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T3–1 Cells: Evidence for Cross-Talk between PKA and Protein Kinase C Pathways1
Endocrinologie Cellulaire et Moléculaire de la Reproduction, Université Pierre et Marie Curie, Unité de Recherche Associeé au Centre National de la Recherche Scientifique (URA CNRS) 1449, Paris, France; Department of Medicine, University of Bristol, Dorothy Crowfoot Hodgkins Laboratories, BS2 8HW Bristol, United Kingdom; and Friedrich Miescher Institute, 4002 Basel, Switzerland
Address all correspondence and requests for reprints to: Dr. Raymond Counis, Endocrinologie cellulaire et Moléculaire de la Reproduction, Université Pierre et Marie Curie, Unité de Recherche Associeé au Centre National de la Recherche Scientifique (URA CNRS) 1449, Case 244, 75252 Paris cedex 05, France. E-mail: Raymond.counis{at}snv.jussieu.fr
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Abstract |
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T3–1 cells). Western blot analysis, using specific immunoaffinity
purified immunoglobulins, revealed expression of catalytic (Cat) and
regulatory type I (RI) and type II (RII) subunits of PKA in these
cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC
with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at
0.5–1 h) and concentration-dependent loss of all PKA subunits.
Forskolin (10–100 µM) reduced Cat and RI by 60% and RII
by 30%, whereas TPA (0.1–1 µM) reduced Cat and RII by
50% and RI by 40%. Simultaneous activation of PKA and PKC caused the
expected dose-dependent reductions in Cat, and the effects of forskolin
or TPA were nearly additive. RI and RII were reduced similarly by 10
nM TPA, whereas 100 nM TPA tended to prevent
the reduction of RI or RII caused by forskolin. GnRH, which activates
phosphoinositidase C and not AC in these cells, caused a clear loss of
Cat or RII at all concentrations tested and of RI at 0.1
nM. Pituitary adenylate cyclase-activating polypeptide 38,
which acts via PVR-1 receptors to stimulate both phosphoinositidase C
and AC in these cells, also caused a clear dose-dependent decrease in
Cat, RI, and RII, although higher concentrations were needed for the
latter effects. Together, the data demonstrate that catalytic and
regulatory subunits of PKA are subject to both hormonal and
receptor-independent regulation in T3–1 cells, reinforcing the
possibility that such effects occur in nonimmortalized gonadotropes.
Whereas the effects of PKA activators very likely involve proteolytic
degradation of the dissociated PKA holoenzyme, the effects of TPA and
GnRH occur in the absence of cAMP elevation by unknown mechanisms.
Whatever the mechanisms involved, the data reveal a mechanism for
cross-talk between phosphoinositidase C and AC-mediated hormonal
signals, in which PKC activation seems to play a pivotal role. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell line
(14). Although GnRH is clearly the major hormonal regulator of gonadotrophs, these cells are also targets for a number of other local or hormonal factors. The latter include pituitary adenylate cyclase activating polypeptide (PACAP), a hypophysiotrophic factor that was first purified from ovine hypothalami using, as a bioassay, its ability to stimulate cAMP accumulation in pituitary cell cultures (15). In the pituitary, PACAP acts predominantly via PVR-1 receptors (16). These are G protein-coupled receptors that bind the two major endogenous forms of PACAP (PACAPs 27 and 38) with comparable affinity (but have much lower affinity for vasoactive intestinal polypeptide) and mediate stimulation by PACAP of both adenylyl cyclase (AC) and PIC (17, 18, 19).
The demonstration that gonadotrophs are direct targets for PACAP action
(14, 20, 21) provides two novel perspectives for consideration of cAMP
signaling in these cells. First, it seems likely that PACAP, rather
than GnRH, is the major stimulus for cAMP production in gonadotropes
and that earlier studies involving PKA activation were mimicking the
action of PACAP (rather than that of GnRH); and second, the probable
coincident activation of AC and of PIC raises the question of the
nature and functional consequences of cross-talk between these
signaling pathways. In this context, we recently have shown that GnRH
inhibits PACAP-stimulated cAMP accumulation in T3–1 cells, an
effect that can be mimicked by PKC-activating phorbol esters and
apparently reflects inhibition of AC, rather than activation of
phospho-diesterase (22).
