From the INSERM, Unité 489, Hôpital
Tenon, 75020 Paris, France and the ¶ School of Biological
Sciences, University of East Anglia, Norwich NR4 7TJ, United
Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We analyzed the expression and regulation of
matrix metalloproteinase 2 (MMP2) and MMP9 gelatinases in a rabbit
kidney collecting duct principal cell line (RC.SVtsA58) (Prié,
D., Ronco, P. M., Baudouin, B., Géniteau-Legendre, M.,
Antoine, M., Piedagnel, R., Estrade, S., Lelongt, B., Verroust, P. J., Cassingéna, R., and Vandewalle, A. (1991) J. Cell
Biol. 113, 951-962) infected with the temperature-sensitive (ts)
SV40 strain tsA58. At the permissive temperature (33 °C), cells
produced only MMP2. Shifting cells to a nonpermissive temperature
(39.5 °C) induced a marked increase in total gelatinolytic activity
due to an increase of MMP2 and an induction of MMP9 synthesis. This
effect was attributed to large-T inactivation at 39.5 °C because it
was abolished by re-infecting the cells with wild-type SV40 strain LP.
Run-on experiments showed that negative regulation of MMP2 and MMP9 by
large-T was transcriptional and posttranscriptional, respectively. MMP2
and MMP9 were also produced by primary cultures of collecting duct cells. In rabbit kidney, both MMP2 and MMP9 were almost exclusively expressed in collecting duct cells, where an unexpected apical localization was observed. Arginine vasopressin and epidermal growth
factor, which exert opposite hydroosmotic effects in the collecting
duct, also exhibited contrasted effects on MMP9 synthesis. Epidermal
growth factor increased but arginine vasopressin suppressed MMP9 at a
posttranscriptional level, whereas MMP2 was not affected. These results
suggest a specific physiological role of MMP2 and MMP9 in principal
cells of renal collecting duct.
Gelatinases matrix metalloproteinase 2 (MMP2)1 and MMP9 belong to
the broad family of MMPs that contain Zn2+, require
Ca2+ for activity, and are active at neutral pH. MMPs are
traditionally subdivided into four classes based on their substrate
specificity: (i) interstitial collagenases; (ii) gelatinases or type IV
collagenases; (iii) stromelysins, matrilysin, and metalloelastase; and
(iv) membrane-type MMPs. This gene family taken as a whole can degrade all extracellular matrix (ECM) components. Therefore, they play a
critical role in tissue remodeling during development and in pathophysiological processes, including inflammation, tissue repair, tumor invasion, and metastasis. However, a growing body of evidence suggests that gelatinases may degrade non-ECM components, such as
myelin basic protein (1) and interleukin-1 In kidney, MMP2 and MMP9 are present in early stages of development
because the mesenchyme of 11-day embryonic kidney already synthesize
both gelatinases (6). MMP2 had no effect on kidney morphogenesis in
organotypic culture. In contrast, blocking MMP9 activity with specific
antibodies or TIMP1 impaired renal morphogenesis by inhibiting growth
and branching of the ureteric bud, the embryonic epithelial precursor
of the collecting duct (6). In adult kidney, gelatinase activity was
detected in glomeruli (7), in epithelial cells that synthesized MMP2
and MMP9 (8, 9), and in mesangial cells that produced only MMP2 (10).
The phenotype of glomerular mesangial cells (11) can be modified by
MMP2 gene expression that is constitutively regulated at a high level
by a specific cell-type enhancer promoter element (12, 13). In
contrast, synthesis of gelatinases by renal tubules is poorly
documented. In vitro cultures of proximal tubule and
collecting duct epithelia showed secretion of both MMP2 and MMP9 (14,
15). However, the collecting duct epithelium is composed of three
different cell types including principal cells and We had previously established a rabbit collecting duct epithelial cell
line (RC.SVtsA58) infected with the temperature-sensitive SV40 strain
tsA58 (16). When cultured at the permissive temperature of 33 °C
(functional large-T antigen), cells display characteristics of
transformed cells. After shifting the cells to the restrictive temperature of 39.5 °C (nonfunctional large-T antigen), cells stop
dividing and acquire a differentiated phenotype characteristic of
collecting duct principal cells including expression of functional type-2 arginine-vasopressin (AVP) receptors (16, 17). We had also shown
that cells shifted to 39.5 °C synthetized a well organized basement
membrane. Profound alterations of the ECM were observed in
dedifferentiated cells at 33 °C, including a marked transcriptional decrease of perlecan, a basement membrane proteoglycan (18). Because
the basement membrane of renal tubules is mainly composed of type-IV
collagen, we asked whether gelatinases could be produced by collecting
duct cells and whether their expression could be increased by
large-T.
We took advantage of the duality of the RC.SVtsA58 cell line, that is
the possibility to switch on or turn off SV40 large-T at each passage
level, to determine the gelatinolytic profile of differentiated
collecting duct principal cells and to analyze the regulation of MMPs
by large-T and by AVP and epidermal growth factor (EGF), two
physiological ligands of collecting duct principal cells. In addition,
we established the in vivo expression of MMP2 and MMP9 at
the apical pole of collecting duct cells, suggesting a specific role of
these MMPs in the physiology of principal cells, irrespective of ECM remodeling.
Materials--
Dulbecco's modified Eagle's medium and Ham's
F-12 were obtained from Life Technologies, Inc. Culture plastic dishes
were purchased from Nunc (Roskilde, Denmark). Acrylamide and
SDS-polyacrylamide gel electrophoresis compounds were from Euromedex
(Souffelweyersheim, France). Hybond-ECL nitrocellulose membrane,
Hybond-N, and Hybond-N+ nylon membrane were obtained from
Amersham Pharmacia Biotech (Little Chalfort, United Kingdom).
