Molecular and Cellular Endocrinology 175 (2001) 141– 148
www.elsevier.com/locate/mce
Extracellular matrix components regulate ACTH production and
proliferation in corticotroph tumor cells
Florian Kuchenbauer a, Ursula Hopfner a, Johanna Stalla a, Eduardo Arzt b,
Günter K. Stalla a, Marcelo Páez-Pereda a,*
b
a
Max Planck Institute of Psychiatry, Department of Endocrinology, Kraepelinstrasse 10, 80804 Munich, Germany
Laboratory Fisiologı́a y Biologı́a Molecular, FCEN, Uni6ersidad de Buenos Aires and CONICET, Ciudad Uni6ersitaria, Pabellon II,
1428 Buenos Aires, Argentina
Received 22 August 2000; accepted 18 December 2000
Abstract
The extracellular matrix (ECM) conveys signals through membrane receptors called integrins producing changes in cell
morphology, proliferation, differentiation and apoptosis. Previous studies suggest that the ECM plays an important role in
pituitary physiology and tumorigenesis. In the present work we studied for the first time the effects of fibronectin, laminin,
collagen I and collagen IV on hormone secretion and cell proliferation in the corticotroph tumor cell line AtT-20 and in normal
pituitary cells, examining the signal transduction mechanisms that mediate these effects. ACTH production in AtT-20 cells was
inhibited by fibronectin, laminin and collagen I. A reporter construct with the POMC promoter showed similar results, indicating
that the effects of the ECM take place at the level of POMC gene transcription. In contrast, ACTH production was not
significantly altered in normal pituitary cells. AtT-20 cell proliferation was stimulated by collagen IV and fibronectin, but inhibited
by collagen I and laminin. In parallel, the cell morphology was modified by the ECM. We found that the production of reactive
oxygen species mediate the effects of laminin and collagen IV. On the other hand, the effect of fibronectin was mimicked by
b1-integrin and Rho activation. These results show for the first time that the ECM controls ACTH biosynthesis and proliferation
in corticotroph tumor cells and suggest a role for the ECM in corticotroph adenoma development. © 2001 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: ACTH; Corticotroph cells; Extracellular matrix; Integrins; POMC; Reactive oxygen species
1. Introduction
The extracellular matrix (ECM) provides cells with
information essential to control general and cell typespecific functions (Lin and Bissell, 1993; Lukashev and
Werb, 1998). Components of the ECM such as laminin,
fibronectin and collagens regulate cell proliferation, differentiation, morphogenesis and hormone production
(Hay, 1993; Lin and Bissell, 1993; Sites et al., 1996;
Bourdoulous et al., 1998; Lukashev and Werb, 1998).
In granulosa cells, for example, fibronectin, laminin,
collagen I and collagen IV increase FSH receptors and
progesterone production (Sites et al., 1996). These
* Corresponding author. Tel: +49-89-30622272; fax: +49-8930622605.
E-mail address: [email protected] (M. Páez-Pereda).
ECM components also regulate the proliferative response of mammary epithelial cells to steroid hormones
(Xie and Haslam, 1997). It has also been demonstrated
that laminin promotes differentiation in fetal pancreatic
cells and human colon adenocarcinoma cells (De Arcangelis et al., 1996; Jiang et al., 1999).
The effects of the ECM are mainly mediated by
surface receptors called integrins, which trigger different cellular responses (Hynes, 1992; Giancotti and Ruoslahti, 1999). Single ECM components are able to
bind to different integrins (Hynes, 1992). Integrins are
formed by different a- and b-subunits, producing more
than twenty different integrin combinations (Hynes,
1992). During ECM signal transduction, integrins activate in parallel multiple downstream effectors following
a complex system of signal integration (Giancotti and
Ruoslahti, 1999; Giancotti, 2000). Integrin signaling
0303-7207/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 3 9 0 - 2
142
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
involves changes in the cell cytoskeleton as well as
GTPase and kinase pathways (Lukashev and Werb,
1998; Giancotti and Ruoslahti, 1999; Giancotti, 2000).
For example, Rho and Rac, members of the small
GTPase family, control the structure of the actin cytoskeleton and cell morphology (Nobes and Hall, 1995;
Hall, 1998; Schoenwaelder and Burridge, 1999). The
cell morphology, in turn, regulates cell proliferation
and apoptosis (Zhu and Assoian, 1995; Chen et al.,
1997). Recent studies have shown that changes in cell
morphology in human capillary cells control proliferation and apoptosis, suggesting that cell morphology
regulates fundamental cell functions (Chen et al., 1997).
