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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.02-1107fje. |
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* Departments of Medicine and Surgery, Department of Veterans Affairs Medical Center, Long Beach, California, University of California, Irvine, California, USA; and
Department of Surgery and Science and
Department of Disaster and Emergency Medicine, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan
2Correspondence: University of California, Irvine, DVA Medical Center, 5901 E. Seventh St., Long Beach, CA 90822, USA. E-mail: michael.jones4{at}med.va.gov
SPECIFIC AIMS
The PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor is a dual specificity phophatase that negatively regulates Akt, the effector of PI 3-kinase. The PI 3-kinase/Akt signaling pathway is integral to processes essential to tissue injury healing including cell proliferation, migration, survival, and angiogenesis. However, the relationship of PTEN to injury and injury healing remains unexplored. Portal hypertensive (PHT) gastropathy, a severe complication of portal hypertension, has impaired gastric mucosal healing but the underlying causes remain unknown. The aim of the present study was to determine whether injured PHT gastric mucosa has abnormal expression/activation of PTEN and/or impaired PI 3-kinase/Akt signaling. Because PTEN expression can be directly induced by the early growth response transcriptional factor (Egr-1), which is itself regulated by TNF-
, we investigated the possible involvement of TNF- and Egr-1 in PTEN expression and PI 3-kinase/Akt signaling during the healing of injured PHT gastric mucosa.
PRINCIPAL FINDINGS
1. Active PTEN protein levels are significantly increased in PHT vs. normal or sham-operated (SO) gastric mucosa after ethanol-induced injury while PI 3-kinase activity and Akt activation are significantly impaired
Phosphorylation of the PTEN carboxyl-terminal regulatory domain results in its inactivation. Unphosphorylated PTEN therefore is the active form of PTEN. We examined total PTEN protein levels and PTEN phosphorylation in PHT vs. normal or SO gastric mucosa of rats before (at baseline) and after ethanol-induced injury. Although the PTEN phosphorylation ratio (% phosphorylated to total PTEN) was significantly higher by 59% (P<0.05) in PHT vs. SO gastric mucosa at baseline (reflecting significantly less active PTEN), at 6 h after ethanol-induced injury not only was the level of total PTEN protein significantly (P<0.01) increased in PHT vs. SO gastric mucosa, but the level of active PTEN (as reflected by a lower phosphorylation ratio) was significantly increased by 2.4-fold (Fig. 1
). We also examined the activation state of Akt in PHT vs. SO gastric mucosa after ethanol-induced injury. Unlike PTEN, phosphorylation of Akt at serine 473 is required for its activation and is used as an indicator of the activation state of Akt. We found that 6 and 12 hours after ethanol-induced injury, the level of phosphorylated Akt in PHT gastric mucosa was significantly (P<0.05) reduced vs. SO gastric mucosa by twofold. Activity of phosphatidylinositol 3-kinase (PI 3-kinase), the upstream effector of Akt activation, was significantly reduced in PHT vs. SO gastric mucosa 6 h after ethanol-induced injury by 3.5-fold. We next examined expression of the early growth response factor-1 (Egr-1) in PHT vs. SO gastric mucosa after ethanol-induced injury. In PHT gastric mucosa 6 h after ethanol-induced injury, Egr-1 protein levels were significantly (P<0.05) greater by 3.3-fold (P<0.05) vs. SO gastric mucosa (data shown in full-length online article).