Proteinase inhibitors, type IV collagen,
p-aminophenylmercuric acetate, levamisole, bovine skin
gelatin, 3-amino-9-ethylcarbazol, epidermal growth factor and arginine
vasopressin were purchased from Sigma. Anti-human TIMP1 monoclonal
antibody was obtained from Oncogene Science (Cambridge, MA).
Horseradish peroxidase- and alkaline phosphatase-conjugated anti-sheep
antibodies were from Serva (Heidelberg, Germany). We used previously
described IgGs from anti-human MMP2 and anti-pig MMP9 sheep sera (19,
20).
Cell Culture and Experimental Protocol--
Primary cultures of
rabbit collecting duct cells were obtained as described (21) and
provided by M. Tauc (CNRS, Sophia Antipolis, Nice, France). The
RC.SVtsA58 renal collecting duct cell line was generated by infection
of a primary culture of isolated rabbit renal cortical cells with the
SV40 temperature-sensitive mutant tsA58 (22). Experiments were
performed between the 30th and 60th passages following the protocol
previously described (16). Briefly, cells were seeded in uncoated Petri
dishes or in 12-well plastic trays at a concentration of 2 × 104 cells/cm2 and cultured to confluency (day
5) at the permissive temperature (33 °C) to allow functional
expression of the large-T oncogene. The medium used was a serum-free
hormonally defined medium (Dulbecco's modified Eagle's medium-Ham's
F-12 1:1 (v/v); transferrin, 5 µg/ml; sodium selenate, 30 nM; glutamine, 2 mM; dexamethasone, 5 × 10
To analyze the effects of temperature per se irrespective of
the functional status of SV40 large-T oncogene on metalloproteinase synthesis, we used as control a rabbit collecting duct cell line of the
same cell type origin (RC.SV3), previously established in our
laboratory after infection of renal cells with the wild-type SV40
strain LP (23). This cell line was studied under the same culture
conditions as RC.SVtsA58 and analyzed at 33 and 39.5 °C.
Finally, the effects of SV40 large-T oncogene on the gelatinase profile
were also analyzed by re-infecting RC.SVtsA58 cells with a wild-type
SV40 strain at the time of temperature shifting. Cells were seeded as
described above in 12-well plastic trays, and after 5 days of culture,
they were washed and either incubated with 300 µl of a stock solution
containing SV40 strain LP (multiplicity of infection, 100 plaque-forming units/cell) or mock-infected (control cell population).
After 3 h at 37 °C, 800 µl of hormonally defined medium were
added, and cells were transferred for 48 or 72 h at 33 or
39.5 °C with one medium change at 24 h. Gelatinolytic activity
was analyzed 48 and 72 h after re-infection or mock infection.
Substrate Gel Electrophoresis (Zymography)--
Cell-associated
and secreted proteinases were detected by zymography. 48-h conditioned
media collected from cultures grown at 33 or 39.5 °C were
centrifuged to remove cell debris. Cell layers of the same culture were
lysed in SDS-polyacrylamide gel electrophoresis sample buffer (50 mM Tris, 1% SDS, 5% glycerol, 0.002% bromphenol blue).
Both media and cell layers were then stored at
In some experiments, the active forms of gelatinases were induced by
incubating the samples for 3 h at 37 °C with 1 mM
p-aminophenyl mercuric acetate (APMA).
All experiments were done at least 10 times.
14C-Acetylated Gelatin and TIMP
Assays--
14C acetylated gelatin assay and measurement
of TIMP activity were performed exactly as described previously (24).
The unit of TIMP activity is the amount that will cause 50% inhibition of 2 units (µg collagen degraded/min) of collagenase.
Immunoblotting--
Conditioned media from cells cultured at
33 °C and 39.5 °C were concentrated approximately 70 times using
Amicon microconcentrators with a 30-kDa cutoff. Samples were submitted
to SDS-polyacrylamide gel electrophoresis in a 8% polyacrylamide gel
under nonreducing conditions and electrotransferred to nitrocellulose
for 90 min at a constant current of 190 mA. Afterward, the
nitrocellulose sheet was saturated with 5% dry milk in 0.1% PBS-Tween
and 1 mM levamisole for 1 h at 37 °C, washed in
Tris-buffered saline with 0.1% Tween, and incubated overnight at
4 °C with anti-human MMP2 (2 µg/ml) or anti-pig MMP9 (2 µg/ml)
sheep IgGs. This step was followed by a 2-h incubation at room
temperature with an anti-sheep IgG antibody (0.2 µg/ml) conjugated to
alkaline phosphatase. Alkaline phosphatase activity was revealed by
adding the nitro blue tetrazolium substrate (nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate complex in 100 mM Tris-HCl, 100 mM NaCl, and 5 mM
MgCl2, pH 9.5). The reaction was stopped in 20 mM Tris-HCl, 5 mM EDTA, pH 8.0.
Immunoblotting experiments were done at least three times.
