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From the Division of Renal and Inflammatory Disease,*
Department of Medicine, University Hospital, Nottingham, United
Kingdom; the Wellcome Centre for Cell-Matrix
Interactions,
School of Biological Sciences,
University of Manchester, Manchester, United Kingdom; and the
Department of Biological Structure,
University of Washington, Seattle, Washington
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, despite a central role in regulation of the glomerular inflammatory response,13-16 remarkably little is known of the mechanisms by which mesangial cell survival is signaled. Our own work has shown that soluble survival factors such as insulin-like growth factor-1 (IGF-1) can suppress apoptosis induced in mesangial cells by serum deprivation as a model of diminished survival factor supply, or by etoposide used as a model of cell death triggered by DNA damage.17 Preliminary data are also available on possible roles for mesangial cell survival signals derived from adherence to extracellular matrix. Singhal and colleagues, building on earlier work,18 showed that despite the presence of serum, murine mesangial cells deprived of adherence exhibited greater than 10-fold increases in apoptosis compared with cells grown on plastic.19 Surprisingly, type IV collagen (a normal constituent of mesangial ECM) and Matrigel (a heterogeneous preparation of ECM from the murine Engelbreth-Holm-Schwarm tumor) appeared to induce mesangial cell apoptosis in this system.19 This finding contrasted with preliminary data on rat mesangial cells from Sugiyama and colleagues20 in which qualitative assays of apoptosis induced by serum deprivation suggested that Matrigel provided survival signals, partial blockade by antisense oligonucleotides to the integrin ß1 chain implicating such receptors in Matrigel-mediated survival.
The current study used a well-established quantitative assay of apoptosis to assess the effects of defined ECM components on apoptosis induced in mesangial cells by serum deprivation or etoposide. Because of difficulty in obtaining human tissue, we used rat mesangial cells, which in previous studies have displayed survival properties similar to those observed in human mesangial cells.17,21 We report that mesangial cell apoptosis was suppressed by normal constituents of the mesangial ECM (collagen IV and laminin), but not by ECM proteins which are expressed in the diseased glomerulus (collagen I, osteonectin/secreted protein acidic and rich in cysteine (SPARC), and plasma-type fibronectin) by a ß1 integrin-dependent but arg-gly-asp (RGD)-independent mechanism. These findings imply that alterations in mesangial ECM seen in progressive glomerular inflammation are likely to dysregulate the signaling of survival to glomerular cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents were from Sigma Chemical Co. (St Louis, MO) unless otherwise stated. Culture media (Dulbecco's modified Eagle medium nutrient mix f-12) and supplements (100 U/ml penicillin, 100 µg/ml streptomycin, 10% fetal calf serum) were from Gibco Laboratories (Grand Island, NY). Sterile tissue culture plastic ware was from Falcon Plastics (Cockeysville, MD).
Culture of Mesangial Cells
A well characterized rat mesangial cell clone was used in all
experiments.17,21-23
Cells were cultured in Dulbecco's
modified Eagle medium nutrient mix f-12 with 10% fetal calf serum and
supplements as above, and underwent serial trypsinization/subculture on
achieving confluency. The cells were used up to passage 15 after
subculture into 24- or 96-well plates. Considerable care was taken to
verify the phenotype of these mesangial cells, as in our previous
studies.17,24
Mesangial cells exhibited typical stellate
morphology when subconfluent, while on becoming confluent they adopted
the well recognized elongated conformation; if cultured beyond
confluence, typical hillocks were seen.25
On electron
microscopy, mesangial cells exhibited characteristic features such as
abundant microfilaments and, on immunofluorescence, contained organized
-smooth muscle actin, emphasizing the myofibroblast-like nature of
these cells. These cells did not exhibit macrophage markers such as
acetylated low density lipoprotein uptake, nor did they exhibit
macrophage functional properties such as phagocytosis of opsonized
zymosan particles. Furthermore, in addition to lack of acetylated low
density lipoprotein uptake, these cells showed a lack of immunostaining
for factor VIII-related antigen and cytokeratin, as expressed by
glomerular endothelial and epithelial cells respectively.
