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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 5, 2001 as doi:10.1096/fj.00-0450fje. |
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* Departments of Medicine and Surgery, Department of Veterans Affairs Medical Center, Long Beach, California, University of California, Irvine, California 90822, USA; and
Department of Surgery II, Kyushu University, Fukuoka, Japan
2Correspondence: Gastroenterology Section (111G) DVA Medical Center, 5901 E. Seventh Street, Long Beach, CA 90822. E-mail: atarnawski{at}yahoo.com
SPECIFIC AIMS
Portalhypertensive (PHT) gastric mucosa has increased susceptibility to injury and impaired mucosal healing. As our previous study showed that mitogen-activated protein (MAP) kinase (ERK) plays a pivotal role in gastric mucosal healing, in this study we examined whether portal hypertension alters ERK signaling in the gastric mucosa after injury.
PRINCIPAL FINDINGS
1. PHT gastric mucosa has reduced activation of ERK2 in response to
alcohol-induced injury
To determine whether ERK signaling in response to alcohol injury
is altered in PHT gastric mucosa, we investigated phosphorylation and
activity levels of ERK2 in alcohol-injured gastric mucosa of PHT and
sham-operated (SO) rats (control) (Fig. 1
). In noninjured PHT gastric mucosa (at baseline), ERK2 phosphorylation
and activity were significantly higher by 87% and 30%, respectively,
than in SO rats. Following alcohol injury, ERK2 phosphorylation and
activity were significantly increased in SO rats only: phosphorylation
by 79%, 120%, and 221%; and activity by 65%, 88%, and 137%,
respectively, at 3, 6, and 24 h vs. baseline. In contrast, in PHT
rats following alcohol-induced injury, ERK2 phosphorylation and
activity were not significantly increased vs. baseline. In fact, both
ERK2 phosphorylation and activity were significantly lower in PHT than
in SO rats by 31% and 36%, respectively, at 24 h after
alcohol-induced injury. These changes were not due to alterations in
ERK2 protein synthesis because total ERK2 protein levels remained
unchanged between both PHT and SO gastric mucosa.
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2. MKP-1 is overexpressed at baseline and further increased
following alcohol-induced injury in PHT gastric mucosa
To investigate the mechanism of reduced ERK2 activation in PHT
gastric mucosa in response to alcohol-induced injury, we studied the
expression of MAP kinase phosphatase-1 (MKP-1), an enzyme that
dephosphorylates and thereby inactivates ERK. In PHT gastric mucosa,
MKP-1 mRNA and protein were overexpressed at baseline vs. SO
(Fig. 2A
). After alcohol-induced injury, MKP-1 expression was
increased further in PHT gastric mucosa. Although MKP-1 expression did
increase in SO rats following alcohol injury, this occurred only at
24 h (Fig. 2B
). By contrast, in PHT gastric mucosa, the
increase in MKP-1 expression occurred earlier following alcohol-induced
injury (at 3 h for MKP-1 mRNA and at 6 h for MKP-1 protein)
(Fig. 2C
). Furthermore, at 24 h following
alcohol-induced injury, expression of MKP-1 mRNA and protein was
increased significantly, by 
40% in PHT gastric mucosa vs. SO rats.
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CONCLUSIONS AND SIGNIFICANCE
PHT is a frequent complication of liver cirrhosis, the fourth leading cause of death in Americans between the ages of 35 to 54. Upper GI bleeding from either esophageal varices or abnormal gastric mucosa—PHT gastropathy—leads to death in approximately 30% of these patients. Clinical and experimental data indicate that the PHT gastric mucosa has the increased susceptibility to injury and impaired healing. Our previous study demonstrated that ERK plays a pivotal role in mucosal repair and healing. Our present study demonstrates for the first time that: 1) activation of ERK is reduced significantly in PHT gastric mucosa during the reparative processes after alcohol-induced injury compared with SO (normal) gastric mucosa; 2) expression of MAP kinase phosphatase-1 (MKP-1) is increased significantly in noninjured PHT gastric mucosa; and 3) reduction of ERK activation correlates with the increased expression of MKP-1 occurring before injury, as well as the further increase in this expression following injury. Therefore, these findings indicate that reduction of ERK2 activation following alcohol injury in PHT gastric mucosa is likely involved in impaired mucosal healing.
The molecular mechanisms underlying impaired mucosal healing following
injury in PHT gastric mucosa remain unexplored. In some nongastric,
cellular systems, TNF-
and oxidative stress have been shown to
activate ERK through activation of tyrosine kinase receptors and
subsequently to induce MKP-1 expression resulting in the
dephosphorylation and inactivation of ERK. Our previous studies
demonstrated that PHT causes increased generation of TNF- and oxygen
free radicals in the gastric mucosa, which indicates that they may be
involved in the increased ERK2 activity and MKP-1 overexpression of
noninjured PHT gastric mucosa. Increased MKP-1 expression in PHT
gastric mucosa could be caused by sustained activation of ERK2. This
contention is supported by the observation that PC12 cells transfected
with the EGF receptor have increased ERK2 activation accompanied by
increased MKP-1 levels upon stimulation with EGF. Under such
conditions, phosphorylated/activated ERK and MKP-1 might be partially
localized to different subcellular compartments (the cytoplasm and the
nucleus, respectively), and this condition could explain why both ERK
activity and MKP-1 expression are increased relative to normal
noninjured gastric mucosa.