A novel signaling pathway that mediates the effects of
ECM-induced morphological changes on gene transcription has been recently described (Kheradmand et
al., 1998). According to this mechanism, fibronectin
and integrins regulate the Rac small GTPase and determine fibroblast morphology. Rac, is coupled to the
NADPH oxidase complex, which generates reactive
oxygen species (ROS). ROS, in turn, activate the NFkB
transcription factor (Kheradmand et al., 1998). An
alternative pathway that transduces fibronectin and
integrin signals involves Rho; another member of the
small GTPase family (Cussac et al., 1996; Bourdoulous
et al., 1998; Clark et al., 1998; Hall, 1998; Hotchin et
al., 1999). This pathway can be activated in an integrinindependent way by lysophosphatidic acid (LPA) and
has been shown to control cell cycle progression in
CHO cells (Bourdoulous et al., 1998; Hall, 1998). These
signaling pathways have not been studied in pituitary
cells yet.
ECM and integrins are not only involved in physiological functions, but also in tumorigenesis. They can
modulate tumor cell proliferation and survival (Varner
and Cheresh, 1996; Giancotti and Ruoslahti, 1999;
Sethi et al., 1999). During cell immortalization, the
process of proliferation and survival becomes independent from integrin-mediated cell adhesion to the ECM.
This is one of the critical steps of tumor development
(Lukashev and Werb, 1998; Giancotti and Ruoslahti,
1999). During the process of pituitary adenoma development fibronectin isoforms are differentially expressed
(Farnoud et al., 1995). Pituitary adenoma cell transformation also correlates with alterations in b1-integrin
expression (Farnoud et al., 1996). a-integrins seem not
to be involved in pituitary tumorigenesis except for a3,
which is coexpressed with b1 and therefore probably
transduces signals through b1-activation (Farnoud et
al., 1996). In line with these results we have previously
shown that the expression of the protooncogene c-fos in
pituitary adenomas depends on the integrity of the
ECM (Páez Pereda et al., 1996). We have also recently
demonstrated that matrix metalloproteinase activity in
pituitary tumor cells regulates tumor cell proliferation
and hormone secretion (Páez Pereda et al., 2000a). All
together these results suggest a regulatory role for the
ECM in pituitary adenoma pathogenesis. On the other
hand, it has been shown that laminin, fibronectin and
collagen IV are present among the epithelial cells forming the Rathke’s pouch during pituitary development
(Horacek et al., 1993). This suggests that ECM proteins
could also be involved in the normal development and
differentiation of the pituitary gland. The functional
role of the ECM in pituitary cells remains, however,
largely unexplored except for the description of the
effects of laminin on prolactin and gonadotropin secretion (Denduchis et al., 1994). In the present work we
examine for the first time the effects of purified ECM
components on the corticotroph tumor cell line AtT-20
and on normal corticotroph cells and we characterize
the molecular and biochemical mechanisms that mediate these effects.
2. Materials and methods
2.1. Materials
Unless stated, all reagents were from Sigma (St.
Louis, MO), Roche Molecular Biochemicals (Penzberg,
Germany) or Pharmacia (Uppsala, Sweden). The monoclonal anti-b1-integrin activating antibody (Clone:
MAR4) was from BD Pharmingen (Heidelberg, Germany). This antibody has been shown to activate
CD4 + T cells (Tanaka et al., 1998).
2.2. Cell culture
Pituitary cell culture was performed as previously
described (Arzt et al., 1992). Pituitary glands were
obtained from adult male Sprague–Dawley rats (180–
250 g) after decapitation. The tissue was washed with
preparation buffer [137mM NaCl, 5mM KCl, 0.7 mM
Na2HPO4, 10mM glucose, 15mM HEPES (pH 7.3)].
Sliced fragments were dispersed in preparation buffer
containing 4 g/l collagenase (Cooper Biochemicals,
Malvern, PA), 10 mg/l DNAse II, 0.1 g/l soybean
trypsin inhibitor, and 1 g/l hyaluronidase. Dispersed
cells were centrifuged and resuspended in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with
2 mM essential vitamins, 5 mg/l insulin, 20 mg/l selenium, 5 mg/l transferrin, 30 pm triiodothyronine (Henning, Berlin, Germany), 10% fetal calf serum and 10 000
U/ml penicillin–streptomycin. Cell preparations with a
viability of at least 95%, as assessed by acridine orange–ethidium bromide staining, were distributed in
extracellular matrix-coated plates and incubated at
37°C under 5% CO2. Normal pituitary cell cultures
were used for a maximum of 24 h in order to avoid the
contribution of endogenous ECM produced by contaminating fibroblasts. This technical limitation precluded
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
the measurement of cell proliferation in normal pituitary cells. AtT-20, a corticotroph tumor cell line, was
obtained from the American Type Culture Collection
(Rockville, MD) and cultured in DMEM with 2 mmol/l
L-glutamine and 10 000 U/ml penicillin– streptomycin,
containing 10% fetal calf serum.