Morphological Studies--
Expression of gelatinases was
analyzed on cryostat rabbit kidney sections. Briefly, a New Zealand
White rabbit was anesthetized, and its kidneys were perfused with PBS
and snap-frozen in liquid nitrogen. Tissue sections (4 µm) were fixed
in 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature,
for 30 min in 50 mM NH4Cl, and further
incubated for 30 min in a peroxidase suppressor reagent (Pierce) to
neutralize endogenous peroxidases. They were then saturated with 10%
dry milk diluted in PBS. Afterward, sections were incubated overnight
at 4 °C with sheep anti-MMP2 or anti-MMP9 IgG (10 µg/ml) diluted
in PBS supplemented with 5% dry milk. They were then incubated for
2 h at room temperature with horseradish peroxidase-conjugated
rabbit anti-sheep IgG antibody (dilution, 15 µg/ml). Enzyme activity
was revealed with the 3-amino-9-ethylcarbazol substrate dissolved in 50 mM acetate buffer, pH 5.0, supplemented with 0.015%
H2O2. Preparations were counterstained with
hematoxylin and examined in a Leitz microscope.
RNA Isolation and Analysis by Northern Blot--
Confluent cells
at 33 or 39.5 °C, cultured in standard conditions or stimulated with
EGF or AVP, were lysed in 4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl, 100 mM
Northern blots were performed at least four times.
Run-on--
Experiments were performed as described previously
(18). Briefly, cells cultured in standard conditions or treated with EGF or AVP for 24 h were trypsinized and lysed in ice-cold 0.5% Nonidet-P40 lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol, 10
Run-on experiments were performed at least three times.
Cultured Principal Cells Synthesize and Secrete MMP2 and
MMP9--
At the permissive temperature (33 °C), gelatin zymograms
revealed a major zone of lysis at 68 kDa in conditioned media (Fig. 1A, lane 1) and in
cell extracts (Fig. 1B, lane 1). This zone corresponded to the migration of MMP2 identified by immunoblotting (Fig. 1D, lane 1). MMP2 activity normalized per
cells number was markedly increased in conditioned media compared with
cell extracts (Fig. 1B, lane 1), suggesting that
the enzyme was predominantly secreted. In conditioned media, the 68-kDa
band of lysis was associated with a faint band at 62 kDa that became
predominant upon incubation with APMA (Fig. 1A, lane 2), an
organomercurial compound that initiates conversion of the proenzymatic
forms of MMPs into their active lower molecular weight forms.
Therefore, the upper band of the doublet is most likely the latent
form, whereas the lower is the active form of the enzyme.
At the restrictive temperature (39.5 °C), cell-associated MMP2
activity increased (Fig. 1B, lane 2), and the
profile of secreted gelatinases was profoundly modified (Fig.
1A, lanes 3 and 4). In addition to a
2-fold increase of MMP2 confirmed by Western blotting (Fig.
1D, lane 2), zymograms showed the appearance of a
lytic zone at 90 kDa (Fig. 1A, lane 3). The
anti-MMP-9 antibody identified a doublet at 90 and 88 kDa (Fig.
1D, lane 4). APMA treatment induced lower
molecular mass bands, corresponding most likely to active forms of MMP9
as shown in Fig. 1A, lane 4. The gelatinolytic
pattern of RC.SVtsA58 conditioned media at 39.5 °C was similar to
that of collecting duct cells in primary culture (Fig. 1C),
a finding that is in keeping with the differentiation process induced
by shifting RC.SVtsA58 cells from 33 to 39.5 °C.
Gelatinase activity was also measured in conditioned media using the
14C-gelatin assay. No activity was detected in samples from
RC.SVtsA58 cells kept at 33 °C, most likely because of lower
sensitivity compared with zymography. By contrast, enzymatic activity
was readily measurable in the media of cells shifted to 39.5 °C
(0.75 units/ml/106 cells, n = 2). TIMP
activity was 2-3-fold lower at 33 °C (0.32 units/ml/106
cells, n = 2) than at 39.5 °C (0.73 units/ml/106 cells, n = 2). This result was
confirmed by Western blot analysis of conditioned media with an
antibody to TIMP1, the main inhibitor of MMP9 (data not shown).
Taken as a whole, these results show that both transformed (RC.SVtsA58
at 33 °C) and differentiated (RC.SVtsA58 at 39.5 °C and primary
cultures) collecting duct cells secrete MMP2, but MMP9 is produced only
when cells are differentiated. The differentiation process is
associated with a marked increase in gelatinolytic activity despite
higher TIMP activity.
Appearance of MMP9 at 39.5 °C in RC.SVtsA58 Cells Is Due to
Inactivation of SV40 Large-T--
To rule out an effect of the
increase in temperature (irrespective of large-T functional activity)
on MMP expression, we first studied the control cell line RC.SV3
derived from the same renal tubule cell population, but infected with a
wild strain of SV40. Gelatinase activity in conditioned media of cells
grown at 33 °C (Fig. 2A,
lane 1) or at 39.5 °C (Fig. 2A, lane
2) was restricted to the proteolytic zone of MMP2, and it was not
influenced by the culture temperature. In particular, shifting RC.SV3
cells to 39.5 °C did not induce the expression of MMP9, in keeping
with the sustained activity of wild-type large-T oncogene at
39.5 °C.
To confirm the inhibitory effect of large-T on MMPs, we re-infected
RC.SVtsA58 cells with the wild-type SV40 strain LP at the time of
temperature shifting. In cells transferred to 39.5 °C, induction of
MMP9 was totally inhibited as early as 48 h after the temperature
shift in SV40 strain LP-infected cells (Fig. 2B, lane
4) compared with mock-infected cells (Fig. 2B,
lane 3). In parallel, the increase in MMP2 activity was
markedly blunted (Fig. 2B, lane 3 versus lane 4). Re-infection did not affect the
gelatinase pattern in cells maintained at 33 °C (Fig. 2B,
lanes 1 and 2). Similar data were observed at
72 h (not shown). These results thus indicate that the changing
pattern of gelatinase activity in RC.SVtsA58 cells does not result from
a direct effect of temperature but is induced by SV40 large-T
functional status.
