Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37°C. Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods. We observed a 50-70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture. We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death. A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes. Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase. Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa. Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined. Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death.
apoptosis; intestine; phosphatase; epithelial; pervanadate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ACTIVATION OF INTRACELLULAR KINASES by growth factors, hormones, and cytokines mediates many cell responses. Protein tyrosine kinases (PTKases) phosphorylate proteins on tyrosine residues. Protein tyrosine phosphorylation is a tightly regulated process, and the action of PTKases is counterbalanced by the ability of specific phosphatases, protein tyrosine phosphatases (PTPases), to dephosphorylate tyrosine. Indeed, specific phosphatase inhibitors, such as sodium vanadate, must be added to cell extracts to prevent tyrosine dephosphorylation during cell homogenization. Although the role of tyrosine phosphorylation in cell growth and differentiation has been extensively studied, very little is known about the role of phosphorylation in the regulation of programmed cell death (6, 7, 23, 25-27), which plays critical physiological and pathological roles in many tissues, including the small intestine (21).
The renewing epithelium of the small intestine has the highest rate of cell proliferation in the intact adult animal (20). To maintain its normal cell mass, the intestinal epithelium eliminates a large number of dying cells daily by releasing them into the intestinal lumen. Remarkably, the average human sheds his body weight in dying cells during a 6- to 9-mo period (14). In addition to regulating intestinal cell mass, apoptosis also plays an important role in intestinal injury, such as ischemic-reperfusion injury (5, 11) or intestinal barrier dysfunction (22). In contrast to the cell proliferation cycle, which extends over several hours and includes several well-defined stages, the study of intestinal programmed cell death in the intact animal is problematic for several reasons. The cell death pathway is short; some cells may detach from the intestinal villus early in the death pathway; and, finally, the biochemical and molecular stages of the cell death pathway are just being described.
We have recently reported that ex vivo cell suspensions of mouse intestinal epithelial cells in serum-free DMEM undergo massive, rapid, and highly synchronized programmed cell death (18). Similar models have been established using dissociated human intestinal epithelial cells (3, 4) and rat colonic crypt cells (7). These models synchronize cell death over a short time frame, allowing one to analyze the role of various signal transduction pathways in dying intestinal tissue.
In our original paper (18), we reported that dying enterocytes downregulate several tyrosine kinases and their modulators, including the epidermal growth factor receptor (EGFR), the Erb-B2 tyrosine kinase, and the son of sevenless (Sos) guanine nucleotide exchangers. This led us to speculate that the elimination of proteins involved in Ras-convergent signaling pathways may contribute to the initiation of apoptosis. We hypothesized that increased tyrosine phosphorylation achieved by inhibiting PTPase activity might prevent apoptosis. To examine the importance of protein tyrosine phosphorylation in intestinal epithelial cell death, we exposed cells to peroxovanadate, a potent and irreversible inhibitor of PTPases. This compound is generated by vanadate peroxidation in the presence of H2O2 and inhibits PTPases in excess to the sum of either vanadate or H2O2 (16). When injected in mice, peroxovanadate induces a striking increase in protein tyrosine phosphorylation in intact tissues.
In this report, we show that intestinal epithelial cell cultures undergo a rapid reduction in tyrosine phosphorylation within 10 min of culture at 37°C, preceding our previously reported downregulation in EGFR, Erb-B2, and Sos protein levels (>30 min). Treatment of these cultures with peroxovanadate resulted in striking increases in tyrosine phosphorylation and in active mitogen-activated protein (MAP) kinase, an important regulator of mitogen signaling that inhibits apoptosis in some cell models (10, 27). Peroxovanadate also decreased intestinal cell death and DNA fragmentation; however, to be effective, it had to be added to the culture medium within the first 5 min when protein tyrosine phosphorylation initially declined.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. B6D2F1 male mice were obtained from Jackson Laboratory (Bar Harbor, ME). Nitrocellulose was obtained from Micron Separation (Westboro, MA). Prestained protein standards were from Amersham Life Sciences (Arlington Heights, IL) or Bio-Rad Laboratories (Hercules, CA). The anti-phosphotyrosine antibodies for blotting (RC20H) and immunohistochemistry (pAb) were obtained from Transduction Laboratories (Lexington, KY). The rabbit polyclonal antibody against the dually phosphorylated MAP kinase was obtained from Promega (Madison, WI). Horseradish peroxidase-conjugated goat anti-rabbit antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence reagent was from New England Nuclear Life Science Products (Boston, MA). Molecular grade agarose was from Bio-Rad Laboratories (Hercules, CA). Sodium vanadate (orthovanadate) was from Fisher Scientific (Pittsburgh, PA). DNAzol was from Molecular Research Center (Cincinnati, OH). DMEM (with high glucose, L-glutamine, 25 mM HEPES buffer, pyroxidine hydrochloride) was obtained from GIBCO BRL (Grand Island, NY). Protease inhibitor cocktail tablets (Complete) were from Boehringer Mannheim (Mannheim, Germany).
Animals. B6D2F1 male mice ranged between 8 and 12 wk of age. Mice were standardized for at least 2 wk before the experiment to a light-dark 12:12-h cycle (lights on at 0600 h; lights off at 1800 h) and provided with food and water ad libitum. Animals were killed between 1000 and 1200 h (17).