2.3. Culture plate coating
Culture plates were prepared by incubation with 5
mg/cm2 of each of the extracellular matrix components
(Becton Dickinson, Bedford, USA) dissolved in PBS.
After 1 h at 37°C, the plates were washed with PBS,
dried and stored at 4°C. These are standard conditions
to efficiently induce attachment, spreading, differentiation and proliferation in several cell types. Previous
studies demonstrated that cells can spread on ECMcoated plates in such a way to make maximum contact
with ECM components even if these components are
present in different amounts (Chen et al., 1997).
2.4. Hormone determination
For ACTH production studies, AtT-20 or rat pituitary cells were seeded on plates coated with different
matrix components and cultured in stimulation medium
(DMEM, pH 7.3, supplemented with 2 mM glutamine,
1 g/l BSA, and 30 mg/l ascorbic acid) for 24 h. For
hormone measurement the supernatants were collected
and ACTH was measured by RIA, as previously described (Arzt et al., 1992).
2.5. POMC-Luc transfection
AtT-20 cells were seeded in 10 cm Petri dishes and
incubated for 24 h. Then the cells were washed and a
mixture of optimem, lipofectamine (Gibco BRL, Life
Technologies, Karlsruhe, Germany) and 12,5 mg
POMC –Luc plasmid was added. This POMC reporter
construct consists of 770 basel pairs (bp) of the rat
promoter, which contains all the necessary sequences
for the correct expression and regulation of POMC in
mouse pituitary cells and 2.6 Kb of the luciferase
cDNA (Liu et al., 1992). After 24 h of incubation the
cells were trypsinized and seeded on six-well plates
coated with laminin, fibronectin, collagen I or collagen
IV. The cells were incubated for 24 h on the matrix
components, washed with PBS and lysed. The total
protein content was determined with the Bradford
method for normalization. Luciferase activity was measured with a Berthold luminometer (Berthold, Wildbad,
Germany).
2.6. Cell proliferation
After three days, 0.1 mCi/ml 3H-thymidine was added
143
to the cultures. After a further 24 h incubation, the
medium was removed and the cells treated with 10%
trichloroacetic acid. 3H-thymidine was measured by
liquid scintillation as previously described (Arzt et al.,
1993; Páez Pereda et al., 2000b). Alternatively, cell
proliferation was measured by the WST-1 proliferation
assay (Roche Molecular Biochemicals, Penzberg, Germany) as previously described (Páez Pereda et al.,
2000a). This assay measures the activity of the mitochondrial succinate dehydrogenase and is similar to the
MTT assay. After 4 days the WST-1 reagent was added
to the cultures to a final dilution of 1:10, which were
incubated for 2 h and then measured with an ELISA
reader at 440 nm according to the manufacturer’s
instructions.
2.7. H2O2 measurement
AtT-20 cells resuspended in DMEM were seeded in
plates coated with 5 mg/cm2 extracellular matrix
proteins as indicated. At different times samples were
collected for H2O2 measurement. H2O2 levels were determined using a colorimetric assay based on the oxidation of ferrous ions and xylenol orange complex
formation following the manufacturers instructions
(R&D Systems, Wiesbaden, Germany). 30% H2O2 was
used to obtain a standard calibration curve. Absorbance was measured at 560 nm with an ELISA plate
reader.
2.8. Statistical analysis
Statistics were performed using one-factor ANOVA
in combination with Scheffe’s test.
3. Results
3.1. Extracellular matrix regulates ACTH secretion
We chose standard conditions that effectively promote cell proliferation, differentiation and spreading in
many different cell types (Lamoureux et al., 1992),
because attachment of cells to ECM substrates does not
follow equilibrium dynamics and the biological responses are not linear. Therefore, to examine the effects
of ECM components on ACTH production, we cultured AtT-20 cells on plates coated with 5 mg/cm2
fibronectin, laminin, collagen I and collagen IV. ACTH
levels were measured by RIA in AtT-20 and normal rat
pituitary cell supernatants after 24 h treatment. Fibronectin, laminin and collagen I produced a statistically significant inhibition of ACTH production in
AtT-20 cells (Fig. 1A). Controls with the WST-1 assay
performed at 24 h indicated no differences in viable cell
numbers, demonstrating that the inhibition was not due
144
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
to differences in cell adhesion, proliferation or cytotoxicity (not shown). To study whether these effects take
place at the level of transcription we transfected AtT-20
Fig. 2. ECM components regulate AtT-20 cell proliferation. AtT-20
cells were cultured on plates coated with 5 mg/cm2 ECM-components
as indicated: control (C), fibronectin (FN), laminin (LN), collagen I
(C I), collagen IV (C IV). After 4 days treatment, cell proliferation
was measured by 3H-thymidine incorporation as described in Section
2.6. *, PB 0.01 as determined by ANOVA in combination with
Scheffe’s test. Similar results were obtained by total cell count and the
WST-1 assay.
cells with a reporter construct consisting of the luciferase gene under the control of the POMC promoter
(Liu et al., 1992) and then measured the luciferase
activity in response to the different ECM components.