MMP2 and MMP9 Are Expressed in Collecting Ducts of Normal Rabbit
Kidney--
To determine whether expression of MMP2 and MMP9 by
differentiated RC.SVtsA58 cells and collecting duct primary cultures
was induced by culture conditions or could be considered as an
additional marker of differentiation, we analyzed their distribution in
the normal kidney by immunohistochemistry with the antibodies
previously used for immunoblotting. Anti-MMP2 as well as anti-MMP9
antibodies stained tubule sections identified as collecting ducts
because of the absence of brush border on phase contrast examination
and their association by bunches of 4-5 units (Fig.
3A). All other nephron
segments including glomeruli did not show appreciable labeling.
However, a faint reactivity was observed in the kidney interstitium
with anti-MMP2 antibody (Fig. 3B). Thus, MMP2 and MMP9 are
essentially expressed in the tubule segment from which the RC.SVtsA58
cell line originates. On higher magnification (Fig. 3, C and
D), the two MMPs, although mostly implicated in ECM
remodeling, were unexpectedly localized at the apical pole of
collecting duct cells, suggesting the presence of receptors and/or
substrates for these enzymes at the luminal pole of the cells.
MMP9 Is Differentially Regulated by Physiological Ligands of
Collecting Duct Cells--
EGF and AVP are physiological ligands of
principal cells that exert antagonistic hydroosmotic effects on the
collecting duct (30, 31). We therefore tested the ability of these
factors to regulate the expression of MMP2 and MMP9 in RC.SVtsA58
cells. MMP2 expression analyzed by zymography (Fig.
4A) and Western blotting (Fig.
4B) in conditioned media of 33 and 39.5 °C cultures was not altered either by 15 ng/ml EGF (Fig. 4, A, lanes
2 and 5, and B, lanes 2 and
4) or by 10
In sharp contrast, EGF and AVP had dramatic effects on MMP9 expression.
EGF induced MMP9 secretion by transformed cells (33 °C) (Fig. 4,
A, lane 2, and C, lane 2).
It also increased enzyme expression in differentiated cells at
39.5 °C (Fig. 4, A, lane 5, and C,
lane 5). Concentration-response experiments indicated that
MMP9 induction or stimulation in the two populations of cells (33 and
39.5 °C) occurred from 0.5 ng/ml (not shown). Cells cultured at the
permissive temperature (33 °C) did not express AVP receptors (16)
and, as expected, the effect of AVP was only observed in differentiated
cells (39.5 °C) in which 10 Posttranscriptional Regulation of MMP9 Gene Expression by SV40
Large-T and Physiological Ligands of Collecting Duct Cells--
The
mechanisms whereby MMP9 was differentially regulated by large-T, AVP
and EGF were further investigated by Northern blotting and run-on
experiments. We used a riboprobe to detect MMP9 mRNA because of
lack of sensitivity of cDNA probe. Northern blot data were in total
accordance with those obtained by zymography and Western blot analysis.
The riboprobe failed to hybridize with RNAs isolated from transformed
cells (33 °C) (Fig. 5, lane
1), whereas it strongly hybridized with an approximately
2.5-kilobase mRNA in differentiated cells (39.5 °C) (Fig. 5,
lane 4). When cells were incubated with EGF (15 ng/ml), MMP9
mRNA hybridization signal was induced in transformed cells
(33 °C) and increased by about 3-fold in differentiated cells
(39.5 °C) (Fig. 5, lanes 2 and 5). By
contrast, AVP (10
To investigate whether MMP9 mRNA levels were transcriptionally
regulated, we performed run-on assays on isolated nuclei of cells
cultured at 33 and 39.5 °C under standard conditions or in the
presence of EGF (15 ng/ml) or AVP (10
In transformed cells, the MMP9 gene was clearly transcribed
(Fig. 6) although the enzyme was not detected in conditioned media and
cell extracts (Fig. 1, A, lane 1, B,
lane 1, and D, lane 3). Surprisingly,
in differentiated cells, the level of MMP9 transcription was not
significantly increased (Fig. 6), in contrast with MMP9 antigen (Fig.
1D, lanes 3 and 4), enzymatic activity
(Fig. 1A, lanes 1 and 3), and mRNA
levels (Fig. 5, lanes 1 and 4). Moreover, EGF did
not significantly modify the level of MMP9 gene transcription (Fig.
6B) although it induced or increased MMP9 antigen (Fig. 4C, lanes 2 and 5), activity (Fig.
4A, lanes 2 and 5), and mRNA (Fig.
5, lanes 2 and 5). AVP, which had the opposite
effects, did not alter either MMP9 transcription (Fig. 6B).
These results suggest that in principal cells of the renal collecting
duct, regulation of MMP9 expression occurs predominantly at a
posttranscriptional level.
On the other hand, run-on analysis showed that transcription of the
MMP2 gene was stimulated by 2-fold after large-T antigen inactivation at 39.5 °C (Fig. 6A), in accordance with the
2-fold increase of MMP2 antigen (Fig. 1D, lanes 1 and 2) and activity (Fig. 1A, lanes 1 and 3). Thus in contrast to MMP9, MMP2 was essentially regulated at the transcriptional level.
