Tissue Transglutaminase Directly Regulates Adenylyl Cyclase Resulting in Enhanced cAMP-response Element-binding Protein (CREB) Activation*
Janusz Tucholski
and
Gail V. W. Johnson
From the
Department of Psychiatry, University of Alabama at Birmingham,
Birmingham, Alabama 35294-0017
Received for publication, April 9, 2003
, and in revised form, May 10, 2003.
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ABSTRACT
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Tissue transglutaminase (tTG) is present in the human nervous system and is
predominantly localized to neurons. Treatment of human neuroblastoma SH-SY5Y
cells with retinoic acid results in increased tTG expression, which is both
necessary and sufficient for differentiation. The goal of the present study
was to determine whether tTG modulates the activation of the cyclic
AMP-response element (CRE)-binding protein, CREB, an event that likely plays a
central role in the differentiation of SH-SY5Y cells. SH-SY5Y cells stably
transfected with active wild type tTG, tTG without transamidating activity
(C277S), an antisense tTG construct that depleted the endogenous levels of
tTG, or vector only were used for the study. Treatment with forskolin, an
adenylyl cyclase activator, increased that activation-associated
phosphorylation of CREB, which was prolonged by tTG overexpression.
CRE-reporter gene activity was also significantly elevated in the tTG cells
compared with the other cells. The enhancement of CREB
phosphorylation/activation in the tTG cells is likely due to the fact that tTG
significantly potentiates cAMP production, and our findings indicate that tTG
enhances adenylyl cyclase activity by modulating the conformation state of
adenylyl cyclase. This is the first study to provide evidence of the mechanism
by which tTG may contribute to neuronal differentiation.
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INTRODUCTION
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Tissue transglutaminase
(tTG)1 is the most
ubiquitously expressed member of the transglutaminase family of proteins
(1). This protein exhibits at
least two distinct enzymatic activities as follows: (i) a
Ca2+-dependent transamidating activity that cross-links
proteins or incorporates polyamines into protein substrates
(2,
3); and (ii) GTPase activity
(46).
Tissue TG is present in the human peripheral and central nervous system and is
localized predominantly in neurons
(7,
8). In rat brain and spinal
cord, TG activity is highest in the late fetal stage
(8,
9). Additionally, tTG protein
levels and TG activity have been shown to be elevated in neurodegenerative
conditions such as Alzheimer's disease
(10,
11) and Huntington's disease
(12,
13).
However, the role of tTG in the pathological processes of these diseases is
not clear. In the nervous system, tTG has been postulated to play a role in
many different processes such as synaptic plasticity
(14,
15), release of
neurotransmitters (16),
long-term potentiation (17),
axonal regeneration (9), and
neuronal death and/or survival
(18). Tissue TG also clearly
plays a key role in neuronal differentiation
(19,
20). Indeed, tTG is both
necessary and sufficient for the neuronal differentiation of human
neuroblastoma SH-SY5Y cells
(20).
The cyclic AMP-response element (CRE)-binding protein, CREB, was originally
identified as a transcriptional activator that responds to cAMP and is
phosphorylated and activated by protein kinase A
(21). CREB belongs to the CREB
transcription factor family that, besides CREB, consists of the cAMP
modulatory protein (CREM) and activating transcription factor 1 (ATF1)
(22,
23). CREB has been postulated
to play a key role in the modulation of neuronal differentiation, neuronal
survival, and synaptic plasticity in the vertebrate nervous system
(24). Indeed, CREB
phosphorylation and CRE reporter gene activity are increased in cortical
neurons during specific periods of nervous system development
(25). Overexpression of CREB
in SK-N-BE cells leads to morphological alterations typical of neuronal
differentiation (26). In
contrast, overexpression of a dominant negative CREB construct induced a
round, poorly differentiated phenotype
(26). Furthermore, CREB has
been described as the first transcription factor to be activated in response
to an elevation in intracellular cAMP levels
(27).
Cyclic AMP has multiple, diverse effects on cellular processes such as gene
expression, cell cycle control, and cell survival-death decisions (for recent
reviews, see Refs.