Morphology of Apoptotic Cultured Mesangial Cells
Cells that had undergone apoptosis were recognized and counted by adding acridine orange (final concentration 10 µmol/L) to culture wells, using inverted fluorescent microscopy to recognize and count total cells and apoptotic cells in randomly selected fields at 200x magnification on a Nikon Optiphot fluorescent microscope. Apoptotic cells were recognized by cell shrinkage, nuclear chromatin condensation, and, occasionally, budding into apoptotic bodies. This fluorescent technique enabled quantitation of changes in apoptotic rates and has previously been carefully verified against mesangial cell apoptosis demonstrated by classical electron and light microscopic features or DNA "laddering" and quantified by assays of DNA fragmentation and flow cytometric demonstration of apoptotic cells.17,21 Furthermore, in contrast to many other methods for quantitation of apoptosis, the method we used has the distinct advantage that the cell culture need not be disturbed before counting, avoiding the potential hazard of losing delicate cells in the later stages of apoptosis and, importantly, preventing the need to disrupt cell-matrix interactions, which were the primary subject of study in this series of experiments.
Induction and Assay of Apoptosis in Cultured Mesangial Cells
To trigger apoptosis, soluble growth factor deprivation was achieved by gentle washing (x3), then incubation of adherent mesangial cell cultures in RPMI 1640 medium with no added supplements. In some experiments, etoposide 50 µmol/L was added to inhibit topoisomerase II and induce DNA strand breaks as an alternative pro-apoptotic stimulus. In each experiment some wells were returned after washing to full medium (ie, RPMI 1640 plus 10% fetal calf serum and insulin/selenium/transferrin growth supplement) as a control. Cells were then incubated at 37°C/5%CO2 for 8 hours routinely, but in some experiments for 24 hours. Apoptosis was quantified by inverted fluorescent microscopy after addition of acridine orange to otherwise undisturbed wells. Total cell number and apoptotic cells were counted first in the plane of the monolayer and then, by altering the plane of focus, in the supernatant above. However, almost invariably apoptotic cells were resting immediately on top of, or next to, their healthy neighbors. Three randomly selected fields in each well were counted, and three wells were examined for each culture condition at every time point (a total of 800-1000 cells per time point).
Assessment of Survival Effects of Matrix Proteins
To assess the effects of matrix proteins, tissue culture-treated
wells were treated with solutions of up to 1 mg/ml of the following
proteins dissolved in phosphate-buffered saline; collagen I (rat tail
purified), collagen IV (purified from human placenta), laminin (from
basement membrane of Engelbreth-Holm-Schwarm mouse sarcoma),
fibronectin (from bovine plasma), and osteonectin/SPARC (recombinant
human protein, prepared as previously described26, 27
).
The protein solution was added to the well in an amount sufficient to
cover the bottom of the well, after which the culture dish was
incubated at 37°C in 5% CO2 for 16 hours. The
excess protein solution was then aspirated, the well washed, and
105
mesangial cells seeded on top in full medium,
plus rabbit polyclonal anti-murine ß1 integrin
blocking antibody 100 µg/ml, rabbit polyclonal anti rat
1 integrin blocking antibody 100 µg/ml
(Cambridge Bioscience), rabbit polyclonal anti rat
2 integrin blocking antibody 100 µg/ml
(Cambridge Bioscience), murine monoclonal anti rat
3 integrin blocking antibody (Chemison, GA),
or control antibody or preimmune serum, or arginine-glycerine-aspartate
serine tetrapeptide (RGDS) at varying concentrations in some
experiments. Sixteen hours after seeding, the cells were subjected to a
pro-apoptotic stimulus as above.
Quantification of Protein Adherent to Wells
Wells of a 96-well tissue culture dish were treated with each matrix protein (100 µg/ml) and incubated at 37°C for 16 hours. Excess fluid was aspirated and discarded and wells treated with 40 µl acetonitrile to strip protein from the plastic. Proteins were treated with acetonitrile to hydrolyze peptide bonds then amino acid composition was determined on a ABI gas phase amino acid analyzer, and quantities compared to known amino acid compositions. In each case, a well was coated with phosphate-buffered saline containing no matrix protein and amino acid content of this well was subtracted from the amount of protein measured in the positive wells to give the final amount, which was comparable in all cases (fibronectin, 0.12 µg/well; laminin, 0.12 µg/well; collagen IV, 0.10 µg/well; collagen I, 0.08 µg/well).