PKA, the major cellular target for cAMP, is a heterotetrameric protein
containing two regulatory and two catalytic subunits (23, 24). Four
major isoforms of the regulatory subunits (RI, RIß, RII, and
RIIß) and three major isoforms of the catalytic subunits (Cat,
Catß, and Cat
) exist, having distinct patterns of distribution
between cell types and within cells (25, 26). In the rat pituitary,
Northern blotting has revealed the expression of all - and
ß-isoforms of RI, RII, and Cat (27); and Western blotting (using
antibodies that do not distinguish the - and ß-isoforms) has
revealed the presence of the corresponding RI, RII, and Cat proteins
(28). Binding of cAMP to the regulatory subunits of PKA reduces their
affinity for the catalytic subunits that are released and activated. In
several cell types, PKA is subject to regulation (by hormones or by
cAMP itself) of PKA subunit transcription or degradation (29, 30, 31).
Indeed, PKA activation is very often associated with a reduction in
cellular levels of the catalytic subunit, because liberation from the
holoenzyme exposes it to proteases (32), and a membrane protease has
been described that specifically degrades the free (dissociated)
catalytic subunit (33).
In rat pituitary cell cultures, 8-Br-cAMP causes a marked dose- and
time-dependent loss of catalytic subunit (28), with a less pronounced
reduction in RI and increase in RII. These changes presumably reflect
alterations in both degradation and synthesis, but because the loss of
catalytic subunit is associated with an increase in Cat mRNA (27),
the loss of protein is most likely caused by proteolysis. Surprisingly,
the effects of 8-Br-cAMP on catalytic subunit levels were mimicked by a
PKC-activating phorbol ester and by GnRH (28), raising the possibility
that PKC activation by GnRH might enable the releasing hormone to
influence cAMP signaling by altering cellular levels of this effector
molecule. However, these studies leave a number of key questions
unresolved. It is not clear, for example, whether the effects of GnRH
or tetradecanoyl phorbol acetate (TPA) were caused by elevation of cAMP
or to what extent the observed changes occurred in gonadotropes
(because the work was performed using a heterologous cell population
containing approximately 10% gonadotropes). The current study was
therefore undertaken to establish whether PKA subunit levels are
subject to direct regulation by receptor-mediated and/or
receptor-independent stimulators of AC or PIC in T3–1 cells.
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Materials and Methods |
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T3–1 cells were cultured in
serum-supplemented DMEM, as described (14, 34). For experiments, T75
flasks of cells at 50–75% confluence were washed in a physiological
salt solution (PSS) containing 127 mM NaCl, 1.8
mM CaCl2, 5 mM KCl, 2
mM MgCl2, 0.5 mM
NaH2PO4, 5 mM NaHCO3,
10 mM glucose, 0.1% BSA, and 10 mM HEPES (pH
7.4). They were then incubated for varied periods at 37 C in PSS
supplemented with forskolin, PMA, or peptides, as indicated. After
incubation, the cells were washed in PSS at 4 C and processed as
described below.
Preparation of cell extracts and Western blotting
Preparation of cell protein extracts and Western blotting
analysis of PKA subunits were performed essentially as previously
described (28). Cells were lysed by successive freezing/thawing cycles
in 10 mM Tris-HCl, pH 7.4, containing 2 mM
EDTA, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, and 20 µg/ml leupeptine, then scraped off the plate and
thoroughly homogenized in a Potter homogenizer (Poly Labo, Strasbourg,
France). The homogenates were centrifuged for 45 min at 37,000 x
g at 4 C, the supernatant collected, and protein
concentration determined according to the method of Bradford (35).
Protein extracts (15 µg) were subjected to slab gel electrophoresis (36) using a 10% polyacrylamide-separating gel and a 4.5% polyacrylamide-stacking gel. Colored protein molecular weight markers (Rainbow marker, Amersham, Arlington Heights, IL) were coelectrophoresed.
After electrotransfer onto nitrocellulose membrane (Hybond-ECL 0.45-µm pore size; Amersham), RI, RII, or Cat subunits were immunodetected using specific, affinity-purified antibodies (32, 37) at dilutions 1:200 for RI or RII and 1:500 for Cat and the Enhanced Chemiluminescence System (ECL-Western blotting system, Amersham). Blots were exposed to Kodak XAR-5 films (Eastman Kodak, Rochester, NY).
Data analysis
Western blots were analyzed with a computer image processing
system (COHU high performance CCD camera and One-Dscan software,
Scanalytics, Billeria MA). Data are expressed as percentage of
appropriate control and are mean ± SEM of three
separate experiments, typically with three replicates for each
experimental group. Differences between means were assessed by ANOVA,
followed by Dunnett’s t test. P 
0.05 was
considered significant.