2831).
Cyclic AMP also promotes neuronal survival in a manner independent of
neurotrophic factors
(3234).
Although cAMP has been shown to have other functions in the cell, the primary
mechanism by which it acts as a second messenger is by activation of protein
kinase A (35), and CREB is a
primary target of protein kinase A
(21). In a previous study
(20), we demonstrated that tTG
is required for development of the neuronal morphology of human neuroblastoma
SH-SY5Y cells. Indeed, treatment of SH-SY5Y cells with the transglutaminase
inhibitor monodansylcadaverine inhibited the retinoic acid-induced
differentiation of SH-SY5Y cells
(36). Although it is clear
that tTG is necessary for neuronal differentiation, the molecular mechanisms
by which tTG contributes to this process are unclear. Therefore, the goal of
the present study was to determine whether tTG modulates CREB
phosphorylation/activation, which likely plays a significant role in the
differentiation of SH-SY5Y cells
(37,
38). To address this question,
SH-SY5Y cells stably transfected with active wild type tTG, tTG without
transamidating activity (C277S), an antisense tTG construct that depletes
endogenous tTG, or vector only
(18,
20) were treated with
forskolin, an adenylyl cyclase activator
(39). Forskolin treatment
resulted in a significant enhancement of CREB phosphorylation that lasted
longer in the cells overexpressing tTG compared with the other cell lines.
Furthermore, CRE-reporter gene activity was also significantly elevated in the
tTG-overexpressing cells compared with the other cells. The enhancement of
CREB phosphorylation/activation in the tTG-overexpressing cells is likely due
to the fact that tTG significantly potentiates cAMP production, and our
findings indicate that tTG directly modulates adenylyl cyclase activity, most
likely by facilitating the conversion of adenylyl cyclase into a more active
conformation. This is the first study to provide evidence of the mechanism by
which tTG may contribute to neuronal differentiation.
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EXPERIMENTAL PROCEDURES
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Cell Culture and TreatmentsHuman neuroblastoma SH-SY5Y
stably expressing wild type tTG (tTG), tTG without transamidating activity due
to a point mutation within the active site (C277S), and an antisense tTG DNA
construct (anti-tTG) along with cells stably transfected with vector-only
pcDNA3.1 (SH/pc) were described previously
(18,
20). In some experiments we
used two different tTG cell lines, tTG-1 and tTG-2, and two different C277S
cell lines, C277S-1 and C277S-2. The majority of experiments were carried out
in the tTG-2 and C277S-2 cell lines, which are referred to as tTG cells and
C277S cells, respectively, throughout.
ImmunoblottingCells were washed twice with cold
phosphate-buffered saline, harvested in lysis buffer (50 mM Tris,
pH 7.5, 0.3 M NaCl, 5 mM EGTA, 1 mM EDTA, 1
mM sodium orthovanadate, 1 µM okadaic acid, 0.5%
Triton X-100, 0.5% Nonidet P-40, 0.1 mM phenylmethylsulfonyl
fluoride, and a 10 µg/ml final concentration each of aprotinin, leupeptin,
and pepstatin), and sonicated on ice. Lysates were clarified, and the protein
concentrations were determined using the bicinchoninic acid (BCA) assay
(Pierce). Lysates (10 µg) were electrophoresed on 10% SDS-polyacrylamide
gels, transferred to nitrocellulose, and probed with following antibodies:
anti-phospho (Ser133) CREB or anti-CREB from Cell Signaling
Technology (Beverly, MA); anti-tTG (TG100) from NeoMarkers (Fremont, CA);
anti-adenylyl cyclase (R-32); anti-G
s (K-20) from Santa Cruz
Biotechnology; anti-G
i (a gift from Dr. R. Jope)
(40); or anti-actin from
Chemicon International (Temecula, CA). After incubating with horseradish
peroxidase-conjugated secondary antibody, the immunoblots were developed using
enhanced chemiluminescence (Amersham Biosciences). Immunoblots were quantified
using a Bio-Rad model GS-670 densitometer.