Flow Cytometry
Flow cytometry was used to assess Bcl-2, Bax, Bcl-x, and Bak protein expression by the mesangial cells using antibodies of well-established specificity, as previously described.21 Briefly, cells were incubated in full medium in wells coated with matrix proteins as above for 16 hours. They were dissociated from adherent culture wells by trypsin/EDTA treatment and fixed and permeabilized with Permeafix (Ortho Diagnostics, NJ) for 30 minutes at room temperature. Primary staining was with P-20 (rabbit polyclonal anti-human Bax IgG) 1 µg/ml, S-18 (rabbit polyclonal anti-human Bcl-x IgG) 1 µg/ml, Clone 124 5 µg/ml (monoclonal mouse anti-human Bcl-2 IgG) (DAKO Ltd., Denmark) and rabbit polyclonal anti-human Bak (kind gift of G. I. Evan, Imperial Cancer Research Fund, London) and secondary staining with fluoroscein isothiocyanate-labeled sheep anti-mouse IgG and fluoroscein isothiocyanate-labeled sheep anti-rabbit IgG (Sigma) as appropriate. Negative controls omitted primary antibody and positive controls used rabbit polyclonal or mouse monoclonal anti-human actin (Sigma) as appropriate. Cell staining was assessed on a Becton Dickinson FACScan flow cytometer.
Statistical Analysis
Results were analyzed by analysis of variance, comparing the effect of matrix proteins and etoposide to controls. Significant results are recorded in the figure legends
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether mesangial matrix proteins regulate mesangial
cell apoptosis we studied two ECM proteins, collagen type IV and
laminin, which are expressed in normal glomeruli.25
We
found that cultured rat mesangial cells plated 16h before onto tissue
culture-treated wells pretreated with collagen IV and laminin were
resistant to apoptosis subsequently induced by 8 hours serum
starvation, compared to cells plated directly onto tissue culture
plastic (Figure 1)
. Serum starvation
represents a well-established model of reduction of survival factor
supply as a trigger of apoptosis.17,21
Although 8 hours
was the preferred time point for assay of mesangial cell apoptosis
because no increase in cell necrosis nor any difference in the number
of cells attached could be detected for any substrate (data not shown),
the pro-survival effect of type IV collagen and laminin
versus tissue culture plastic alone was clearly demonstrable
at later time points. For example, in a series of experiments in which
20.8 ± 5.9% (mean ± SE) of mesangial cells had undergone
apoptosis after 24h serum starvation, cells plated on type IV collagen
(9.9 ± 3.0%, P < 0.05) and laminin (10.5
± 2.5%, P < 0.05) were protected against apoptosis.
Furthermore, in the case of both type IV collagen and laminin the
protective effect at 8h serum starvation was dependent on the
concentration of matrix protein used to treat the plate, being maximal
at the highest tested dose of 100 µg/ml. However, significant
protection was seen with protein treatment concentrations as low as 0.1
µg/ml (collagen IV) and 1 µg/ml (laminin). Nevertheless, the
protective effect of laminin was generally less than that observed for
collagen IV (Figure 1, A and B)
.
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Having established that normal ECM proteins could supply survival
signals, we went on to examine ECM proteins which in glomerulonephritis
are characteristically (over)expressed, using an identical protocol;
again, we observed no differences in numbers of cells attaching to
different substrates (data not shown). Osteonectin/SPARC, which is
expressed in experimental self-limited mesangial proliferative
nephritis at a time when proliferation ceases and cell number
falls,28
failed to have any protective effect at the same
treatment concentrations as collagen IV and laminin (Figure 1C)
.
Similarly, no protective effect was observed when plates were treated
with plasma-type fibronectin or collagen I, ECM proteins which are
usually only expressed in glomerulosclerosis (Figure 1, D and E)
.3
To exclude the unlikely possibility that this effect
was due to different quantities of protein adhering to the wells, these
were quantified and found to be comparable, as detailed in Methods.