Our present study showed that MKP-1 expression is increased in PHT
gastric mucosa at baseline and is increased further following alcohol
injury compared with SO rats. The comparison between the kinetics of
ERK activity and MKP-1 protein expression following alcohol-induced
injury suggests that the reduced ERK2 activation following injury in
the PHT gastric mucosa may result from both the increased expression of
MKP-1 occurring before injury and its further increase following
alcohol-induced injury (Fig. 2B, C
). The relationship
between induction of MKP-1 expression and prompt inactivation of ERK
remains controversial but is likely cell type-dependent. Recent
in vitro studies have demonstrated that induction of MKP-1
is not rapid enough to inactivate ERK2 after exposure to extracellular
stimuli and that inhibition of total protein synthesis by cyclohexamide
does not affect the rapid deactivation of ERK2 in some cell types such
as PC 12 and Hela cells. A recent study with human intestinal cells
demonstrated that MKP-1 down-regulates ERK activation, which in turn
leads to inhibition of cell proliferation. This finding is consistent
with our results indicating that, in PHT gastric mucosa, injury-induced
MKP-1 may inhibit ERK2 activation in response to injury. Furthermore,
in transfected cell lines overexpressing MKP-1, ERK activation was
inhibited, which suggests that dephosphorylation of ERK2 relies on
preexisting phosphatases, rather than on inducible MKP-1. Our findings
indicate that the preexisting overexpression of MKP-1 in noninjured PHT
gastric mucosa may indeed be sufficient to cause reduced ERK activation
in response to alcohol injury.
Although increased expression of MKP-1 during gastric mucosal healing may be one of the mechanisms responsible for the reduced ERK activation after injury to PHT gastric mucosa, changes in upstream participants of the ERK signaling pathway may also be involved. There are many steps at which the ERK signaling pathway can be attenuated, including down-regulation of receptor tyrosine kinases, Ras-dependent or -independent Raf activation, and regulation of Raf, MEK and MAPK by protein phosphatases; for example, MKP-1 and protein phosphatase 2A. Further studies are required to address the precise upstream mechanisms contributing to reduced ERK activation in injured PHT gastric mucosa.
A diagrammatic representation of how ERK and MKP-1 may participate in
the increased susceptibility to injury and impaired healing of PHT
gastric mucosa is presented in Fig. 3
.Epithelial cells of PHT gastric mucosa are exposed to continual
extracellular stimuli such as oxidative stress and TNF-, which
activate ERK and induce MKP-1. Consequently, both ERK and MKP-1 might
be increased and localized to the cytoplasm and the nucleus,
respectively, in noninjured PHT gastric mucosa. Following alcohol
injury, epithelial cells of both normal and PHT gastric mucosa become
exposed to extracellular stimuli resulting from the injury. In normal
gastric mucosa of SO rats, these stimuli cause increased ERK
activation, which is essential for the healing process. In contrast, in
PHT gastric mucosa, MKP-1 expression is increased prior to injury. The
preexisting increased expression of MKP-1 at baseline, and its further
and rapid increased expression in response to injury, greatly limit the
amount of activated ERK in PHT gastric mucosa. Thus, the healing of
acute injury in PHT gastric mucosa is impaired, in part, by reduced ERK
activation resulting from the increased expression of MKP-1.
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In conclusion, the ERK signaling pathway is impaired significantly in PHT gastric mucosa following alcohol-induced injury and this condition may be due to increased expression of MKP-1. Altered ERK activation in PHT gastric mucosa in response to injury may explain, at least in part, its impaired healing.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0450fje ; to cite this
article, use FASEB J. (January 5, 2001)
10.1096/fj.00-0450fje 
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) at baseline and following alcohol-induced
injury. Autoradiograph shows myelin basic protein (MBP) phosphorylated
by ERK2 immunoprecipitated with a polyclonal anti-ERK2 antibody. Kinase
activity was assayed by measuring the levels of radiolabeled
[
-32P] ATP incorporated into MBP. Values are
expressed as picomole per minute per milligram protein and represent
mean ± SD (n=6 animals per group for each time
point). *P < 0.05 vs. SO, **P < 0.01 vs. SO,
#P < 0.05 vs. baseline, ##P < 0.01 vs.
baseline.
) and portal hypertensive (PHT) rats (
) at
baseline and following alcohol-induced injury. Quantitative data were
obtained by a computerized video analysis of the Western blots. Values
are expressed as intensity (unit) and represent mean ±
SD (n=6 animals per group for each time point).
*P < 0.05 vs. SO, **P < 0.01 vs. SO,
#P < 0.05 vs. baseline. B) Comparison of ERK2 activity
(•) and MKP-1 protein (