We took advantage of the duality of the RC.SVtsA58 cell line to
analyze the gelatinolytic profile of collecting duct cells and its
regulation (i) by SV40 large-T at the permissive temperature (33 °C)
and (ii) by physiological ligands of collecting duct cells (AVP and
EGF) at the nonpermissive temperature (39.5 °C). We first showed
that differentiated collecting duct cells in culture (39.5 °C) and
in vivo produced the two gelatinases MMP2 and MMP9. Second, we demonstrated that functional expression of large-T antigen reduced
MMP2 and suppressed MMP9 at transcriptional and posttranscriptional levels, respectively. Third, we provided evidence that MMP9, but not
MMP2, was posttranscriptionally regulated by EGF and AVP, suggesting
that MMP9 could play a physiological role in principal cells.
It is generally admitted that MMP2 is regulated differently from the
other MMPs and that it is constitutively expressed at low level in
normal tissues (32). We showed that cultured RC.SVtsA58 cells secreted
large amounts of MMP2 and that, in vivo, the collecting duct
was the segment of the renal tubule to express substantial amounts of
MMP2 antigen. In other nephron segments, MMP2 specific staining was
absent or very faint, although cultured cells from these segments were
shown to produce MMP2 (14). The discrepancy between the in
vivo and in vitro data may be due to rapid secretion of
the enzyme in the extracellular milieu. In culture, the secreted enzyme
can accumulate in the medium, being therefore easily detectable by
zymography. Unlike MMP2, MMP9, which was originally identified in
polymorphonuclear leukocytes and macrophages (33, 34), is produced by a
small number of cell types. However, its expression can be readily
regulated by a number of agents, including growth factors, hormones,
cytokines, and extracellular matrix molecules (35), which explains why
MMP9 is often associated with inflammation, tissue injury and tumor
invasion. In normal rabbit kidney, we showed that MMP2 and MMP9 were
only detected at the apical pole of collecting duct cells. In addition,
both MMPs were secreted partially in active forms as attested by the
presence of a lower molecular weight band on zymograms and Western
blots. This finding suggests the expression of a MT-MMP required for
MMP2 activation (36, 37) at the apical pole of principal cells, as well
as of enzymes activating the pro-enzymatic form of MMP9. Further studies are required to identify the molecular mechanisms involved in
MMP activation by collecting duct cells.
To investigate the effects of SV40 large-T on MMPs, RC.SVtsA58 cells
were either kept at 33 °C or shifted to 39.5 °C. At the permissive temperature, functional large-T antigen induced a
down-regulation of MMP2 and suppressed MMP9 expression. At first
glance, these results were surprising because transformed cells are
usually thought to exhibit high proteolytic activity responsible for
their invasiveness potential. They cannot be accounted for by cell
selection or genetic drift because studies were conducted in parallel
on cells originating from the same passage maintained at 33 °C or shifted to 39.5 °C at various passage levels. Neither can they be
explained by the change in culture temperature, which did not affect
the pattern of gelatinolytic activity in a control cell line
transformed with a wild-type strain of SV40 and grown at 33 or
39.5 °C. Moreover, reinfection of RC.SVtsA58 cells at the time of
cell shifting to 39.5 °C with a wild strain of SV40 suppressed within 48 h the increase of MMP2 and the induction of MMP9, thus demonstrating that when large-T is functional, it has a negative effect
on MMPs expression irrespective of culture temperature.
Decreased expression of MMP2 by SV40 had previously been reported in
human skin fibroblasts (38) and, at the permissive temperature, in
human placenta cells transformed with the temperature-sensitive SV40
strain tsA30.1 (39). We further show that this inhibition is
transcriptional. Like large-T, adenovirus E1a oncogene represses MMP2 gene transcription in human tumor cell lines (40). The effect of E1a is mediated by the AP-2 transcription pathway (41). Binding of AP-2 to the 5'-flanking region of MMP2 gene seems
to be essential for gene activation. Consequently the interaction of
E1a protein with the DNA binding/dimerization region of AP-2 inhibits
MMP2 gene transcription (42). Large-T also prevents AP-2-mediated activation of gene transcription by inhibiting AP-2 binding to DNA (43). However, the mechanisms of the negative transcriptional effect of large-T on MMP2 may be more
complex. Indeed in addition to AP-2, a p53 binding site located in the enhancer region containing the AP-2 regulatory sequence of the MMP2
promoter was recently described by Bian and Sun (44), who demonstrated
a dual direct and indirect effect of p53 on MMP2 transcription resulting in a net activation of MMP2. In
RC.SVtsA58 cells, cascade immunoprecipitation experiments with
anti-large-T and anti-p53 antibodies revealed that p53 was totally
bound to large-T at 33 °C, whereas it was released from the mutated
large-T protein at 39.5 °C
(45).2 Thus, increased
MMP2 transcription at 39.5 °C could be the consequence of
both the sudden release of free p53 and the loss of inhibitory effect
of the mutated large-T on AP-2 binding to DNA. Conversely, the negative
effect on MMP2 transcription of p53 quenching by large-T at
33 °C is corroborated by preliminary experiments showing a
substantial increase in MMP2 activity in RC.SVtsA58 cells transfected with the full-length rabbit p53
cDNA.3 In contrast to
MMP2, MMP9 was essentially regulated at a posttranscriptional level as
shown by Northern blot and run-on experiments. However, we cannot
exclude a minor effect of large-T on MMP9 transcription as
well because AP-2 was shown to induce cell-type specific transcription of MMP9 in rabbit corneal epithelial cells (46).
Gelatinolytic activity was not detectable at 33 °C, and this was
associated with a marked decrease in TIMP activity and TIMP1 antigen.