Dual Luciferase AssayCells were grown in low serum
conditions with 1% Fetal Clone II (HyClone) and 4% horse serum to
30%
confluency prior to transient transfection with the pCRE-Luc reporter
construct (Stratagene) and pRL-TK vector as an internal control (Promega,
Madison, WI) at a 10:1 ratio. Thirty-six hours later, the cells were
transferred to serum free media for 3 h subsequent to incubation for 4 h with
10 µM forskolin. Luciferase levels were measured using the
Dual-Luciferase reporter assay system kit (Promega) and a TD-2021 luminometer
(Turner Designs, Sunnyvale, CA). The Firefly luciferase data was
normalized to the Renilla luciferase values for each sample.
Measurement of cAMP AccumulationThe levels of cAMP in
control and stimulated cells were determined by using an enzyme immunoassay
kit according to the manufacturer's protocol (Amersham Biosciences). Briefly,
104 cells/well were plated in low serum conditions with 1% Fetal
Clone II and 4% horse serum 12 h before being transferred to serum-free media
for 3 h prior to treatment. After each treatment, the cells were rinsed and
then incubated with the lysis reagent (0.5% dodecyltrimethylammonium bromide)
for 15 min at room temperature. Cellular extracts were then transferred to
individual wells of a 96-well plate pre-coated with donkey anti-rabbit
antibody, and then rabbit antiserum against cAMP was added, and the plates
were incubated for 2 h. cAMP-horseradish peroxidase conjugates were added to
each individual well and incubated at 4 °C for 1 h. After extensive
washing, peroxidase substrate was added, incubation was carried out at room
temperature for 1 h, and the plate was read at 450 nm after stopping reaction
with 50 µl of 1 M sulfuric acid.
StatisticsData were analyzed using analysis of variance
(ANOVA), and values were considered significantly different when p
< 0.05. Results were expressed as mean ± S.E.
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RESULTS
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Elevated tTG Expression Leads to Enhanced CREB Phosphorylation and Its
Transcriptional Activation in Response to ForskolinTissue TG is
essential for retinoic acid-induced neuronal differentiation of SH-SY5Y cells
(20,
36). One of the proteins that
likely plays a key role in differentiation and survival processes in neuronal
cells is the CREB protein (24,
41). Phosphorylation of
Ser133, located within a 60-amino acid kinase-inducible domain, is
a prerequisite for CREB transcriptional activation
(21). To enhance CREB
phosphorylation on Ser133, cells were treated with forskolin, a
direct adenylyl cyclase activator
(39). As expected, treatment
with 10 µM forskolin resulted in a significant and transient
increase in CREB phosphorylation on Ser133 in all of the human
neuroblastoma SH-SY5Y cell lines (Fig.
1). However, in the tTG cells, forskolin treatment resulted in a
more robust and sustained CREB phosphorylation
(Fig. 1). To determine whether
the differences in CREB phosphorylation in response to forskolin treatment in
the different SH-SY5Y cell lines resulted in differential CREB transcriptional
activity, studies were carried out with a luciferase CRE-reporter vector. In
the control SH/pc cells, forskolin treatment resulted in a significant
increase in CRE-luciferase activity. CRE-luciferase was also elevated in the
tTG cells in response to forskolin, although the increase was significantly
greater then it was in the SH/pc cells (6.9-fold increase for the tTG cells
compared with a 3.7-fold increase for the SH/pc cells)
(Fig. 2). In contrast,
forskolin-stimulated CRE activity was significantly attenuated in the anti-tTG
cells compared with the SH/pc or tTG cells
(Fig. 2).

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FIG. 1. Forskolin-induced CREB phosphorylation at Ser133 is enhanced
in cells overexpressing wild type tTG. Human neuroblastoma cells stably
transfected with vector only (SH/pc), wild type tTG
(tTG), transglutaminase-inactive tTG mutant (C277S), or
antisense tTG (anti-tTG) were exposed to 10 µM
forskolin for the indicated time periods. A, cell lysates were
immunoblotted for phosphorylated CREB (p-CREB) or total CREB.