Collagen-IV and Laminin Protect Rat Mesangial Cells from Apoptosis Induced by Etoposide
Inhibition of topoisomerase II with etoposide is a standard tool
in cell death studies for inducing DNA damage, which in inflamed
glomeruli might result, for example, from generation of reactive oxygen
species by activated leukocytes.29
Fifty micromolar
etoposide increased the apoptotic signal approximately threefold
compared to serum starvation alone, though sometimes by as much as
sevenfold, resulting in a large spread in the data (Figure 2)
. However, both collagen IV and laminin
clearly protected rat mesangial cells from apoptosis induced by
etoposide.
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Bcl-2 is the index member of a growing family of intracellular
counterregulatory proteins which, via a series of complex
heterodimerizations, regulate cell susceptibility to
apoptosis.30,31
Because in previous studies of
integrin-mediated, ECM protein-dependent survival,
ß1 integrin ligation was associated with
increased Bcl-2 expression,9
we studied the expression of
Bcl-2 family members in rat mesangial cells subcultured onto protective
or nonprotective ECM proteins. Flow cytometry of permeabilized cells is
well established as a functionally relevant, quantitative assay of the
expression of Bcl-2 family members,32
which we have
previously adapted to study of the mesangial cell.21
However, flow cytometric analysis of mesangial cells stained for Bcl-2,
Bax, Bcl-x, and Bak showed no difference in the expression of these
regulatory proteins when subcultured for 16 hours on any of the
ECM proteins (Figure 3)
. Although
in each case there was a subpopulation of cells which expressed low
levels of the pro-apoptotic protein, Bak, the size of this
subpopulation did not vary between different ECM proteins. Because
up-regulation of Bcl-2 has been reported in survival mediated by the
RGD (arg-gly-asp)-dependent integrins
vß37
and
5ß1,9
we
went on to seek confirming evidence that such adhesion receptors were
unlikely to be mediating the pro-survival effects of type IV collagen
and laminin.
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To date, those integrins that mediate survival
(5ß1 and
vß3) by up-regulation
of Bcl-27,9
share the property of recognizing the
arg-gly-asp (RGD) sequence in their ligands; their function can be
inhibited with RGD-bearing peptides such as RGDS at around 1
mmol/L.33
Various concentrations of RGDS were added to the
rat mesangial cells at the time of subculture on ECM protein-coated
plastic in an attempt to inhibit such interactions, and the results are
shown in Figure 4
. The addition of 1
mmol/L RGDS (but not 1 mmol/L arg-gly-glu-ser, RGES) to the
cells inhibited their adhesion to tissue culture-treated plastic.
However, despite the presence of 1 mmol/L RGDS cells adhered normally
to collagen IV- and laminin-treated wells (data not shown), indicating
that binding to the protective proteins did not depend on RGD-mediated
integrin binding. Furthermore, after 8 hours' serum deprivation, RGDS
failed significantly to reduce the protective effect of collagen IV or
laminin compared to untreated wells. Therefore, in view of this strong
evidence against involvement of RGD-dependent integrins, we went on to
investigate roles for other adhesion receptors expressed by mesangial
cells.34
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Two RGD-independent integrins of the ß1
family have been implicated in signaling survival;
2ß1 in kidney
cells10
and
3ß1,35
which has been implicated in glomerular cell survival by impaired
glomerular development in mice with targeted mutation of the -3
integrin gene.36
To seek a role for RGD-independent
integrin ECM receptors of the ß1 family, a
polyclonal rabbit anti-rodent ß1 integrin
antibody of well established specificity37,38
was added to
the rat mesangial cells at the time of subculture and 2 hours later.
Addition of this antibody increased the apoptotic signal of cells
plated onto collagen IV-treated wells to the same level as that seen in
cells plated directly onto tissue culture plastic, completely
abrogating its protective effect (Figure 5)
. Indeed, when added to cells
subcultured onto laminin-treated wells, the apoptotic signal was
increased above control levels by a factor of approximately two.
