that in the appropriate cellular context, intestinal and liver (il)-TMP can mediate density-associated inhibition of proliferation. (i) Accumulation of il-TMP glycoforms precedes terminal differentiation of HT-29-inosine cells and occurs as they undergo density-dependent cessation of growth. il-TMP levels are lower and glycosylation less extensive in HT-29-glucose cells, which do not undergo growth arrest at confluence. (ii) HeLa cells normally do not produce il-TMP. Forced expression of il-TMP inhibits proliferation as cells approach confluence. The extent of il-TMP glycosylation in the transfected cells is similar to that observed in HT-29-inosine cells and greater than in HT-29-glucose cells. (iii) SW480 cells are derived from a human colon adenocarcinoma and do not express il-TMP. Like nontransfected HeLa cells, they do not stop dividing at confluence, whether grown in medium containing glucose or inosine. Expression of il-TMP has no effect on the growth properties of SW480 cells. The extent of il-TMP glycosylation in SW480-glucose cells is similar to that noted in HT-29-glucose cells, lending further support to the notion that il-TMP's activity is related to its state of N-glycosylation.
The lifespan of an epithelial cell encompasses a series of decisions. The cell must decide when to proliferate, to commit to a specific cell lineage, to terminally differentiate, and/or to undergo a programmed death. In the small intestine, proliferation, differentiation, and death programs are expressed along a geographically well defined pathway that extends from the crypt of Lieberkühn to the villus' apical extrusion zone(1) . The mouse and human intestinal epithelium are continuously and rapidly renewed (2, 3) . Thus, the crypt-villus axis represents a model of perpetual development in mammals and provides an opportunity to examine the molecular mechanisms that regulate decision making in epithelial cells.
Most of what is known about intestinal epithelial renewal comes from studies of the mouse. The mouse small intestinal epithelium contains four principal terminally differentiated cell types. Absorptive enterocytes comprise >80% of its cells(4) . Members of the mucus-producing goblet and enteroendocrine cell lineages exhibit remarkable variations in their differentiation programs as a function of their location along the crypt-villus and duodenal-ileal axes (see, e.g., (5, 6, 7) ). Paneth cells secrete a variety of anti-microbial peptides, digestive enzymes, and growth factors(8) . These epithelial lineages arise from a multipotent stem cell located near the base of each crypt. Descendants of active multipotent stem cell(s) undergo several rounds of cell division in the mid-portion of each crypt(9) . Enterocytes, goblet, and enteroendocrine cells undergo terminal differentiation as they cease proliferating and migrate in vertical coherent bands from the crypt to the apex of a surrounding villus(10) . Cells are either phagocytosed or exfoliated into the intestinal lumen at the villus tip. Differentiation and removal is completed in 2-5 days depending upon the lineage and the location of crypt-villus units along the duodenal-to-ileal axis(2, 4, 11, 12, 13, 14) . Members of the Paneth cell lineage complete their differentiation program as they migrate downward to the base of each small intestine crypt where they reside for several weeks(15) .
Given the
spatial complexities of this epithelium, most analyses of the
regulation of its proliferation and differentiation programs have used in vivo models (see, e.g., Refs. 1, 16, and 17).