In agreement with the ECM effects on ACTH production, POMC–Luc expression was significantly inhibited
by fibronectin, laminin, and collagen I (Fig. 1B). However, in normal rat pituitary cells the ECM components
did not significantly affect ACTH production (Fig. 1C).
Therefore, ECM components do not have an effect on
ACTH secretion in the normal rat pituitary in contrast
to tumor AtT-20 cells.
3.2. Extracellular matrix components regulate AtT-20
cell proliferation
Fig. 1. Regulation of ACTH secretion by ECM components. AtT-20
cells were cultured for 24 h at a density of 3 × 105 cells/well on six
well plates coated with 5 mg/cm2 ECM components as indicated:
control (C), fibronectin (FN), laminin (LN), collagen I (C I), collagen
IV (C IV). (A) ACTH production in AtT-20 cells. Supernatants were
collected after 24 h and ACTH measured by RIA as described in
Section 2.4. (B) Regulation of POMC –Luc expression by ECM
components. AtT-20 cells were transfected with a POMC –Luc plasmid and luciferase activity was measured as described in Section 2.5.
(C) ACTH production in normal rat pituitary cells. Rat pituitary cells
were cultured for 24 h in 96-well plates at a density of 15 ×103
cells/well. The supernatants were collected and ACTH was measured
as described. *, P B 0.01 as determined by ANOVA in combination
with Scheffe’s test. These results are representative of three independent experiments.
To examine the possible effects of the ECM components on corticotroph cells we used the corticotroph
tumor cell line AtT-20 and measured cell proliferation
by 3H-thymidine incorporation. An increase in AtT-20
cell proliferation was induced by fibronectin and collagen IV after 4 days (Fig. 2). Collagen I and laminin,
however, produced an inhibitory effect (Fig. 2). Total
cell counts and the WST-1 proliferation assay after 4
days of treatment showed similar results (not shown).
Effects due to differences in cell adhesion were ruled
out by determining the number of viable cells with
acridine orange and ethidium bromide at different time
points during the treatment. The comparison with normal cell proliferation was precluded by the technical
limitations of normal pituitary cell cultures in which
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
145
fibroblasts could synthesize ECM and obscure proliferation measurements.
3.3. Extracellular matrix induces morphology changes
in AtT-20 cells
AtT-20 cells had different ECM-dependent morphology and dispersion patterns. They spread and dispersed
more on a fibronectin substrate (Fig. 3B), whereas on
laminin or collagen I AtT-20 cells formed clusters and
adopted a round shape, clearly different from an apoptotic cell morphology (Fig. 3C and D). On a collagen
IV substrate AtT-20 cells had an intermediate shape
(Fig. 3E). Acridine orange and ethidium bromide staining as well as measurement by WST-1 assay at different
times (Fig. 4B and data not shown) ruled out the
possibility that the rounded cells would be undergoing
apoptosis.
3.4. Reacti6e oxygen species transduce extracellular
matrix signals
To examine the possible role of ROS in ECM signal
transduction in AtT-20 cells we measured H2O2 production under different ECM treatments. Laminin, collagen I and collagen IV strongly stimulated H2O2
production whereas fibronectin did not produce any
significant effect (Fig. 4A). To test whether ROS production mediates the effects of the ECM on proliferation we used N-acetyl-cystein (NAC), a scavenger of
oxygen radicals. NAC reversed the stimulation produced by collagen IV and partially reversed the inhibition produced by laminin (Fig. 4B). However, NAC did
Fig. 4. ROS mediate the effects of ECM on AtT-20 cell proliferation.
(A) AtT-20 cells were seeded on ECM coated plates as indicated:
control (C), fibronectin (FN), laminin (LN), collagen I (C I), collagen
IV (C IV). At different times H2O2 production was measured as
described in Section 2.7. The percentage of increase was calculated
for the different times as compared to the basal level 0.2 90.001 pmol
H2O2/2 ×105 cells ( = 100%). (B) AtT-20 cells were cultered on plates
coated with 5 mg/cm2 ECM components as indicated. 10 − 4 M
N-acetyl-cysteine (NAC) was added and proliferation measured after
four days by WST-1 assay. *, PB0.01; **, P B0.05 as determined by
ANOVA in combination with Scheffe’s test.
not significantly modify the effects of collagen I and
fibronectin suggesting that collagen I activates a parallel signaling pathway besides the production of ROS
(Fig. 4B). These results are in agreement with the ROS
increases induced by laminin and collagen IV, demonstrating that ROS production mediates some effects of
the ECM on proliferation. The fact that ROS can have
stimulatory or inhibitory effects on AtT-20 cell proliferation could be related to the different cell morphology
that these cells adopt under laminin or collagen IV
treatment (Fig. 3).