B, quantitation of the increase in CREB phosphorylation at
Ser133 in response to 10 µM forskolin. Values were
normalized to the total amount of CREB in each fraction. Enhancement of CREB
phosphorylation was observed in the tTG-overexpressing cells compared with the
other cells.
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FIG. 2. tTG significantly increases CREB transcriptional activity in response to
forskolin treatment. SH/pc, tTG, anti-tTG, and C277S cells were
transfected transiently with the pCRE (Firefly luciferase) reporter
construct along with an internal control, pRL-TK vector (Renilla
luciferase), followed by treatment with 10 µM forskolin.
Luciferase activity was significantly greater in cells expressing wild type
tTG and significantly suppressed in the anti-tTG cells compared with control
cells (SH/pc) (mean ± S.E., n = 3 experiments; *, p
< 0.05).
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cAMP Accumulation Is Directly Modulated by tTG Expression in SH-SY5Y
CellsThe data described above suggest that tTG may increase
forskolin-stimulated cAMP production, which, in turn, leads to enhanced CREB
activation. Therefore, basal and forskolin-stimulated cAMP levels were
measured in two different tTG cell lines, tTG-1 and tTG-2, and two different
cell lines overexpressing transglutaminase inactive C277S-tTG, C277S-1 and
C277S-2, along with the control SH/pc cells and the anti-tTG cells. Basal cAMP
levels in all cell lines were in the range of 3070 fmol/well
(Fig. 3). However, after
stimulation with 10 µM forskolin, the different cell lines
exhibited significantly different increases in the levels of cAMP. In both
wild type tTG-expressing cell lines, cAMP levels were elevated to a
significantly greater extent compared with the SH/pc cells. In contrast,
forskolin-stimulated cAMP levels in the anti-tTG and the C277S cells were
significantly lower than what was observed in the SH/pc cells
(Fig. 3). Given the finding
that both tTG cell lines and both C277S cell lines responded similarly, in all
subsequent experiments just the tTG-2 and C277S-2 cells lines were used and
referred to as tTG cells and C277S cells, respectively. These were also the
cell lines used for Figs. 1 and
2.

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FIG. 3. In response to forskolin treatment, cAMP levels increased to a
significantly greater extent in cells overexpressing tTG. Cyclic AMP
levels were measured under basal conditions (con) and, after
stimulation with 10 µM forskolin (FSK), in vector
control cells (SH/pc), two cell lines overexpressing wild
type tTG (tTG-2 and tTG-1), two cell lines expressing C277S
mutant tTG (C277S-2 and C277S-2), and the antisense tTG
cells (anti-tTG). Forskolin-stimulated cAMP production was
significantly greater in both wild type tTG-expressing cell lines compared
with the control SH/pc cells (mean ± S.E., n = 3 experiments;
*, p < 0.05).
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The level of intracellular cAMP is regulated by the balance between the
activities of adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE)
(42,
43). To test whether the
observed enhancement of cAMP accumulation in the tTG cells is due to a
decrease in the levels and/or activity of PDE, cells were pretreated for 3 h
with 30 µM rolipram, a specific inhibitor of the high-affinity
cAMP PDE (44), prior to
treatment with 10 µM forskolin for 5 min. Rolipram treatment
significantly increased cAMP levels only in the tTG cells from a basal level
of 46 ± 12 fmol cAMP/well to 281 ± 69 fmol cAMP/well after
rolipram treatment (Fig.
4A). Treatment with rolipram and forskolin together
resulted in a dramatic synergistic increase of cAMP in all the cell lines
(Fig. 4B).
Furthermore, the extent of cAMP accumulation in the tTG cells was
significantly greater then that observed in the other cell lines
(Fig. 4B). These data
indicate that decreases in PDE are not responsible for enhanced cAMP
production in the tTG cells, as inhibiting PDE did not prevent the enhanced
forskolin-stimulated cAMP accumulation in the tTG cells compared with the
other cell lines.