However, in vitro models would be useful for initially
identifying gene products that may regulate these processes. HT-29
cells are derived from a human colon adenocarcinoma (18) and
represent one such model. If cultured in standard medium containing 25
mM glucose, they proliferate even after reaching confluence
and do not produce proteins synthesized by terminally differentiated
intestinal epithelial cells in vivo(19) . If HT-29
cells are cultured in the absence of glucose, using galactose (19) or inosine (20, 21) as the carbon source,
they cease to proliferate once they reach confluence. If maintained in
this confluent state for 10-14 days, most of the quiescent cells
differentiate into enterocytes; they become polarized, form tight
junctions, and elaborate an apical brush border membrane containing a
variety of hydrolases(19, 21) . If glucose is added
back to confluent cells that have been cultured for many generations in
its absence, they will still terminally differentiate. This suggests
that at some point during exponential growth in the absence of glucose,
HT-29 cells make the decision to differentiate. Huet et al.(22) noted that 10% of differentiated HT-29 cells
synthesize and secrete intestinal mucins, indicative of a goblet
cell-like phenotype. Importantly, they found that a single clone of HT-29-glucose cells can give rise to progeny with enterocyte-
and goblet-like phenotypes when switched to media containing carbon
sources other than glucose. These results indicate that (i) HT-29 cells
cultured in glucose have properties of undifferentiated
multipotent transit cells located in the lower half of intestinal
crypts, (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt,
and (iii) confluent HT-29-inosine cells have features
of terminally differentiating villus-associated enterocytes and goblet
cells. Thus, HT-29 cells cultured in media with different carbon
sources and/or at different growth phases make decisions that may
resemble decisions that occur along the (human) crypt-villus axis.
In this report we describe how subtracted cDNA probes were used to
screen an HT-29-inosine cDNA library to identify an intestinal and
liver tetraspan membrane protein (il-TMP). ()il-TMP can
mediate density-dependent cell proliferation. Moreover, this function
can be directly correlated with the extent of its N-glycosylation.
To determine if the steady state concentration of il-TMP mRNA varies along the duodenal-colonic axis, segments of intestine were recovered at defined points along this axis from three organ donors (age = 32, 36, and 72 years). The entire bowel of each donor was sampled: i.e. duodenum, jejunum, ileum, and proximal, middle, and/or distal colon (n = 2 separate full thickness samples/segment/donor).
Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Corp.), and the membranes were probed with (i) affinity-purified, il-TMP peptide-specific antibodies (final concentration = 170 ng/ml of PBS containing gelatin (1%, w/v), Tween 20 (0.2%, v/v), and sodium azide (0.1%, w/v)), (ii) a mouse mAb directed against actin (C4; (28) ), or (iii) a mouse mAb raised against human sucrase-isomaltase (mAb HSI 9, a gift from Andrea Quaroni, Cornell University; cf. (29) ). il-TMP-antibody complexes were visualized with alkaline phosphatase-conjugated secondary antibodies using the Western Light(TM) kit (Tropix). Blots were stripped for reprobing with additional antibodies(30) .
Focus formation was assessed in parallel cultures of pControl/neo- and pil-TMP/neo-transfected HeLa cells. After 12-14 days of culture, cells were washed twice with ice-cold PBS and stained for 15 min with a solution of 25% isopropanol, 10% acetic acid, 0.025% Coomassie Blue. Flasks were rinsed with 25% isopropanol, 10% acetic acid, allowed to air-dry, and viewed under a dissecting microscope. For each independently transfected pool of cells, focus formation was surveyed in two to three separate experiments.
Surveys of different regions of the entire intestine from each of three adult human organ donors revealed a distinct duodenal-colonic gradient in the concentration of the two transcripts (Fig. 1). Highest levels were noted in the jejunum (7 pg/µg of total cellular RNA). Concentrations in the duodenum, ileum, and colon were 2-4-, 5-10-, and 10-40-fold lower, respectively, than in jejunum. The steady state level of the transcripts in jejunum was 10-20-fold higher than in liver.
Figure 1:
il-TMP mRNA levels vary along the
cephalocaudal axis of adult human intestine. RNA blots containing 5
µg of total cellular RNA/lane were probed with a full-length P-labeled il-TMP cDNA. All samples were derived from
different intestinal segments from a single adult human organ donor.
Note that the highest steady state levels of il-TMP mRNA are
encountered in the jejunum. Similar results are observed with RNA
samples prepared from two other organ
donors.
Southern blots of human genomic DNA, digested to completion with EcoRI, BamHI, or HindIII, indicated that the cDNA was derived from a single copy gene (data not shown).