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Results |
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T3–1 cellsT3–1 cells. As shown on Fig. 1
, using our specific, affinity-purified antibodies, all
three subunits (Cat, RI, and RII) were detected in these cells as
single bands with apparent mol wts of 40–41, 51, and 52–54K,
respectively, as observed previously in several other tissues
[including rat pituitary (28)]. In each case, the revealed material
represents total subunit present in cells, because the - and
ß-isoforms do not resolve on SDS-gels and the antibodies used do not
distinguish between these forms (Hemmings, unpublished data). The most
intense bands were routinely obtained for the catalytic subunit, as
expected from the fact that in many cells, total regulatory and
catalytic subunits are present with a 1:1 stoichiometry (24).
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shows the effects of forskolin (Fig. 2A)
or phorbol ester TPA (Fig. 2B) on PKA subunits after a 6-h incubation.
As shown, forskolin and TPA both induced marked concentration-dependent
and subunit-specific decreases in the levels of all three subunits.
Maximally effective concentrations of forskolin (10 and 100
µM) reduced Cat and RI by approximately 60% and reduced
RII by approximately 30%, whereas maximally effective concentrations
of TPA (100–1000 nM) reduced Cat and RII by 50% and RI by
approximately 40%.
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shows the time-course of changes in PKA subunit
in cells stimulated with 2 µM forskolin or 50
nM TPA. Control values determined at each time point did
not differ significantly and were used for the normalization of data.
As shown, all three subunits declined rapidly after addition of either
forskolin or TPA. In each case, the decline in Cat and RII was
particularly rapid, with minimum levels being reached essentially
within 1 h of stimulation. This reduction was typically followed
by an increase after 1–3 h.
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T3–1 cells
were incubated for 6 h in the presence of graded concentrations of
forskolin and either 10 nM or 100 nM TPA.
Although PKC activation has been shown to alter cyclic nucleotide
signaling in many systems, we have shown that forskolin-stimulated cAMP
accumulation is unaltered by TPA in these cells (22). As shown (Fig. 4), forskolin and TPA both caused the expected
dose-dependent reduction in Cat, and the effects of these stimuli were
nearly additive. Similarly, forskolin and 10 nM TPA had
approximately additive effects on RI and RII levels, but the
interaction between forskolin and 100 nM TPA was more
complex. In the presence of 100 nM TPA, RI was measurably
reduced only by the highest concentrations of forskolin and was
actually increased (30%) by 1 µM forskolin. Similarly,
in the presence of 100 nM TPA, RII was reduced only by the
highest concentrations of forskolin. Overall, the tendency was for
additivity between 10 nM TPA and forskolin but for
inhibition, by 100 nM TPA, of the reduction caused by
forskolin.
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T3–1 exposure to GnRH
(which activates PIC and not AC in these cells) and PACAP 38 (which
activates both PIC and AC in these cells) was examined. As shown (Fig. 5), GnRH caused a clear loss of Cat or RII at all
concentrations tested (maximum decline by 30 and 45%, respectively).
In contrast, the RI level was decreased only at 0.1 nM (by
36%) and was unaffected at higher concentrations. The effects of PACAP
38, PACAP 27, and VIP on PKA subunit levels are shown in Fig. 6. PACAP 38 caused a clear dose-dependent loss of Cat,
reaching 70% at the highest concentration tested. In contrast, RI and
RII levels were essentially unaffected at concentrations under 10
nM and were decreased (40–50%) by 100 and 1000
nM PACAP 38. PACAP 27 and VIP were approximately as
effective as PACAP 38 at 1000 nM, except for Cat, which was
reduced only 40%.
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), GnRH had most pronounced effects on Cat and
RII (these are essentially maximal after 1 h incubation, with a
tendency for elevation thereafter); 10 nM GnRH had no
significant effect on RI. In contrast to GnRH, PACAP 38 caused a marked
decrease in all the PKA subunits, although its effects were somewhat
slower in onset because maximal loss of each subunit was not observed
until 6 h of stimulation with PACAP 38.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells) to
address this possibility more directly. These cells were chosen for
this study as the only model available to us in which the effects of
the stimuli used could be assumed to be exerted directly upon
GnRH-responsive cells and because these cells express both GnRH
receptors and PACAP receptors (PVR-1), enabling receptor-mediated
activation of PIC with or without concomitant activation of AC.