Decrease in gelatinolytic activity in transformed cells (33 °C)
apparently contradict previous literature reporting that transfection
with oncogenes induces protease activity, including MMP9 activity (38,
47, 48), together with increased invasiveness and metastatic potential
(review in Ref. 49). But they are in agreement with a growing number of
reports showing that in human tumoral tissues, MMPs are secreted by
leukocytes, stroma cells, or both, rather than by tumor cells
themselves (50).
Similarly to our kidney principal cell line, shifting of the
temperature-sensitive human placenta cell line to the restrictive temperature increased production of MMPs in parallel with an induction of invasiveness, a characteristic of differentiated placenta
trophoblast-like cells (39). Therefore, we can postulate that high
gelatinolytic activity in principal cells cultured at 39.5 °C could
participate in the expression of a differentiated phenotype.
Because MMP2 and MMP9 are expressed in collecting duct cells in
vivo, we used RC.SVtsA58 cells as a model to test the ability of
EGF and AVP, two physiological ligands of collecting duct, to influence
MMPs production. AVP is the principal hormone regulating water and
electrolyte transport in the collecting duct via cAMP production and
protein kinase A activation (Ref. 51 and review in Ref. 52). It
increases water reabsorption by inducing insertion of AQP2 water
channels in the apical membrane of principal cells (53). It also raises
sodium reabsorption by apical epithelial sodium channels (54). EGF
inhibits the hydroosmotic effects of AVP by acting at a post-cAMP level
(30). Conversely, AVP inhibits the EGF-stimulated mitogen-activated
protein kinase cascade (55), indicating that the signal transduction
pathways of AVP and EGF are closely connected in collecting duct cells.
We found that MMP2 was not regulated by either AVP or EGF. In sharp
contrast, MMP9 was markedly but differentially modulated by the two
ligands: AVP decreased whereas EGF increased MMP9 protein, activity,
and mRNA in collecting duct principal cells cultured at 39.5 °C.
This regulation occurred at a posttranscriptional level. Regulation of
MMPs by cAMP is poorly documented. cAMP up-regulated TIMP1, TIMP2, and
MMP2 expression both at mRNA and protein levels in the human
fibrosarcoma cell line HT1080, but MMP9 was not affected (56).
Stimulation of MMP9 by EGF has been reported in other cell types
(57-62), but the regulation was not posttranscriptional. On the other
hand, EGF induced MMP1 and MMP3 in cultured human fibroblasts by
increasing mRNA stability (63). Rat, rabbit, and human MMP1
mRNA have a 3'-untranslated region that contains three repeats of
the AUUUA motif (64) that are implicated in the regulation of mRNA
stability (65). Similar sequences might be involved in the regulation
of MMP9 mRNA stability by EGF and AVP and during the cell
differentiation process induced by temperature shift.
The strong expression of MMP2 and MMP9 at the apical pole of collecting
duct cells in vivo, as well as the opposite
posttranscriptional regulation of MMP9 by physiological ligands with
antagonistic hydroosmotic effects, suggests that MMP9 plays a role in
the physiology of principal cells, irrespective of its collagenase
activity. A variety of nonextracellular matrix macromolecules are
cleaved by MMP9, including myelin basic protein (1),
galactoside-binding proteins CBP30 and CBP35 (66, 67), and
interleukin-1 In conclusion, we have demonstrated that MMP2 and MMP9 are produced by
cultured collecting duct cells and are specific markers of this nephron
segment in vivo. Large-T dramatically reduces MMP2 and MMP9
expression at transcriptional and posttranscriptional levels,
respectively. Only MMP9 is regulated by AVP and EGF, which induce
opposite posttranscriptional effects. Further studies are needed to
determine the renal physiological consequences of MMP9 and
MMP2 gene invalidation and the physiological substrates of these enzymes in principal cells.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(2), may process the
tumor necrosis factor-
precursor (3), and may be involved in a
variety of physiological processes, such as platelet aggregation (4).
Except for MMP2, which is considered to be constitutively expressed,
MMPs are highly regulated by growth factors, cytokines, hormones and
extracellular matrix components. This regulation occurs at
transcriptional and posttranscriptional levels and also involves
natural tissue inhibitors of MMPs (TIMPs) (5).
- and
-intercalated cells, making it difficult to identify the cell type
responsible for MMP secretion.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
8 M; insulin, 5 µg/ml; HEPES, 20 mM, pH 7.4). On day 5, the medium was changed,
and cell cultures were separated in two batches for the next 48 h:
one batch was maintained at the permissive temperature, whereas the
other one was transferred to the restrictive temperature of 39.5 °C.
In some experiments, confluent cells were incubated for the last
24 h of culture with EGF (15 ng/ml) or AVP (10
7
M). Cells and 48-h conditioned media were separately
sampled on day 7 and kept at
20 °C until further analysis.
20 °C until further
analysis. Lysates and conditioned media of ~10,000 cells were
subjected to electrophoresis under nonreducing condition in 8%
SDS-polyacrylamide gels copolymerized with 1 mg/ml gelatin or type IV
collagen. Gels were washed twice for 30 min in 2.5% Triton X-100 to
remove SDS, incubated in substrate buffer (50 mM Tris-HCl,
5 mM CaCl2, 1 µM
ZnCl2, 0.01% NaN3, pH 7.5) overnight at
37 °C, stained in 0.5% Coomassie Blue G in 40% methanol, 10%
acetic acid for 30 min at room temperature and destained in 40%
ethanol, 1% acetic acid. Clear proteolytic zones indicated the
presence of gelatinases at their respective molecular weights.