Fig. 2A presents the nucleotide sequence of the 1385-base pair cDNA. A polyadenylation signal (ATTAAA) is located 27 nucleotides upstream of its poly(A) tail. A second polyadenylation signal (AATAAA) spans nucleotides 953-958 and is likely to account for the shorter mRNA species observed in human intestine, liver, and cultured HT-29 cells (see below). The cDNA encodes a primary translation product of 202 amino acids with a calculated mass of 21,396 Da. There is no site near the N terminus likely to undergo cotranslational cleavage by signal peptidase(36) . Cysteine and glycine residues are clustered in three regions. The hexapeptide Cys-Cys-Gly-Cys-Cys-Gly appears in two of these regions (residues 74-79 and 192-197 in Fig. 2A).
Figure 2: Nucleotide and deduced amino acid sequences of il-TMP. Panel A, nucleotide and amino acid numbers are indicated to the left and right, respectively, of the sequences. Two potential polyadenylation signals are boxed. Two potential sites for N-linked glycosylation are circled. Cys-Cys-Gly motifs are underlinedtwice. The peptide sequences used for generation of polyclonal antibodies are underlinedonce. PanelB, Kyte-Doolittle hydropathy profile of il-TMP. Negative values indicate hydrophilic regions, while positive values indicate hydrophobic areas. Note the presence of four hydrophobic domains (graylines) and the presence of two predicted extracellular domains (boldblacklines). The peptide sequences used to raise polyclonal antibodies against il-TMP are indicated by the filledboxes within the blacklines labeled A and B. The upwardpointingarrows show the location of potential sites for N-linked glycosylation of il-TMP.
A hydropathy plot (37) predicts
that the protein contains four hydrophobic domains, each sufficiently
long to span cellular membranes (Fig. 2B). The
hydrophilic region between the third and fourth hydrophobic domains has
two potential sites for N-linked glycosylation (Asn and Asn
; Fig. 2, A and B).
A search of nonredundant protein data bases using the BLAST network (38) revealed that the protein is 50% identical to L6, an antigen expressed on the surface of colon, lung, breast, and ovarian carcinoma cells(39, 40) . The protein also shows significant homology to members of a superfamily of tetraspan membrane proteins (TMPs; (41) ). Most TMPs are rich in cysteines and glycines, contain at least two copies of a Cys-Cys-Gly motif, and have four transmembrane domains. TMPs also contain a sequence of variable length and hydrophilicity positioned between the third and fourth hydrophobic domains that typically has sites for N-linked glycosylation. Based on these sequence comparisons, we named the 202 residue polypeptide intestinal and liver tetraspan membrane protein or il-TMP.
Frozen sections of adult human jejunum were incubated with each of the four antibody preparations. There is a dramatic increase in il-TMP levels at the crypt-villus junction (Fig. 3, A and B). High levels are maintained as nonproliferating, differentiated epithelial cells complete their migration to the villus tip (Fig. 3A). Less intense staining is present in the crypt (Fig. 3, A and B). Staining is limited in all cases to the apical borders of epithelial cells. No staining was detected with preimmune sera (data not shown) or if the antibodies were preincubated with the appropriate peptide (e.g.Fig. 3C). None of the antibodies reacted with any mesenchymal cell populations (Fig. 3, A and B). These findings suggest that if il-TMP affects proliferative status in crypt-villus units, it does so as a negative regulator.