In our first series of experiments, we established by Western blotting
that T3–1 cells, like rat pituitary cells, express catalytic and
regulatory (RI and RII) subunits of PKA, and this was confirmed by
Northern blotting, which revealed the expression of the mRNA encoding
the -isoform of the Cat and the ß-isoform of RII (not shown). In
these cells, activation of PKA by stimulation with forskolin caused a
dose- and time-dependent reduction in each of the subunits (Cat, RI,
and RII). This effect is similar to that previously observed using
8-Br-cAMP to stimulate PKA in primary cultures of rat pituitary cells
(28) and supports the interpretation that cellular levels of PKA
subunits are regulated in gonadotropes. In other systems, dissociation
of the regulatory and catalytic subunits has been shown to make these
available for proteolysis (25, 32). Because the reduction in subunits
must reflect the net effect of any alterations in degradation and
synthesis, and 8-Br-cAMP is known to alter subunit mRNA in primary
cultures of pituitary cells (27), we have also assessed possible
regulation of Cat and RIIß mRNA levels in T3–1 cells (not
shown) but found that treatment for 6 h with 2 mM
8-Br-cAMP did not measurably alter Cat or RIIß mRNA (92 ± 10
and 87 ± 8% of control, respectively). Although we cannot
exclude the possibility of posttranscriptional regulation (or indeed,
of transcriptional regulation of other subunits), these data, together
with the rapidity of the response to forskolin (maximal reduction
within 60 min of stimulation), support cAMP-induced holoenzyme
dissociation and subsequent proteolytic degradation as the most likely
explanation of the observed effects of forskolin. The observation that
PKC activation by TPA caused a similar dose- and time-dependent
reduction in each of the subunits measured is, however, less easily
explained. Again the response was rapid (maximal reduction of Cat at 60
min), and 6 h exposure to 50 nM TPA did not measurably
alter Cat or RIIß mRNA (106 ± 11 and 97 ± 8% of
control, respectively), but TPA does not increase cAMP levels in these
cells (22). The TPA effect, therefore, cannot reflect cAMP-induced
dissociation of the holoenzyme as shown in other systems (38) but
implies instead, either: 1) that TPA causes holoenzyme dissociation
without increasing cAMP; 2) that TPA stimulates subunit proteolysis
without holoenzyme dissociation; or 3) that subunit synthesis can be
rapidly reduced without alterations in steady-state levels of Cat
and RIIß mRNA. Irrespective of the mechanism involved, this effect of
TPA indicates a novel means by which PIC-activating ligands might
influence signaling through AC-coupled receptors.
When the effects of combined stimulation with forskolin and TPA were assessed, complex patterns of dose-dependent and subunit-specific interactions were observed. For the catalytic subunit, the reductions caused by 10 nM and 100 nM TPA were essentially additive with the effects of forskolin. The fact that the maximal effect of forskolin on Cat levels was further enhanced by addition of TPA is indicative of distinct mechanisms, as would be expected if, for example, forskolin had caused dissociation-induced proteolytic loss of Cat and TPA had caused a dissociation-independent proteolysis. For the regulatory subunits, effects of 10 nM TPA and forskolin were approximately additive, whereas the higher concentration of TPA prevented the inhibitory effects of 0.1–10 µM forskolin. The reasons for these effects are unknown, but they may, of course, be related to a TPA-induced loss of PKC (39) or to combined effects of TPA and forskolin on subunit synthesis, as well as degradation.
Having established that receptor-independent regulation of PKA subunits
occurs in T3–1 cells, we continued to test for hormonal regulation
of subunit expression. We observed that GnRH and PACAP are both able to
cause dose- and time-dependent reductions in each of the subunits
measured, providing the first demonstrations that hormonal regulation
of PKA subunit levels occurs in GnRH-responsive pituitary cells and
that PACAP is able to regulate its own effector system. In general, the
effects of PACAP were more pronounced than those of GnRH
(e.g. 30% and 70% reductions in Cat caused by 100
nM GnRH and 1000 nM PACAP 38, respectively),
but the effects of GnRH were more rapid in onset (e.g.