Fig. 3. AtT-20 cell morphology on different ECM substrates. AtT-20
cells were cultured for 48 h on plates coated with 5 mg/cm2 ECM
components as indicated. (A) AtT-20 cells on plastic, (B) fibronectin,
(C) laminin, (D) collagen I, (E) collagen IV. Magnification 100 × .
3.5. Rho acti6ation stimulates AtT-20 cell proliferation
Fibronectin stimulates AtT-20 cell proliferation without inducing ROS production. Therefore, we studied
146
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
whether the activation of the Rho GTPase instead of
Rac, NADPH oxidase and ROS plays a part in the
control of corticotroph cell proliferation (Varner and
Cheresh, 1996; Clark et al., 1998). For this purpose, we
stimulated AtT-20 cells with 200 mg/ml LPA, which
activates Rho through an integrin-independent mechanism (Bourdoulous et al., 1998; Hall, 1998). LPA stimulated proliferation on its own whereas in combination
with fibronectin no further increase was observed (Fig.
5). This indicates a role for Rho in AtT-20 cell proliferation, which could be a possible mechanism for the
stimulatory effect of fibronectin (Varner and Cheresh,
1996; Clark et al., 1998).
3.6. Role of the i1 -integrin
Fibronectin, laminin and collagens bind to different
integrins composed of the b1-integrin, which is abundantly expressed in all types of pituitary adenomas
(Farnoud et al., 1996). To study the role of the b1-integrin in corticotroph cell proliferation we used a specific
activating antibody. The anti-b1-integrin activating antibody stimulated AtT-20 cell proliferation on its own
(Fig. 6). NAC did not reverse the stimulation produced
by the anti-b1-integrin antibody suggesting that the
effect of the anti-b1-integrin antibody is not mediated
by ROS (Fig. 6). Therefore, the stimulatory effect of
the anti-b1-integrin antibody is similar to the effect of
fibronectin, which stimulates proliferation without inducing ROS production. The combination of
fibronectin and the anti-b1-integrin antibody did not
further stimulate proliferation. These results indicate
that the b1-integrin can stimulate AtT-20 cell prolifera-
Fig. 5. Rho regulates AtT-20 cell proliferation. AtT-20 cells were
cultured on plastic plates (C) or plates coated with 5 mg/cm2
fibronectin (FN). After 24 h, 200 mg/ml lysophosphatidic acid (LPA)
was added. The cells were treated for further 3 days and then
proliferation was measured by WST-1 assay as described in Section
2.6. *, PB 0.01 as determined by ANOVA in combination with
Scheffe’s test.
Fig. 6. Regulation of AtT-20 cell proliferation by the b1-integrin.
AtT-20 cells were cultured on plastic plates (C) or plates coated with
5 mg/cm2 fibronectin (FN). After 24 h, 5 mg/ml anti-b1-integrin
antibody (b1-Ab) or 10 − 4 M N-acetyl-cysteine (NAC) were added.
After further 3 days proliferation was measured with the WST-1
reagent. *, PB 0.01 as determined by ANOVA in combination with
Scheffe’s test.
tion and suggest that the effect of fibronectin on AtT20 cell proliferation could be meditated by the
b1-integrin.
4. Discussion
In the present work we show that ECM components
induce functional and morphological changes in corticotroph tumor cells. We found that laminin, collagen I
and collagen IV inhibit ACTH biosynthesis at the level
of POMC gene transcription. On the other hand,
laminin and collagen I inhibited cell proliferation
whereas fibronectin and collagen IV had a stimulatory
effect. These effects probably involve two alternative
signal transduction pathways that include the Rho or
Rac small GTPases. The differential activation of Rho
or Rac and ROS production together with the corresponding morphological changes induced by the different ECM components result in different proliferation
rates. This model in which the ECM activates different
small GTPases and in parallel modifies cell morphology, gene transcription and proliferation is in line with
the models proposed for other cell types (Chen et al.,
1997; Kheradmand et al., 1998). Therefore our results
indicate that these mechanisms could also be extended
to some hormone-secreting epithelial cells.