Figure 3: Cellular distribution of il-TMP in adult human intestine and liver. PanelA, frozen section of adult human jejunum incubated with antisera raised against residues 134-146 of il-TMP (final dilution = 1:2000). Antigen-antibody complexes were visualized with Cy3-conjugated donkey anti-rabbit Ig. The arrow points to a crypt/villus junction. PanelB, higher power view of a crypt-villus junction located in the jejunum. The section was processed as in panelA. Note that the concentration of il-TMP in the apical membranes of crypt epithelial cells is lower than in the apical membranes of villus-associated enterocytes. PanelC, frozen section prepared from the same sample of human jejunum as used for panelsA and B. The primary antisera was preincubated with its peptide antigen (GDYLNDEALWNKC) (blocking control). PanelD, frozen section of adult human liver stained with affinity-purified antibodies raised against residues 134-146 of il-TMP followed by Cy3-conjugated donkey anti-rabbit Ig. Nuclei (blue) were revealed by counterstaining with bisbenzimine. The closedarrow points to immunoreactive il-TMP associated with the canalicular membranes of hepatocytes. The openarrow points to a cluster of il-TMP-positive cells whose identity has not been established. Shorter photographic exposure times revealed that il-TMP is concentrated in the plasma membrane of these cells. PanelE, frozen section of liver incubated with an affinity-purified preparation of rabbit antibodies to residues 134-146 of il-TMP and a mouse monoclonal antibody raised against human DPP-IV. Antigen-antibody complexes were visualized with Cy3-conjugated donkey anti-rabbit Ig and FITC-conjugated donkey anti-mouse Ig. Arrows point to the il-TMP-positive (orange) canalicular membrane of hepatocytes located in zone 1 of the acinus (zone 1 = periportal area). The arrowhead points to representative DPP-IV-positive hepatocytes located in zones 2 and 3 of the acinus. Immunoreactive DPP-IV (green) is associated with the cannicular membrane. PanelF, higher power view of the section shown in panelE. Note that DPP-IV (green) but not il-TMP (orange) is present in the apical border of bile duct epithelial cells (closedarrow; cf. Ref 42). il-TMP but not DPP-IV is expressed in the unidentified population of cells (e.g.openarrow). Bars = 25 µm.
Frozen sections were also prepared from intestinal segments harvested at various positions along the duodenal-ileal axis of a single organ donor. The cellular distribution and levels of il-TMP are similar in duodenal and jejunal crypt-villus units. The apical border of epithelial cells are also stained in ileal villi, but the intensity of staining is lower (data not shown). These cephalocaudal differences are consistent with the observed regional variations in steady state il-TMP mRNA concentrations (Fig. 1).
The structural and functional unit of the liver is the acinus. Each acinus can be divided into three zones. Zone 1 is defined as the periportal region. Zone 3 surrounds the central vein. Zone 2 is positioned between zones 1 and 3. Tritiated thymidine labeling studies suggest that stem cells located in zone 1 give rise to populations of hepatocytes that terminally differentiate as they slowly migrate toward the central vein(43) . Incubation of frozen sections of adult human liver with each of the four il-TMP antibody preparations revealed a ``chicken wire'' pattern of staining in zone 1 (Fig. 3, D-F). Blocking controls and studies with preimmune sera established the specificity of this reaction (data not shown). The chicken wire pattern of il-TMP staining is similar to that observed with DPP-IV, a protein known to be targeted to the canalicular membrane of ``mature'' hepatocytes positioned in zones 2 and 3 (Fig. 3E; cf.(42) and (44) ). There is an inverse relationship in the expression of these two proteins along the zone 1-3 axis: multilabel confocal microscopic studies revealed that il-TMP and DPP-IV are only coexpressed in a narrow band of hepatocytes positioned between zones 1 and 2 and that il-TMP is not present in zones 2 and 3 (data not shown). The fact that il-TMP is only expressed in the nondividing population of hepatocytes with highest proliferative potential is consistent with the notion that this protein could play a role in suppressing proliferation in the acinus as well as in crypt-villus units.