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FIG. 4. Inhibition of PDE with rolipram in cells overexpressing tTG results in
cAMP accumulation. A, cAMP levels under basal conditions
(con) and after treatment with 30 µM rolipram
(Rol) were measured in SH/pc, tTG, anti-tTG, and C277S cells. The
cAMP levels in the tTG cells after rolipram treatment were significantly
increased when compared with control SH/pc cells (**, p < 0.001).
B, cAMP levels were measured in the same cells in either the presence
of rolipram alone (Rol) or rolipram and 10 µM forskolin
(Rol + FSK). When cells were treated with rolipram and
forskolin together, the accumulation of cAMP was significantly greater in the
tTG cells compared with SH/pc cells (**, p < 0.001). All data is
mean ± S.E., n = 3 separate experiments.
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Several studies indicate that there are two conformations of adenylyl
cyclase for forskolin binding. There is a lower affinity binding state,
wherein forskolin binds and activates adenylyl cyclase at concentrations of
10 µM, and a higher affinity binding state, in which
forskolin can activate adenylyl cyclase at a concentration of
0.1
µM (45).
Furthermore, the higher affinity forskolin binding state of adenylyl cyclase
is usually due to the binding of G
s
(46), which results in a
conformational change in adenylyl cyclase. Treatment of the cells with 0.1
µM forskolin for 5 min significantly increased cAMP levels only
in the tTG cells from a basal level of 30 ± 20 fmol cAMP/well to 192
± 26 fmol cAMP/well after forskolin treatment
(Fig. 5A). In
contrast, no changes in cAMP levels were observed in either the SH/pc or C277S
cells (Fig. 5A). This
suggests that adenylyl cyclase in the tTG cells may be in a conformation that
allows forskolin to bind with a high affinity.

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FIG. 5. Treatment of tTG cells with 0.1 µM forskolin
(FSK) or CTX alone results in a significant elevation of cAMP
levels. A, cAMP levels were measured in SH/pc, tTG, or C277S
cells after incubation in the absence or presence of 0.1 µM
forskolin. An elevation of cAMP in response to 0.1 µM forskolin
was only observed in the tTG cells. B, cells were incubated in the
absence or presence of 20 µM CTX prior to the measurement of
cAMP. A significant CTX-induced increase in cAMP was only observed in the tTG
cells. C, cells were incubated with 20 µM CTX in the
absence or presence of 0.1 µM forskolin. Although cAMP levels in
cells treated with CTX and 0.1 µM forskolin were significantly
greater than cAMP levels in cells treated with CTX alone, the levels of cAMP
in the tTG cells were significantly greater than those observed in the other
cell lines. D, cAMP levels were measured in cells incubated with 20
µM CTX alone, 10 µM forskolin alone, or in cells
treated with CTX and, subsequently, 10 µM forskolin. CTX
pretreatment of SH/pc and C277S cells, but not tTG cells, resulted in a
synergistic increase in cAMP production in response to forskolin (mean
± S.E., n = 3 experiments; *, p < 0.05).
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To assess whether the G
s subunit may be involved in the
observed enhancement of forskolin-stimulated adenylyl cyclase activity in the
tTG cells, cholera toxin (CTX) was used in the next set of experiments. CTX
catalyzes the NAD-dependent ADP-ribosylation of the G
s
subunit (47), which results in
inhibition of the intrinsic GTPase activity of G
s and
stabilization of the activated conformation
(48). CTX-induced
stabilization of the activated conformation of G
s results in
a long-lived activation of adenylyl cyclase
(49,
50). Treatment with 20
µM CTX alone for 4 h did not result in any significant increases
in cAMP levels in either the SH/pc or C277S cells
(Fig. 5B). However,
CTX treatment of the tTG cells increased cAMP levels from 30 ± 20 fmol
cAMP/well under basal conditions to 331 ± 83 fmol cAMP/well
(Fig. 5B). Treatment
with CTX and 0.1 µM forskolin together resulted in a dramatic
synergistic increase in cAMP levels in all the cell lines
(Fig. 5C); however,
the extent of cAMP accumulation in the tTG cells was significantly greater
then than that observed in the other cell lines
(Fig. 5C). In the next
set of experiments, cAMP accumulation was measured in the different cells
lines after treatment with CTX alone for 4 h, 10 µM forskolin
for 5 min, or CTX for 4 h and, subsequently, 10 µM forskolin for
an additional 5 min (Fig.