Fibronectin and laminin have different localization in
pituitary adenomas as compared to the normal anterior
pituitary (Farnoud et al., 1994, 1995). We found that
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
fibronectin, laminin and collagen I inhibit ACTH
biosynthesis in the corticotroph tumor cell line AtT20. The effects on the POMC gene transcription at 24
h might indicate a progressive effect of the ECM on
ACTH biosynthesis. However, in normal rat pituitary
cells no significant changes were observed. This difference in the regulation of ACTH production could be
related to the differences in ECM and integrin expression between normal and adenomatous cells. Moreover, these results, together with the fact that
pituitary adenomas contain less laminin than normal
pituitary tissue (Farnoud et al., 1994), suggest the hypothesis that ACTH secreting adenomas might produce high levels of ACTH due, in part, to a
reduction of a laminin-induced inhibition.
On a fibronectin substrate AtT-20 cells are spread
and dispersed, whereas on laminin, collagen I and
collagen IV the cells are round and grow in clusters.
This indicates possible functional correlates between
substrate-induced changes in cell morphology and cell
function, similar to the ones occurring in endothelial
cells (Chen et al., 1997). Endothelial cells that adopt
different morphologies according to their interaction
with the substrate undergo either apoptosis or proliferation (Chen et al., 1997). Here we show that the
production of ROS in combination with different cell
morphologies produce different proliferative responses. Therefore, our results provide further support for this model. The production of ROS in
parallel with cell rounding has also been shown to
regulate gene transcription in fibroblasts (Kheradmand et al., 1998). In these cells, cell rounding occurs
in parallel with Rac activation, which in turn is coupled to an increase in ROS production by NADPH
oxidase. Granulosa cells have also been shown to respond differently in the presence of different ECM
components. In granulosa cells, ECM components
produce changes in the spreading pattern and cell
morphology (Sites et al., 1996). In the presence of
laminin, granulosa cells form clusters of round cells,
similar to the ones we observed in AtT-20 cells. In
the same model, ECM components regulate progesterone production and FSH receptor expression (Sites
et al., 1996). Our results provide, therefore, further
insight in the mechanisms connecting substrate-dependent cell morphology and the function of endocrine
cells.
Fibronectin, on the other hand, does not produce
any significant change in ROS production in AtT-20
cells. This fact together with the spread cell morphology on fibronectin suggest the involvement of an alternative signal pathway for the effects of fibronectin
on cell proliferation in AtT-20 cells (Clark et al.,
1998; Hotchin et al., 1999). Such a pathway could
involve the activation of the Rho GTPase instead of
Rac and NADPH oxidase, as demonstrated in other
147
cell types (Bourdoulous et al., 1998; Clark et al.,
1998; Hall, 1998; Hotchin et al., 1999). AtT-20 cells
treated with LPA showed a significant increase of
proliferation demonstrating that the activation of the
Rho GTPase can stimulate AtT-20 cell proliferation.
The fact that LPA in combination with fibronectin
does not have any further effect could indicate that
AtT-20 cells on fibronectin have already reached the
level
of
maximum
stimulation.
Alternatively
fibronectin might facilitate the entrance into the cell
cycle, through the phosphorylation of the EGF receptor (Moro et al., 1998). Our results show that
fibronectin and collagen IV significantly stimulate
AtT-20 cell proliferation. This suggests a role for
these ECM components in the progression of corticotroph adenomas. The anti-b1-integrin activating antibody also stimulates AtT-20 cell proliferation in
agreement with the stimulation produced by
fibronectin. However, this antibody does not further
stimulate proliferation in the presence of fibronectin.
This could suggest that fibronectin already produces a
maximal stimulation through b1-integrins. The proliferative action of b1-integrin is in agreement with the
fact that only this integrin is expressed in most cases
of pituitary adenomas (Farnoud et al., 1996). In line
with this, it has been shown that paracrine and autocrine factors such as transforming growth factor-b1
(TGF-b1), a cytokine suspected to be involved in corticotroph adenoma pathogenesis (Ray and Melmed,
1997; Asa and Ezzat, 1998; Arzt et al., 1999), can
regulate the expression of individual integrin subunits
in other cell types (Heino et al., 1989). This mechanism could contribute to the change of integrin expression and the consequent change in proliferation in
corticotroph adenomas.
In conclusion, we have found for the first time that
purified components of the ECM induce changes in
morphology, proliferation and hormone production in
a corticotroph tumor cell line. The signal transduction
mechanisms mediating these effects follow a complex
model that involves the activation of the b1-integrin,
the Rho GTPase, the production of ROS and the
regulation of the POMC gene transcription. Our results provide, therefore, a basis to understand the role
of the ECM during the progression of corticotroph
adenomas.