Figure 4:
il-TMP expression precedes differentiation
in HT-29-inosine cells. PanelA, HT-29-inosine or
HT-29-glucose cells were plated in 25-cm T-flasks
containing glucose-free DMEM supplemented with 2.5 mM inosine
or DMEM containing 25 mM glucose, respectively. At the
indicated times after plating, cells were recovered, lysed, and
aliquots of the lysate assayed for protein content. The arrows point to the time when cells first become 100% confluent. The shadedboxes encompass the period of increasing
steady state levels of il-TMP glycoforms. PanelsB and C, aliquots containing 5 µg of cellular protein
were also analyzed by Western blotting. Triplicate protein blots were
probed with affinity-purified rabbit antibodies to residues
134-146 of il-TMP, a mouse mAb raised against human
sucrase-isomaltase, or a mouse mAb raised against actin. Bound
antibodies were visualized using alkaline phosphatase-conjugated goat
anti-rabbit or goat anti-mouse Ig. The arrows point to the day
when cells first become 100% confluent. Note that although the
steady-state levels of il-TMP glycoforms increase in postconfluent
cells, longer exposures of the il-TMP blots indicate that the
electrophoretic mobilities of the glycoforms do not change during the
course of the experiment.
Figure 5: Glycosylation of il-TMP is different in HT-29-inosine and HT-glucose 29 cells. Lysates were prepared from confluent HT-29-inosine (Ino) or HT-29-glucose (Glc) cells and then incubated with (+) or without(-) peptide N-glycosidase F or endo H. Aliquots of the glycosidase reaction containing 0.6 µg of cellular protein were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The protein blots were probed with affinity-purified antibodies raised against residues 134-146 of il-TMP. Antigen-antibody complexes were detected with alkaline phosphatase-conjugated secondary antibodies.
Since most of the il-TMP in the small intestine is present in non-dividing villus-associated enterocytes, Western blot analysis of lysates prepared from full thickness samples of human small intestine should reflect the state of il-TMP glycosylation in these cells. Protein blots revealed that il-TMP levels along the cephalocaudal axis mimic the variations noted during our immunocytochemical and RNA analyses. The size distribution of il-TMP glycoforms in human duodenal, jejunal, and ileal extracts indicates that the protein is more extensively glycosylated than in HT-29-glucose cells (Fig. 6).
Figure 6: Glycosylation of il-TMP in villus-associated epithelial cells is similar to that in HT-29-inosine cells. Lysates were prepared from HT-29 cells cultured in glucose (Glc) or inosine (Ino) and from full thickness segments of adult human small intestine. Western blots of cellular proteins (4 µg/lane) were probed with affinity-purified antibodies to residues 134-146 of il-TMP.
Together these findings suggest that (i) il-TMP oligosaccharides do not accumulate as high mannose forms in either HT-29-glucose or -inosine cells, (ii) glycosylation in the proliferating glucose-treated cells is probably impaired at a step distal to the activity of mannosidase II, and (iii) glycosylation may influence il-TMP's ability to affect proliferation as cells approach confluence.
Figure 7: Expression of il-TMP in cultured cell lines. PanelA, proliferating HT-29-inosine cells were harvested during log phase and stained with affinity-purified polyclonal antibodies to residues 134-146 of il-TMP plus FITC-conjugated donkey anti-rabbit Ig. Note the intense staining of the cell periphery as well as the punctate pattern of staining of the plasma membrane of these polarized cells. PanelB, postconfluent, differentiated HT-29 inosine cells fixed and stained with the same il-TMP antibody used in panelA plus Cy3-conjugated donkey anti-rabbit Ig. Note the evenly distributed, punctate staining of the entire cell surface. PanelC, postconfluent HT-29-glucose cells fixed and stained as in panelB. Note thesimilar patterns of staining in these nonpolarized cells and in the proliferating HT-29 inosine cells shown in panelA. PanelD, HeLa cells were transfected with pil-TMP/neo. Three days later, cells were fixed in methanol and stained for il-TMP using affinity-purified antibodies to residues 134-146 of il-TMP and Cy3-conjugated donkey anti-rabbit Ig (red). Nuclei were counterstained with bisbenzimine (blue). Note the high levels of il-TMP expression in a subset of cells. Bars = 25 µm. PanelsE and F, Coomassie-stained flasks containing a pool of HeLa cells stably transfected with pControl/neo (panelE) or pil-TMP/neo (panelF). Cells were stained 14 days after plating in glucose-containing medium and viewed under the dissecting microscope. The arrow in each panel points to a focus of cells. Comparison of panelsE and F reveals that focus number and size is greater in cells containing the il-TMP expression vector. Note that the magnification factor is 20-fold lower in panelsE and F compared to panelD.