5D). CTX treatment alone had no effect on the cAMP levels
in the SH/pc and C277S cells; however, as indicated above, CTX treatment did
significantly increase cAMP levels in the tTG cells. In the SH/pc cells,
treatment with 10 µM forskolin increased cAMP levels to 1088
± 62 fmol cAMP/well, and pretreatment with CTX followed by treatment
with 10 µM forskolin resulted in a synergistic increase in cAMP
levels to 2570 ± 165 fmol cAMP/well. Similarly, in the C277S cells,
cAMP levels increased from 452 ± 102 fmol cAMP/well in the presence of
10 µM forskolin to 1174 ± 24 fmol cAMP/well after
treatment with CTX and 10 µM forskolin. Surprisingly though, a
similar synergistic enhancement of cAMP accumulation in the tTG cells was not
observed. In the tTG cells, cAMP levels were 2348 ± 198 fmol cAMP/well
in the presence of 10 µM forskolin and 3351 ± 67 fmol
cAMP/well in the presence of CTX and forskolin
(Fig. 5D). This
indicates that adenylyl cyclase in the tTG cells is already in a more
activated state, and, thus, pretreatment with the CTX is not as effective at
increasing forskolin-induced cAMP production as it is in the other cell
lines.
Expression of Adenylyl Cyclase, G
s,
and G
i Are Unaltered by tTG
ExpressionTo evaluate whether the observed differences in the rate
of cAMP accumulation may be associated with differences in the expression of
adenylyl cyclase, G
s, and/or G
i, an
immunoblot analysis was performed. The results indicate that there are no
significant differences in the protein expression levels of these proteins in
SH/pc, tTG, anti-tTG, and C277S cells that correlate with the enhanced cAMP
production in the tTG cells (Fig.
6).
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DISCUSSION
|
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The aim of this study was to gain insight into the mechanism by which tTG
contributes to neuronal differentiation processes
(19,
20). The results presented in
this study clearly demonstrate that tTG greatly facilitates adenylyl cyclase
activation, resulting in enhanced cAMP production and subsequent CREB
phosphorylation and activation. Furthermore, the data indicate that tTG
directly or indirectly facilitates the transition of adenylyl cyclase into an
activated state. These findings are significant given the fact that the
cAMP-CREB pathway has been shown to play a critical role in neuronal
differentiation processes (33,
51).
It is well established that treatment of human neuroblastoma SH-SY5Y cells
with retinoic acid results in differentiation
(52). The retinoic
acid-induced neuronal-like phenotype is accompanied by significant increases
in tTG expression (3,
20). This characteristic of
SH-SY5Y cells makes them an ideal model for investigating the role of tTG in
neuronal differentiation. Previously, we demonstrated that tTG appears to be
necessary and sufficient for the development of the neuronal morphology of
SH-SY5Y cells. Overexpressing wild type tTG in SH-SY5Y cells results in
spontaneous differentiation in 5% serum conditions. In contrast,
overexpression of an antisense tTG construct that suppresses the expression of
endogenous tTG results in cells that are unresponsive to retinoic acid in that
they continue to proliferate and do not extend neurites even after extended
retinoic acid treatment (20).
This fact clearly indicates that tTG is an essential component of the
differentiation process of human neuroblastoma SH-SY5Y cells. The finding that
treatment of SH-SY5Y cells with the transglutaminase inhibitor
monodansylcadaverine inhibited retinoic acid-induced differentiation has
further substantiated this conclusion
(36). Although it is clear
that tTG is essential for differentiation to a neuronal phenotype, the cell
signaling events modulated by tTG that regulate this process are not
known.