effects of GnRH on Cat and RII were maximal at 60 min, whereas the
effects of PACAP 38 on all subunits were maximal at 6 h). The
effects of PACAP 38 on these cells are exerted primarily via PVR-1
receptors, which bind the two endogenous forms of PACAP (PACAP 38 and
27) with comparable affinity, and which bind VIP with much lower
affinity. It was therefore not unexpected that the effects of PACAP 38
were mimicked by 1000 nM PACAP 27 or VIP, because all three
stimuli can increase cAMP accumulation by 10- to 100-fold in these
cells (14). It is interesting, however, to note that whereas forskolin
caused parallel dose-dependent reduction of all three subunits, low
concentrations of PACAP 38 (0.1–10 nM) reduced Cat without
measurably altering RI or RII. This may reflect the difference in
efficiency of coupling of PVR-1 receptors to AC and PIC. In T3–1
cells, PACAP 38 stimulates cAMP accumulation with an EC50 of
approximately 3 nM, and IP accumulation with an EC50 of
approximately 20 nM (14). The pronounced reduction in RI
and RII seen between 10 and 100 nM PACAP 38 (Fig. 6
),
therefore, probably is not associated with any major increase in PKA
activation but might, instead, reflect the additional activation of
PIC. If so, this would imply that the two signaling pathways activated
by PVR-1 receptors in these cells act cooperatively to regulate PKA
subunit expression. One intriguing consequence of the differential
regulation of subunits by PACAP 38 is an imbalance between the
catalytic and regulatory subunits. In most cells, the stoichiometry of
catalytic subunits to (total) regulatory subunits is 1:1 (24), although
deviation from this ratio has been observed in a number of tumor cell
lines (40). As shown in Fig. 6, 10 nM PACAP 38 reduced the
catalytic subunit to approximately 50% of control without measurably
altering RI and RII and, therefore, must have increased the ratio of
regulatory to catalytic subunits. In other systems, overexpression of
regulatory subunits has been shown to dramatically inhibit effects of
AC-coupled receptors (29), implying that PACAP may cause
desensitization of AC signaling by reducing both the amount of
catalytic subunit available and the proportion of that subunit that is
dissociated on activation of AC. It remains to be seen, however,
whether such changes have important effects on hormonal regulation of
these cells.
Because GnRH activates PKC and does not increase cAMP in these cells
(22), the effects of this hormone are very likely mediated by PKC
activation rather than by cAMP-induced holoenzyme dissociation. The
alternative possibility, that the GnRH effects are mediated by
elevation of cytosolic Ca2+, seems unlikely, because we
know of no system in which Ca2+ elevation alters PKA
subunit levels, and because GnRH and PACAP had dissimilar effects on
PKA subunit levels (Fig. 7) but have very similar effects on cytosolic
Ca2+ concentration in these cells (14).
In summary, we have shown that the catalytic and regulatory subunits of
PKA expressed in T3–1 cells are subject to regulation by hormonal
and pharmacological activation of PKA and PKC, reinforcing the
possibility that such effects also occur in nonimmortalized
gonadotropes (28). Whereas the effects of PKA activators very likely
involve proteolytic degradation of the dissociated PKA holoenzyme (26, 29), the effects of TPA and GnRH occur in the absence of cAMP elevation
by completely unknown mechanisms. Irrespective of the mechanism
involved, it is of interest that GnRH and TPA are both able to reduce
PKA subunit levels and PACAP-stimulated cAMP production in these cells,
because this implies a key role for PKC in cross-talk between the PIC
and AC signaling systems in gonadotropes. The observations indicate
that activation of PKC by GnRH enables the releasing hormone to
modulate PACAP action by coordinated inhibitory effects on cAMP
generation and action.
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Footnotes |
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Received December 23, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 pituitary gonadotrope
cells. Endocrinology 136:1361–1366[Abstract]
T3–1 cell line. Endocrinology 134:315–323T3–1 cells.
Endocrinology 134:2599–2605- and ß-isoforms of cyclic
adenosine 3',5'-monophosphate-dependent protein kinase subunits present
in the anterior pituitary. Regulation of RIIß and C gene
expression by the cyclic nucleotide and phorbol ester. Endocrinology 133:1010–1019This article has been cited by other articles:
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 M. Hezareh, W. Schlegel, and S. R. Rawlings Stimulation of Ca2+ influx in alpha T3-1 gonadotrophs via the cAMP/PKA signaling system Am J Physiol Endocrinol Metab, November 1, 1997; 273(5): E850 - E858. [Abstract] [Full Text] [PDF]
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), RI
(
), and RII (
), the intensity of bands on the autoradiographs was
determined by densitometry. The figure shows data pooled from three
separate experiments (mean ± SEM) and normalized as a
percentage of the internal control group incubated without forskolin or
TPA. *, P 