Acknowledgements
We would like to thank Y. Grübler for excellent
technical assistance and Dr P. Lohrer for revising the
manuscript for English usage. This work was supported by grants from the Volkswagen Foundation
(I/76 803) and the Deutsche Forschungsgemeinschaft
(Sta 285/7-3).
148
F. Kuchenbauer et al. / Molecular and Cellular Endocrinology 175 (2001) 141–148
References
Arzt, E., Stelzer, G., Renner, U., Lange, M., Müller, O.A., Stalla, G.K.,
1992. Interleukin-2 and interleukin-2 receptor expression in human
corticotrophic adenoma and murine pituitary cell cultures. J. Clin.
Invest. 90, 1944 – 1951.
Arzt, E., Buric, R., Stelzer, G., Stalla, J., Sauer, J., Renner, U., Stalla,
G.K., 1993. Interleukin involvement in anterior pituitary cell
growth regulation: effects of interleukin-2 and interleukin-6. Endocrinology 132, 459 –467.
Arzt, E., Páez Pereda, M., Perez Castro, C., Pagotto, U., Renner, U.,
Stalla, G.K., 1999. Pathophysiological role of the cytokine network
in the anterior pituitary gland. Front. Neuroendocrinol. 20, 71 – 95.
Asa, S., Ezzat, S., 1998. The cytogenesis and pathogenesis of pituitary
adenomas. Endocrine Rev. 19, 798 –827.
Bourdoulous, S., Orend, G., MacKenna, D.A., Pasqualini, R., Ruoslahti, E., 1998. Fibronectin matrix regulates activation of Rho
and CDC42 GTPases and cell cycle progression. J. Cell Biol. 143,
267 – 276.
Chen, C.S., Mrksich, M., Huang, S., Whitesides, G.M., Ingber, D.E.,
1997. Geometric control of cell life and death. Science 70, 1425 –
1428.
Clark, E.A., King, W.G., Brugge, J.S., Symons, M., Hynes, R.O., 1998.
Integrin-mediated signals regulated by members of the rho family
of GTPases. J. Cell Biol. 142, 573 –586.
Cussac, D., Leblanc, P., L%Heritier, A., Bertoglio, J., Lang, P., Kordon,
C., Enjalbert, A., Saltarelli, D., 1996. Rho proteins are localized
with different membrane compartments involved in vesicular
trafficking in anterior pituitary cells. Mol. Cell. Endocrinol. 119,
195 – 206.
De Arcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger,
M., Simon-Assmann, P., 1996. Inhibition of laminin alpha 1-chain
expression leads to alteration of basement membrane assembly and
cell differentiation. J. Cell Biol. 133, 417 – 430.
Denduchis, B., Rettori, V., McCann, S., 1994. Role of laminin on
prolactin and gonadotrophin release from anterior pituitaries of
male rats. Life Sci. 55, 1757 –1765.
Farnoud, M.R., Derome, P., Peillon, F., Li, J.Y., 1994. Immunohistochemical localization of different laminin isoforms in human
normal and adenomatous anterior pituitary. Lab. Invest. 70,
399 – 406.
Farnoud, M.R., Farhadian, F., Samuel, J.L., Derome, P., Peillon, F.,
Li, J.Y., 1995. Fibronectin isoforms are differentially expressed in
normal and adenomatous human anterior pituitaries. Int. J. Cancer
61, 27 – 34.
Farnoud, M.R., Veirana, N., Samuel, J.L., Derome, P., Peillon, F., Li,
J.Y., 1996. Adenomatous transformation of the human anterior
pituitary is associated with alterations in integrin expression. Int.
J. Cancer 67, 45 – 53.
Giancotti, F.G., Ruoslahti, E., 1999. Integrin signaling. Science 285,
1028 – 1032.
Giancotti, F.G., 2000. Complexity and specifity of integrin signalling.
Nature Cell Biol. 2, E13 –E14.
Hall, A., 1998. Rho GTPases and the actin cytoskeleton. Science 279,
509 – 513.
Hay, E., 1993. Extracellular matrix alters epithelial differentiation.
Curr. Opin. Cell Biol. 5, 1029 –1035.
Heino, J., Ignotz, R.A., Hemler, M.E., Crouse, C., Massague, J., 1989.
Regulation of cell adhesion receptors by transforming growth
factor-(. J. Biol. Chem. 264, 380 – 388.
Horacek, M.J., Thompson, J.C., Dada, M.O., Terracio, L., 1993. The
extracellular matrix components laminin, fibronectin, and collagen
IV are present among the epithelial cells forming Rathke’s pouch.
Acta Anat. 147, 69 – 74.
Hotchin, N.A., Kidd, A.G., Altroff, H., Mardon, H.J., 1999. Differen-
tial activation of focal adhesion kinase, Rho and Rac by the ninth
and tenth FIII domains of fibronectin. J. Cell Sci. 112, 2937 –2946.
Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling.
Cell 69, 11 – 25.
Jiang, F.X., Cram, D.S., DeAizpurua, H.J., Harrison, L.C., 1999.
Laminin-1 promotes differentiation of fetal mouse pancreatic beta
cells. Diabetes 48, 722 – 730.
Kheradmand, F., Werner, E., Tremble, P., Symons, M., Werb, Z., 1998.
Role of Rac1 and oxygen radicals in collagenase-1 expression
induced by cell shape change. Science 280, 898 – 902.
Lamoureux, P., Zheng, J., Buxbaum, R.F., Heidemann, S.R., 1992. A
cytomechanical investigation of neurite outgrowth on different
culture surfaces. J. Cell Biol. 118, 655 – 661.
Lin, C.Q., Bissell, M.J., 1993. Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 7, 737 – 743.
Liu, B., Hammer, G.D., Rubinstein, M., Mortrud, M., Low, M.J.,
1992. Identification of DNA elements cooperatively activating
proopiomelanocortin gene expression in the pituitary glands of
transgenic mice. Mol. Cell Biol. 12, 3978 – 3990.
Lukashev, M., Werb, Z., 1998. ECM signaling: orchestrating cell
behavior and misbehavior. Trends Cell. Biol. 8, 437 – 441.
Moro, J., Venturino, M., Bozzo, C., Silengo, L., Altruda, F., Beguinot,
L., Tarone, G., Defillipi, P., 1998. Integrins induce activation of
EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 17, 6622 – 6632.
Nobes, C.D., Hall, A., 1995. Rho, Rac, and Cdc 42 GTPases regulate
the assembly of multimolecular focal complexes associated with
actin stress fibers, lamellipodia, and filopodia. Cell 81, 53 –62.
Páez Pereda, M., Goldberg, V., Chervin, A., Carrizo, G., Molina, A.,
Andrada, J., Sauer, J., Renner, U., Stalla, G.K., Arzt, E., 1996.
Interleukin-2 (IL-2) and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants. Mol. Cell. Endcrinol.
124, 33 – 42.
Páez Pereda, M., Ledda, M.F., Goldberg, V., Chervin, A., Carrizo, G.,
Molina, H., Müller, A., Renner, U., Podhajcer, O., Arzt, E., Stalla,
G., 2000a. High levels of matrix metalloproteinases regulate proliferation and hormone secretion in pituitary cells. J. Clin. Endocrinol.
Metab. 85, 263 – 269.
Páez Pereda, M., Missale, C., Grübler, Y., Arzt, E., Schaaf, L., Stalla,
G.K., 2000b. Nerve growth factor and retinoic acid inhibit proliferation and invasion in thyroid tumor cells. Mol. Cell. Endocrinol.
167, 99 – 106.
Ray, D., Melmed, S., 1997. Pituitary cytokine and growth factor
expression and action. Endocrine Rev. 18, 206 – 228.
Schoenwaelder, S.M., Burridge, K., 1999. Bidirectional signaling
between the cytoskeleton and integrins. Curr. Opin. Cell Biol. 11,
274 – 286.
Sethi, T., Rintoul, R.C., Moore, S., MacKinnon, A.C., Salter, D.,
Choo, C., Chilvers, E.R., Dransfield, I., Donnelly, S.C., Strieter, R.,
Haslett, C., 1999. Extracellular matrix proteins protect small cell
lung cancer cells against apoptosis: a mechanism for small cell lung
cancer growth and drug resistance in vivo. Nat. Med. 5, 662 –668.
Sites, C.K., Kessel, B., LaBarbera, A.R., 1996. Adhesion proteins
increase cellular attachment, follicle-stimulating hormone receptors, and progesterone production in cultured porcine granulosa
cells. Proc. Soc. Exp. Bio. Med. 212, 78 – 83.
Tanaka, Y., Ogawa, M., Nishimura, Y., Matsushita, S., 1998. Efficient
induction of human CD4 + T cell lines with a self-K-ras-derived
peptide in vitro, using a mAb to CD29. Hum Immunol. 59,
343 – 351.
Varner, J.A., Cheresh, D.A., 1996. Integrins and cancer. Curr. Opin.
Cell Biol. 8, 724 – 730.
Xie, J., Haslam, S.F., 1997. Extracellular matrix regulates ovarian
hormone-dependent proliferation of mouse mammary epithelial
cells. Endocrinology 138, 2466 – 2473.
Zhu, X., Assoian, R.K., 1995. Integrin-dependent activation of MAP
kinase: a link to shape-dependent cell proliferation. Mol. Biol. Cell
6, 273 – 282.