We first isolated pools of transiently transfected HeLa cells to determine whether the vector could direct production il-TMP. Cells were fixed in methanol 72 h after transfection and stained for il-TMP with the affinity-purified antibody preparations. As expected, cells transfected with pControl/neo did not express the protein. Cultures transfected with pil-TMP/neo contained a subpopulation of cells with readily detectable amounts of il-TMP (Fig. 7D).
HeLa cells were then stably transfected with pil-TMP/neo or pControl/neo. We were concerned that if il-TMP plays a role in promoting cell contact-associated inhibition of growth, the selection for neomycin resistance would favor amplification of cells that either produced less il-TMP or were less sensitive to its putative growth inhibitory effects. Therefore, HeLa cells were maintained in a state where cell-cell contacts were minimized throughout the course of G418 selection, i.e. they were trypsinized and replated every 2-3 days until stably transfected pools were obtained. The data presented in Fig. 8(A and B) confirm transcription of integrated plasmid DNA in two independently derived pools of pControl/neo and pil-TMP/neo-HeLa cells. Three mRNA species are present (Fig. 8A). The 3.0-kilobase transcript encodes both il-TMP and neomycin phosphotransferase. The 1.0- and 1.4-kilobase transcripts presumably arise from use of either one of the two polyadenylation signals present in the 3`-untranslated region of il-TMP mRNA (Fig. 8C). The relative concentrations of the three species are similar in both cellular pools.
Figure 8:
il-TMP mRNA levels in stably transfected
HeLa cells. PanelsA and B, Northern blots
containing RNAs prepared from two independently derived stably
transfected pools of HeLa cells (10 µg of total cellular RNA/lane).
Duplicate blots were probed with a full-length P-labeled
il-TMP cDNA (panelA) or the neomycin
phosphotransferase gene (Neo; panelB). PanelC, explanation of the origins of the multiple
transcripts observed in pil-TMP/neo-HeLa cells shown in panelA. The structure of the expression vector is shown. The upwardpointingarrows indicate the
positions of the two polyadenylation signals present in
il-TMP. When either is utilized, the IRES/Neo portion of the
RNA transcript is eliminated.
The growth rates of stably transfected pControl/neo- and pil-TMP/neo-HeLa cells were essentially identical at low density in medium containing glucose and G418 (doubling time = 26 h; Fig. 9A). pControl/neo cells achieve confluence 8 days after plating. They then continue to divide and form numerous multilayered foci (e.g.Fig. 7F). pil-TMP/neo cells do not reach confluence, even 12 days after plating. On day 12, virtually all pil-TMP/neo HeLa cells were distributed in clusters containing a single layer of cells rather than in multilayered foci (e.g.Fig. 7E) (n = 3 independent transfection experiments). These differences in cell number are reflected by differences in total cellular protein/T-flask/time point surveyed (Fig. 9A). Parallel measurements of total cellular protein/flask, DNA content/flask, or cell number/flask provide similar results when assessing HeLa cell growth(24) .