Therefore blocking ß1 integrin interactions
rendered rat mesangial cells growing on laminin more susceptible to
serum starvation induced apoptosis. The antibody was equally effective
whether added at the time of subculture or 2 hours later, when cell
adhesion and spreading had begun. Control preimmune serum had no
detectable effect when added at the same concentration. Furthermore, it
is particularly important to note that (i)
anti-ß1 antibody did not increase mesangial
cell apoptosis when cells were attached to plastic alone (Figure 5, A and B)
and (ii) under no conditions was anti-ß1
antibody observed to cause a reduction in cell attachment to the plate,
as would have been the case had anti-ß1
antibody merely induced cell detachment and so-called secondary
apoptosis.19
Thus, when the number of cells attached to
plastic alone was counted in each well after 8 hours' serum starvation
and standardized as 100% of control, anti-ß1
antibody did not diminish attachment to tissue culture-treated plastic
(106 ± 11%, mean ± SE, n = 3), or to type
IV collagen- (109 ± 16% of control) or laminin- (95.1 ±
8.0% of control) treated tissue culture-treated plastic. The data
strongly implied that laminin- and collagen IV-mediated survival is
dependent on ß1 integrin ligation of a non-RGD
peptide sequence.
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6ß440
However, attempts to use our antibody blockade approach to define which
ß1 integrin mediated the pro-survival effects
of type IV collagen- and laminin-treated substrates were inconclusive.
Because 5 and v
appeared unlikely to partner ß1 given the
RGD-independence of survival signaling on type IV collagen and laminin
(see above), we examined the effects of function-blocking antibodies to
rat 1, 2, and
3 chains. No antibody affected cell attachment
after 8 hours' serum starvation and neither 1
nor 2 antibodies abrogated type IV collagen-
or laminin-mediated mesangial cell survival (data not shown). However,
although 3 antibody appeared to inhibit these
effects (in a series of experiments in which 8 hours' serum starvation
induced 10.3 ± 5.3% apoptosis, 3
antibody increased apoptosis on type IV collagen-treated substrate from
3.7 ± 3.3% to 8.6 ± 4.2% and on laminin-treated
substrates from 4.7 ± 6.6% to 8.7 ± 5.1%, mean ±
SE, n = 3) no firm conclusions could be drawn because
3 antibody also increased apoptosis in cells
cultured on plastic alone (in the same series of experiments, from
10.3 ± 5.3% to 16.5 ± 0.7%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 vitronectin
receptor and 5ß1
fibronectin receptor. In contrast to other cell systems in which these
RGD-dependent integrins are implicated, ECM protein-mediated
suppression of apoptosis did not correlate with changes in
intracellular levels of apoptosis regulatory proteins of the Bcl-2
family. However, it was not possible to characterize which integrin
chain(s) partner(s) ß1 in survival signaling
under the conditions used. From these data we draw a number of conclusions which will help to elucidate mechanisms controlling mesangial cell numbers in inflamed glomeruli. First, although cell adhesion is recognized to have generally suppressive effects on apoptosis, particular ECM proteins can supply additional survival signals to mesangial cells. It is important to note that mesangial cell apoptosis in our culture system is not merely an example of apoptosis that follows cell detachment; as previously established,17,21 the vast majority of apoptotic cells were found in the plane of the cultured monolayer, apparently still attached to substrate rather than floating in the medium. Our second conclusion is that ECM-derived survival signals may be particularly important in regulating susceptibility of mesangial cells to pro-apoptotic stimuli, because blocking antibody to ß1 integrins induced massive apoptosis (approximately 50% at 8 hours) in serum-deprived mesangial cells on laminin-bearing substrate. Third, this suggests in turn that a search for interruption of ECM-derived survival signals might be productive in instances of unscheduled mesangial cell apoptosis in glomerular disease. Although not addressed in the current study, our data suggest the interesting possibility that in progressive glomerular scarring the accumulation of nonprotective abnormal ECM constituents might disrupt survival signaling from normal ECM proteins, perhaps promoting undesirable glomerular apoptosis as glomerular inflammation progresses to hypocellular scar.