-mercaptoethanol. Total RNAs were then extracted as
described by Chomczynski and Sacchi (25). 20 µg of RNA for each
condition were then analyzed by Northern blot using a
NorthernMaxTM kit (Ambion, Austin, TX). Briefly, RNAs were
electrophoresed in 1% agarose gel under denaturing conditions and
transferred for 2 h onto Hybond-N nylon membrane. The bluescript
plasmid containing the rabbit MMP9 cDNA (26) was
digested with smaI to excise a 1971-base pair cDNA
fragment of MMP9. The resulting plasmid containing a
352-base pair 3' fragment of rabbit MMP9 cDNA was used
as a template to produce an antisense riboprobe. Transcription of
MMP9 antisense RNA was carried out with T7 polymerase using
a RNA transcription kit (Ambion). Transferred RNAs were then hybridized
overnight at 65 °C in a hybridization buffer provided with the kit,
washed, and exposed to Fuji medical x-ray films at
80 °C.
6 M
antipain). Nuclei were isolated by centrifugation (700 × g) and kept at
80 °C in glycerol storage buffer (50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 40% glycerol). For each culture condition, ~107 nuclei were transcribed
in 50 mM Tris-HCl, pH 8.0; 5 mM
MgCl2; 150 mM KCl; 0.1 mM EDTA; 1 mM dithiothreitol; 10 mM creatine phosphate; 10 µg/ml creatine phosphokinase; 1500 units/ml RNasin; 1 mM
each ATP, GTP, and CTP; 100 µCi of [
-32P]UTP (3000 Ci/mmol). Reactions were terminated by treating nuclei with DNase I and
proteinase K and labeled RNAs were extracted by phenol/chloroform.
Plasmid Bluescript containing cDNA probes were linearized, blotted,
and immobilized on Hybond N+ nylon membranes. cDNAs
used were the full-length human MMP2 (27), the full-length rabbit MMP9
(26), the full-length rat GAPDH (28), and the full-length mouse actin
(29). Membranes were hybridized, washed, and exposed to Fuji medical
x-ray films.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (42K):
[in a new window]
Fig. 1.
Gelatin zymograms (A-C) and
Western blot (D) of conditioned media (A, C,
and D) and cells lysates (B) from the
collecting duct cell line RC.SVtsA58 (A, B, and
D) and primary cultures of collecting duct cells
(C). RC.SVtsA58 cells were either cultured at 33 °C
(permissive temperature) (large-T +) or shifted to
39.5 °C (restrictive temperature) (large-T -) in the last
48 h. Zymograms were carried out on 8% SDS-polyacrylamide gels
copolymerized with gelatin. The medium and cells lysate samples applied
to each lane corresponded to ~10,000 cells. Concentrated media (70×)
from RC.SVtsA58 cells were also analyzed by Western blotting with
anti-MMP2 (D, lanes 1 and 2) and anti-MMP9
(D, lanes 3 and 4) sheep IgG as described under
"Experimental Procedures." Zymograms yielded a main 68-kDa band of
lysis at 33 °C (A, lane 1) and two bands at 90 and 68 kDa
at 39.5 °C (A, lane 3). These lytic areas were ascribed
to MMP2 (68 kDa) and MMP9 (90 kDa) by Western blotting (D).
The temperature shift increased MMP2 (A, lanes 1 and
3, and D, lanes 1 and 2) and induced
MMP9 (A, lanes 1 and 3, and D, lanes 3 and 4). Note that the gelatinolytic profile of
differentiated RC.SVtsA58 cells at 39.5 °C (A, lane 3)
was similar to that of primary cultures (C). Samples of
conditioned media of RC.SVtsA58 cells shown in lanes 1 and
3 (A) were treated with APMA to generated the
active forms of the enzymes at 82 kDa for MMP9 and 62 kDa for MMP2
(A, lanes 2 and 4). Note that these active forms
were secreted at 39.5 °C (A, lane 3, and D, lane
4).
View larger version (59K):
[in a new window]
Fig. 2.
Zymographic analysis of large-T effects on
MMP2 and MMP9 secretion by collecting duct cells. A,
RC.SV3 is a collecting duct cell line transformed with wild-type SV40
strain LP. Note the absence of MMP9 secretion at 33 °C and
39.5 °C (A) (large-T is functional at both temperatures).
RC.SVtsA58 cells were re-infected (B, lanes 2 and
4) or mock-infected (B, lanes 1 and 3)
on day 5, and conditioned media were studied after additional 48 h
period of culture at 33 °C (B, lanes 1 and 2)
or 39.5 °C (B, lanes 3 and 4). Re-infection
induced a substantial increase in cell number at 39.5 °C (820,000 versus 530,000 cells/well in mock-infected cells) and had no
effect at 33 °C (1300,000 versus 1400,000 cells). Samples
of conditioned media deposited onto the gel were normalized for cell
number (~10,000 cells). Note induction of MMP9 and marked increase of
MMP2 at 39.5 °C in mock-infected cells (B, lane 3 versus lane 1). In contrast, at 39.5 °C, re-infected
cells produced trace amounts of MMP9, and less MMP2 than mock-infected
cells (B, lane 4 versus lane 3). There
was no effect of cell re-infection at 33 °C.
View larger version (175K):
[in a new window]
Fig. 3.
Expression of MMP9 and MMP2 in vivo
in the normal rabbit kidney. Cryostat tissue sections were
processed for immunohistochemistry as indicated under "Experimental
Procedures" and incubated with same anti-MMP9 (A and
C) and anti-MMP2 (B and D) antibodies
as for Western blotting. Note staining of collecting duct sections by
both anti-MMP9 (A and C) and anti-MMP2
(D) antibody. B and D, on higher
magnification, MMP9 (B) and MMP2 (D) antigens
were localized at the apical pole of collecting duct cells.