Figure 9:
Glycoforms of il-TMP produced in
transfected HeLa and SW480 cells. PanelA, stably
transfected pools of HeLa cells containing pil-TMP/neo or pControl/neo
were plated in 25 cm T-flasks containing DMEM (25 mM glucose). All attached cells were harvested at the indicated times
from each T-flask, and the amount of total cellular protein/flask was
determined. Two flasks were assayed/time point. PanelB, pools of HeLa or SW480 cells stably transfected with
pil-TMP/neo or pControl/neo were grown in DMEM (25 mM glucose). The transfected cells were harvested during late log
phase (
80% confluent). HT-29-inosine (Ino) or
HT-29-glucose (Glc) cells were harvested during late log phase
(not shown) or 10 days after reaching confluence and used as controls.
Cells were lysed and il-TMP was immunoprecipitated from 60 µg
HT-29-inosine and HT-29-glucose cell protein, 800 µg of HeLa cell
protein, and 1200 µg of SW480 cell protein. Immunopurified proteins
were fractionated by SDS/PAGE, transferred to PVDF membranes, and the
protein blots probed with affinity-purified antibodies raised against
residues 134-146 of il-TMP. Bound antibodies were detected using
alkaline phosphatase-conjugated goat anti-rabbit Ig. The il-TMP
glycoforms present in pil-TMP/neo-HeLa cells have a migration profile
similar to those present in (i) nonproliferating postconfluent
HT-29-inosine cells (lane1) and (ii) proliferating
preconfluent HT-29-inosine cells (data not shown). The glycoforms
present in pil-TMP/neo-SW480 cells have a migration profile similar to
those produced in pre- and postconfluent HT-29-glucose cells (data not
shown and lane4,
respectively).
There is a 3-fold reduction in cell number produced by il-TMP 12 days after plating (Fig. 9A). This is likely to be an underestimate of the magnitude of the protein's effect on proliferation as cells reach confluence. If il-TMP functions to inhibit growth, any population of cells in the pool that produces low levels of il-TMP would have a proliferative advantage over a population that expresses relatively higher levels. The result would be a progressive increase in the fractional representation of the latter population and a ``masking'' of il-TMP's growth inhibitory properties. In addition, foci of pControl/neo HeLa cells detach from the T-flask and remain suspended in the medium. This fraction is not scored in our assay of cell density, which only considers cells that remain attached after refeeding.
Our results with transfected
HeLa cells establish that il-TMP can participate in cell
density-related inhibition of proliferation. A number of TMPs have been
implicated in the regulation of proliferation. TAPA-1 is a cell surface
protein expressed in many human cell lines (52) . Incubation of
several lymphoma-derived lines with TAPA-1 mAbs inhibits their
proliferation(52) . Antibodies to CD37, a glycoprotein produced
by human B-lymphocytes, can have a positive or negative effect on
proliferation, depending upon how the B-cells are
stimulated(53) . mAbs to CD82 can inhibit the mitogenic
activation of peripheral B-lymphocytes (54) or act
synergistically with mAbs to CD3 to stimulate T-cell
proliferation(55) . OX-44 (the rat ortholog of the human
leukocyte TMP known as CD53) identifies the subset of thymocytes that
proliferate in response to alloantigens and lectins(56) . mAbs
to OX-44 induce proliferation of T-lymphocytes(57) . TI-1 is a
transforming growth factor--inducible TMP produced in mink lung
epithelial cells whose expression changes with their state of
proliferation(58) . CD9/DRAP 27, an integral membrane protein
produced by a variety of epithelial and hematopoietic cell lineages,
potentiates the juxtacrine growth factor activity of the
membrane-anchored, heparin-binding epidermal growth factor-like
precursor/diphtheria toxin receptor(59) .