However, some caution in interpretation of the data are required. First, although cultured mesangial cells adopt the smooth muscle-like phenotype observed in diseased glomeruli in vivo, our data were obtained with a simple two-dimensional culture model. Nevertheless, data obtained in two-dimensional culture have proved predictive of mesangial cell behavior in vivo, for example in defining the roles of platelet-derived growth factor and transforming growth factor-ß in mesangial cell proliferation and excess matrix accumulation respectively.41-43 Secondly, the experiments were performed on rat mesangial cells, which necessitated the use of some ECM proteins derived from human or bovine rather than rodent sources. However, the primary, secondary, and tertiary structures of the various ECM proteins are well conserved between species, as is their relative expression in disease states.3 For example, the amino acid sequence of human osteonectin/SPARC is 96% identical with the rat protein, and there is strong evidence of functional equivalence.28 Nevertheless, differences in reagents and experimental conditions appear likely to account for differences between the current data and those reported in studies of SV40-transformed murine mesangial cells.19 However, such discrepancies emphasize that it will be necessary to devise approaches by which to dissect ECM-mediated signaling of glomerular cells in vivo.
The current data add importantly to previous work in which we showed
that mesangial cell survival is also under cytokine control, especially
by the autocrine/paracrine actions of secreted
IGF-I.21
Further studies will be required to
dissect how these different survival signals are integrated, but
it is worth noting that inhibition by ß1
integrin-blocking antibody is very strong evidence of a direct effect
of ECM proteins and mitigates against the unlikely possibility that
survival effects of ECM represent an artifact of bound passenger
cytokines. Furthermore, our blocking antibody data extend and confirm
Sugiyama and colleagues' antisense oligonucleotide studies implicating
ß1 integrins in Matrigel-mediated survival of
serum-stained rat mesangial cells.20
However, neither the
current study nor previous work20
has succeeded in
identifying the integrin chain(s) which partner(s) the
ß1 chain in signaling mesangial cell survival
on ECM-treated substrates. Nevertheless, taken together with mesangial
cell defects in mice deleted for the 3
integrin chain,36
our data suggest that future studies
should develop approaches beyond antibody blockade to learn whether a
dominant role in this process exists for the
3ß1 integrin, because
3 antibody increased apoptosis (without
affecting cell attachment) on all substrates, including plastic.
Although we did not set out to dissect completely the integrin-initiated intracellular pathways by which matrix proteins signal survival of mesangial cells, we obtained evidence indicating that survival signaling could be independent of changes of intracellular expression of Bcl-2 family members (unlike reported increases in Bcl-2 expression in ß1 integrin-mediated survival signaling in Chinese hamster ovary cells9 ). However, the data do not exclude more subtle effects such as changes in intracellular location of these regulatory proteins or small but functionally important alterations in expression of Bcl-2 family members which might not be detected by the techniques used. Clearly, future studies will need to delineate more specifically intracellular signaling of ECM-mediated survival of mesangial cells, but such work is beyond the scope of this report.
To conclude, we have shown that the normal mesangial matrix proteins collagen IV and laminin promote mesangial cell survival in vitro by inhibition of apoptosis, via a ß1 integrin-dependent but RGD ligation-independent mechanism. We propose that evidence of interruption of such survival signaling should be sought in postinflammatory glomerular scarring in which unscheduled mesangial cell apoptosis appears to lead to undesirable glomerular hypocellularity. Furthermore, this should be considered as a generally applicable candidate mechanism for unscheduled and undesirable loss of resident cells from tissues undergoing postinflammatory scarring.
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Acknowledgements |
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Footnotes |
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A. M. received a Medical Research Council Clinical Training Fellowship; R. B. was supported by the National Kidney Research Fund. Additional support came from The Wellcome Trust (047273).
Accepted for publication April 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994, 79:1157-1164[Medline]
5ß1 integrin supports survival of cells on fibronectin, and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 1995, 92:6161-61652ß1 integrin expression results in reduced cyst formation, failure of hepatocyte growth-factor scatter factor-induced branching morphogenesis, and increased apoptosis. J Cell Sci 1995, 108:3531-3540[Abstract]
5 subunit in HT29 colon-carcinoma cells suppresses apoptosis triggered by serum deprivation. Exp Cell Res 1996, 224:208-213[Medline]
(V) integrins in mesangial cell adhesion to vitronectin and von Willebrand factor. Kidney Int 1997, 51:1900-1907[Medline]
3ß1 integrin has a crucial role in kidney and lung organogenesis. Development 1996, 122:3537-3547[Abstract]
6ß4 and laminin-5 extracellular matrix. Biochem Biophys Res COmmun 1998, 251:49-55[Medline]
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