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2. Neutralization of TNF- normalizes the increased protein levels of Egr-1 and PTEN, and restores PI 3-kinase and Akt activity in PHT gastric mucosa after ethanol-induced injury
Uninjured PHT gastric mucosa has elevated tumor necrosis factor- (TNF-) levels, and injury has been reported to further increase PHT blood plasma levels of TNF-. When we examined TNF- protein levels in gastric mucosa, we found not only a significant (P<0.00001) twofold greater level of TNF- in PHT vs. SO gastric mucosa at baseline but, at 6 h after ethanol-induced injury, TNF- levels were further increased in PHT vs. SO gastric mucosa by an additional 2.4-fold (P<0.0001). We examined PTEN protein levels and phosphorylation in gastric mucosa after treatment of PHT and SO rats with a TNF- neutralizing antibody. TNF- neutralization normalized total PTEN protein levels and PTEN phosphorylation (reflecting normalization of active PTEN levels) in PHT gastric mucosa to the levels of SO gastric mucosa at baseline and at all time points after injury (Fig. 2
). We next examined the effect of TNF- neutralizing antibody treatment on Akt activation (reflected by Akt phosphorylation levels) in PHT vs. SO gastric mucosa. As with PTEN, neutralization of TNF- with the neutralizing antibody restored levels of activated Akt in PHT gastric mucosa to those of SO gastric mucosa both at baseline and at all times after injury. Furthermore, treatment with the TNF- neutralizing antibody restored PI 3-kinase activity in PHT gastric mucosa to the levels of SO gastric mucosa at baseline and after ethanol-induced injury. This indicates that the elevated levels of TNF- after ethanol-induced injury impair activation of Akt both by increasing the levels of PTEN and inhibiting PI 3-kinase activity. In addition, treatment with the TNF- neutralizing antibody normalized Egr-1 protein levels in PHT gastric mucosa to those of SO gastric mucosa. When we examined the effect of TNF- neutralizing antibody treatment in PHT rats on ethanol-induced gastric mucosal injury itself, we found that not only did TNF- neutralization significantly (P<0.005) reduce the area of macroscopic mucosal damage by twofold but also resulted in a significant (P<0.001) twofold increase in the extent of mucosal regeneration by 24 h.
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CONCLUSIONS AND SIGNIFICANCE
Although loss of PTEN has been implicated in cancer growth and progression, a role for PTEN during tissue injury healing remains unexplored. Our present study demonstrates for the first time that PHT gastric mucosa has elevated levels of active PTEN and impaired activation of PI 3-kinase and Akt after ethanol-induced injury compared with normal gastric mucosa. Our demonstration that ethanol-induced injury to PHT gastric mucosa results in elevated levels of TNF- and Egr-1, and that treatment with a TNF- neutralizing antibody normalizes Egr-1 and PTEN levels and restores PI 3-kinase activity and Akt phosphorylation levels to those of normal gastric mucosa after injury, indicates that the abnormalities in PTEN levels and PI 3-kinase/Akt signaling in injured PHT gastric mucosa are the result of elevated TNF- and Egr-1. As PTEN is not a direct regulator of PI 3-kinase activity per-se, the mechanism of PI 3-kinase inhibition in PHT gastric mucosa remains uncertain. Studies indicate that TNF- can inhibit PI 3-kinase activity in certain cell types and that this inhibition is dependent on both TNF- concentration and activation of the TNF- receptor TNFR1. It is possible that TNF- directly inhibits PI 3-kinase activity via TNFR1 in injured PHT gastric mucosa. Because PI 3-kinase is the upstream effector of Akt activation, its inhibition during PHT gastric mucosal injury healing, in addition to elevated PTEN, most likely contributes to impaired Akt activation/function. The intimate involvement of Akt signaling in processes essential to tissue injury healing, together with the findings of our present study, supports the model shown in Fig. 3
. Injury to PHT gastric mucosa results in highly elevated TNF- levels that induce Egr-1 expression. This, in turn, leads to increased PTEN levels. The increased PTEN prevents activation of Akt and thus impairs essential processes of healing. Our findings extend the current knowledge of PTEN function during tissue injury healing and point out a novel mechanism whereby elevated TNF- levels play a primary role in the impaired healing of injured PHT gastric mucosa via inhibition of PI 3-kinase and Akt activation. These findings likely extend broadly to injury healing in any organ tissue where mediators of injury (e.g., hemorrhagic shock) result in highly elevated TNF- levels.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1107fje; doi; 10.1096/fj.02-1107fje 
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