7 M AVP (Fig.
4A, lanes 3 and 6), which induces a
peak cyclic AMP response (16).
View larger version (36K):
[in a new window]
Fig. 4.
Zymographic (A) and Western blot
(MMP2 (B) and MMP9 (C)) analysis of EGF and AVP
effects on MMP2 and MMP9. RC.SVtsA58 cells cultured at 33 °C
(large-T +) or shifted at 39.5 °C (large-T )
were incubated for the last 24 h of culture with 15 ng/ml EGF or
10
7 M AVP. EGF induced MMP9 at 33 °C
(A, lane 2; C, lane 2) and markedly increased
MMP9 at 39.5 °C (A, lane 5; C, lane 5). AVP
had no effect on MMP9 at 33 °C (A, lane 3; C, lane
3) but almost totally inhibited the expression of MMP9 at
39.5 °C (A, lane 6; C, lane 6). MMP2 was
substantially increased when large-T was inactivated at 39.5 °C
(A, lane 4; B, lane 3) but was not affected at
either temperature by EGF (A, lanes 2 and 5;
B, lanes 2 and 4) or AVP (A, lanes 3 and 6).
7 M AVP
markedly inhibited MMP9 activity and antigen shown by zymography (Fig.
4A, lane 6) and Western blotting (Fig.
4C, lane 6), respectively.
7 M) strongly reduced the
amount of MMP9 mRNA in differentiated cells (Fig. 5, lane
6).
View larger version (63K):
[in a new window]
Fig. 5.
Northern blot analysis of large-T, EGF (15 ng/ml), and AVP (10 7 M) effects on
MMP9 mRNA. RC.SVtsA58 cells cultured at 33 °C (lanes
1-3) and 39.5 °C (lanes 4-6) were treated with
hormonally defined medium alone (lanes 1 and 3),
EGF (lanes 2 and 5), or AVP (lanes 3 and 6). 20 of µg RNA immobilized on Hybond-N nylon
membrane were hybridized with MMP9 riboprobe. Ethidium bromide staining
of 28S ribosomal RNA was used to document equal loading of RNA in each
lane.
7 M)
(Fig. 6). In addition to actin, we used
GAPDH as housekeeping gene because we had previously shown that actin
gene transcription was increased by about 2-fold in transformed
RC.SVtsA58 cells maintained at 33 °C (18). Such modulation was
confirmed in the present experiments, in which only GAPDH signal was
unaltered by large-T functional activity, EGF, and AVP (Fig. 6). In
contrast, actin signal was reduced by 2-3-fold compared with GAPDH in
differentiated cells (39.5 °C), but it was not appreciably altered
by EGF and AVP.
View larger version (47K):
[in a new window]
Fig. 6.
Nuclear run-on analysis of gelatinase gene
transcription in nuclei from RC.SVtsA58 cells cultured at 33 or
39.5 °C and treated with EGF (15 ng/ml) and AVP
(10 7 M). Nuclei were isolated from cells
cultured at 33 and 39.5 °C treated with the hormonally defined
medium alone, EGF, or AVP. Nuclei were transcribed in vitro
in the presence of 32P-UTP, and labeled transcripts were
hybridized to immobilized cDNA for MMP2, MMP9, actin, and GAPDH.
Linearized bluescript was used as a control for nonspecific
hybridization. A, effect of large-T antigen on
MMP2 and MMP9 gene transcription. Note reduced
transcription of MMP2 and unchanged level of MMP9
transcription compared with the housekeeping GAPDH gene when mutated
large-T is functional. Large-T also increases actin transcription.
B, effect of EGF and AVP on MMP9 gene
transcription. Note unchanged level of MMP9 transcription in
the presence of either ligand.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(2). In the collecting duct, other proteases have been
implicated in important physiological functions. It was shown that a
glycosylphosphatidylinositol-anchored serine protease named CAP1
regulated apical epithelial sodium channel activity (68) and that a
membrane metalloendopeptidase could degrade the V2-type AVP receptor
(AVP-R2) and thus regulate its function (69). Considering the apical
localization of MMP2 and MMP9, and MMP9 regulation by AVP and EGF,
potential substrates include apical ion and water channels, apical
receptors and their ligands, and urine constituents. To identify these
substrates, it is essential to establish whether MMPs are located in
the plasma membrane or in submembranous vesicles as shown for MMP9 in
human microvascular endothelial cells (70).
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to M. Tauc (CNRS and University of Nice-Sophia Antipolis, Nice, France) for the gift of primary cultures, to M. Harrison for performing the 14C-acetylated gelatin and TIMP assays, to C. Bazaud for secretarial assistance, and M. Delauche for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by INSERM, the Association pour la Recherche sur le Cancer, and the Arthritis and Rheumatism Campaign, United Kingdom.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.
§ To whom correspondence should be addressed. Tel.: 33-1-56-01-65-12; Fax: 33-1-56-01-62-17. E-mail: remi.piedagnel{at}tnn.ap-hop-paris.fr.
The abbreviations used are: MMP, matrix metalloproteinase; APMA, p-aminophenyl mercuric acetate; AVP, arginine vasopressin; ECM, extracellular matrix; EGF, epidermal growth factor; GAPDH, glyceraldehyde phosphodehydrogenase; TIMP, tissue inhibitor of matrix metalloprotainase; ts, temperature-sensitive; PBS, phosphate-buffered saline; AP-2, activator protein-2.
2 M. L. Cittanova, unpublished results.
3 F. Le Goas and R. Piedagnel, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|