Some TMPs also appear to be involved in mediating cell-cell and/or membrane-membrane adhesion. A CD9 mAb induces adhesion(60) . OX-44 associates with the cell adhesion receptor, CD2(57) . TAPA-1 mAbs induce cell-cell adhesion(61, 62, 63) . CD82 (KAI1) can suppress metastasis of human prostate cancer cells(64) . RDS(65) , also known as peripherin (66) , is located in the outer segment disc membranes of retinal photoreceptor cells. In mice, a truncation mutant of RDS produces abnormal development of the outer segment discs followed by slow degeneration of the photoreceptor and blindness (retinal degeneration slow; rds). Studies in transgenic animals indicate that this disease can be corrected by expressing the normal protein in rds photoreceptors(67) . Travis et al.(68) proposed that RDS/peripherin may function as an adhesion molecule that stabilizes outer segment discs through homophilic or heterophilic interactions with adjacent discs.
Experiments employing monoclonal antibodies to various TMPs suggest
that members of this superfamily participate in cellular signal
transduction cascades. For example, incubation of human platelets with
mAbs against CD9 results in an increase in intracellular
Ca(69, 70, 71) .
Intracellular Ca
also increases when U937 cells are
exposed to CD82 mAbs(54) . OX-44 mAbs produce elevations in
inositol phosphates, stimulate tyrosine phosphorylation of cellular
proteins, and increase Ca
in RNK-16
cells(57) . TAPA-1 mAbs also increase tyrosine
phosphorylation(63) . These effects appear to require the
participation of other cell surface proteins (e.g. Refs. 49,
57, and 62).
Although we have operationally defined il-TMP as a participant in the regulation of contact-associated inhibition of HeLa cell growth, its function in cultured HT-29-inosine cells, or in the human intestinal epithelium, may be as a mediator of cell-cell contacts (e.g. adhesion), or inter- or intracellular signal transduction cascades.
N-Glycosylation of other TMPs appears to be important for expression of their biological functions. Treatment of isolated retinas with tunicamycin produces a phenotype similar to that observed in rds mice(72) . Some forms of retinitis pigmentosa in humans (which is similar to retinal degeneration slow) are associated with rds gene mutations(73, 74) . Most of these mutations occur in the glycosylated domain located between the protein's third and fourth transmembrane domains. These observations suggest that the structure of this extracellular domain and its state of glycosylation are critical for the homotypic or heterotypic interactions of RDS. The extent of N-glycosylation of CD82 fluctuates dramatically with human T-cell lymphotrophic virus, type I transformation or phytohemagglutinin activation of T-lymphocytes(51, 75) . Fukudome and co-workers have suggested that extensive N-glycosylation of CD82 may be important for reducing self-fusion of T-cells already infected with human T-cell lymphotrophic virus, type I by reducing cell-cell interactions.
The mechanism by which glycosylation affects TMP
function is not known. The combinatorial model of (il-)TMP regulation
may have to consider each glycoform as having unique biological
properties. il-TMP contains two potential sites for N-glycosylation. HeLa cells offer an opportunity to assess the
relative activities of wild type il-TMP versus mutants with
Asn
Gln and/or Asn
Gln
substitutions. However, results obtained in HT-29-glucose cells and in
pil-TMP/neo-transfected SW480 cells suggest that the nature of
the carbohydrate moiety rather than its mere presence or absence
modulates il-TMP function. This latter notion can be assessed with the
transfected HeLa and SW480 cell lines described in this paper. For
example, pil-TMP/neo and pControl/neo-transfected HeLa cells can be
treated with inhibitors of carbohydrate processing enzymes and the
effects on the ability of the il-TMP pathway to modulate cell growth
monitored. In addition, glycosyltransferases associated with
oligosaccharide branching can be introduced into pil-TMP/neo- (and
pControl/neo) SW480 cells to generate new patterns of (il-TMP)
glycosylation and assess the effects on proliferation. This, of course,
will require that the appropriate nucleotide sugars are available to
the glycosyltransferase and that the carbohydrate moiety already
present on il-TMP can serve as an acceptor site. In this sense,
regulation of specific il-TMP glycoform production may need to be
viewed as a composite of the regulation of il-TMP expression,
(glycosyl)transferase expression, and expression of enzymes involved in
sugar metabolism.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31449[GenBank].