Previous studies have provided evidence that the cAMP-CREB pathway is of a
critical importance for appropriate neuronal differentiation
(26,
53). Indeed, increasing cAMP
levels have been shown to induce neuronal differentiation in several cell
models (33,
51,
54). Furthermore, a critical
role for CREB in neuronal differentiation has been clearly illustrated by the
fact that overexpression of CREB in SK-N-BE cells leads to morphological
alterations typical of neuronal differentiation
(26). In contrast,
overexpression of a dominant negative CREB construct results in a poorly
differentiated phenotype (26).
Moreover, in another neuronal-like cell system, PC12 cells, neuronal
differentiation has been documented to be tightly associated with CREB
transcriptional activation
(53), and elevation of cAMP in
PC12 cells results in morphological changes similar to those observed in
response to nerve growth factor treatment
(53). Additionally it has been
shown that, in CREB null mice, axonal growth is severely compromised
(55,
56). Thus, it is readily
evident that any processes that result in sustained CREB activation are likely
to contribute significantly to the neuronal differentiation process.
Therefore, it is reasonable to hypothesize that the tTG-induced potentiation
of CREB activation may be one mechanism by which tTG facilitates neuronal
differentiation.
Because both CREB and the CREB-binding protein have glutamine-rich regions
(22,
57), we originally postulated
that these proteins were substrates of tTG. However, to date we have not been
able to demonstrate that either CREB or the CREB-binding protein are
substrates of tTG in situ. Given this situation, we focused on
signaling events upstream of CREB activation. The data in this study strongly
suggest that tTG is either directly or indirectly modulating adenylyl cyclase.
Even though the molecular mechanism of activation of adenylyl cyclase by tTG
remains to be elucidated, our findings indicate that tTG probably affects the
conformational state of adenylyl cyclase, leading to a more responsive state.
This mechanism appears to be most probable given the activation of adenylyl
cyclase in the tTG cells with a very low forskolin concentration (0.1
µM) and the fact that there was the fact that there was a
significant accumulation of cAMP upon treatment with rolipram only in the tTG
cells. Indeed, it is clear that adenylyl cyclase exists in multiple
conformational states, elicited by binding of effectors such as G
subunits, forskolin, or by occupancy of the ATP binding site
(42,
58). Therefore, we postulate
that increased tTG expression in SH-SY5Y cells results in a change in the
conformational state of adenylyl cyclase, which leads to an increased
sensitivity to the activating agents.
Our findings as to the role of tTG in the modulation of adenylyl cyclase
activity and the accumulation of intracellular cAMP are in contrast to the
findings of Gentile et al.
(59). In this previous study
it was postulated that wild type tTG or its TG-inactive mutant, C277S,
inhibits adenylyl cyclase activity in human fibroblast Balb-C 3T3 and bovine
aorta endothelial cells. The apparent discrepancy between the present study
and the study by Gentile et al.
(59) may be associated with
different cell models and experimental approaches. One the other hand, the
present finding may emphasize the complexity of tTG function in different cell
lineages. In conclusion, we believe that the present findings add a new
dimension to our understanding of the functions of tTG in the nervous system
and clearly indicate that tTG plays a central role in regulating neuronal
signaling processes.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grant AG12396. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Supported by a fellowship from the Wills Foundation. 
To whom correspondence should be addressed: Dept. of Psychiatry, University of
Alabama at Birmingham, 1720 7th Ave. S., SC1061, Birmingham, AL 35294-0017.
Tel.: 205-934-2465; Fax: 205-934-3709; E-mail:
gvwj{at}uab.edu.
1 The abbreviations used are: tTG, tissue transglutaminase; TG,
transglutaminase; CRE, cAMP response element; CREB, CRE-binding protein; PDE,
cyclic nucleotide phosphodiesterase; CTX, cholera toxin. 
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ACKNOWLEDGMENTS
|
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We thank Dr. R. Jope for helpful advice and discussions during the course
of this project as well as the gift of the G
i antibody.
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