Preparation of peroxovanadate and cell suspension cultures. Peroxovanadate was prepared within 15 min of use as previously described (16) and then added to DMEM. Mice were anesthetized with CO2 and then killed by cervical dislocation. The last 12 cm of the distal small intestine (ileum) were removed, and the mucosa was detached by gentle scraping with a microscopic slide. Cells were then placed in 2.4 ml of DMEM (4°C) on ice and suspended by gentle pipetting. Two-hundred-microliter cell aliquots were placed in 2 ml of DMEM in polypropylene tubes at 37°C. The tubes were capped and then immediately incubated without agitation in a 37°C water bath for varying periods of time. Zero time tubes were also placed in DMEM at 4°C, but they were immediately centrifuged at 13,000 g for 20 s at 4°C. The incubated tubes were also centrifuged later at 4°C. After centrifugation, the medium overlying the cell pellet was aspirated. The cells were quick-frozen in liquid nitrogen or immediately homogenized, and then membrane homogenates or DNA extractions were performed as described in Membrane preparation and DNA preparation and agarose eletrophoresis, respectively.
Membrane preparation. Tissue was homogenized in 250-500 µl of buffer (10 mM Tris, pH 7.2, 1 mM EGTA, 200 µM sodium vanadate, 50 µM sodium molybdate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 protease inhibitor cocktail tablet from BRL per 15 ml of homogenization buffer) with a tissue Tearor at setting 4 for 15 s. Sample tubes were kept on ice afterward. In some cases the homogenate was centrifuged at 18,000 g for 60 min, and the resultant pellet was resuspended in homogenization buffer. The supernatant fraction was defined as the cytosol. The homogenate, membrane, and cytosolic fractions had final protein concentrations ranging between 1 and 5 mg/ml.
Western blotting. Homogenate, membrane, or cytosolic samples were heated in Laemmli buffer at 95°C for 5 min, separated by 7% SDS-PAGE, and transblotted to nitrocellulose at 100 V for 90 min at 4°C. Transfer efficiency was confirmed by use of prestained protein standards, Ponceau S staining of the transblot, or Coomassie staining of gel after electrophoresis. After blocking the blot for 90 min with 5% defatted milk (or BSA for RC20H) and 0.05% Tween-20, the immunoreactive proteins were exposed for 90 min at room temperature to primary antibodies. RC20H was used at a dilution of 1:4,000, whereas anti-active MAP kinase was used at a dilution of 1:2,000. An autoradiographic signal was generated by the enhanced chemiluminescence method as described by the manufacturer (Amersham), using the relevant goat anti-rabbit-peroxidase-linked IgG (1:5,000 dilution). RC20H does not require a second antibody. Molecular weights were determined by analysis of the relative migration (Rf) of the protein bands in relation to known standard proteins. Laser densitometry incorporating the Gel-Pro densitometric software was used to quantify the chemiluminescent signal for some blots.
DNA preparation and agarose electrophoresis. DNA was prepared from pelleted cells using the DNAzol genomic DNA isolation reagent following the manufacturer's instructions. DNA was resuspended after ethanol precipitation in 8 mM NaOH. The sample was neutralized as recommended with 1 M HEPES, and the DNA concentration was determined by spectrophotometric readings at 260 nm. Three micrograms of DNA were loaded per lane on a 2% agarose gel. The samples were electrophoresed for 2 h at 95 V, stained with ethidium bromide, transilluminated, and photographed.
Immunohistochemistry. Intestinal pieces or cell pellets were fixed in fresh 4% paraformaldehyde at 4°C for 4 h. Tissue pieces were then rinsed several times in 70% ethanol at 4°C, embedded in paraffin, and cut into 5-µm sections. Slides were deparaffinized by placing them first in xylene and then in a series of graded ethanol solutions. Sections were processed by the manufacturer's protocol (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched by placing the slides in 0.3% H2O2 in methanol for 30 min. After blocking the slides in a 3.0% goat serum solution for 1 h, the tissue sections were incubated with rabbit anti-phosphotyrosine antibodies (0.5 µg/ml) in PBS for 90 min in a humidified chamber. The sections were then washed, incubated with biotinylated secondary antibody, rinsed in PBS, and incubated with the Vectastain Elite ABC reagent. Diaminobenzidine was used as the peroxidase substrate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tyrosine phosphorylation declines rapidly in ileal
cultures. To determine whether tyrosine phosphorylation
of membrane and cytosolic proteins changed during ex vivo cell culture,
we analyzed phosphorylation at various times within the first 15 min of
cell culture in DMEM at 37°C. Cells were homogenized, separated
into membrane and cytosolic fractions, and then resolved by SDS-PAGE. As shown in Fig. 1, immunoblot
analysis of proteins (3 min chemiluminescent exposure) resolved on a
7% SDS polyacrylamide gel indicated a rapid and marked decrease in
tyrosine phosphorylation. This decline was readily apparent for many of
the individual proteins in both membrane and cytosolic fractions and
was confirmed by measuring the total phosphotyrosine signal by laser
densitometry. Total phosphotyrosine decreased between 50 and 70%
within the first 10 min of culture. The reduction in phosphotyrosine
signal was especially rapid for two as yet unidentified membrane
proteins of ~87 and 90 kDa (Fig. 1, arrow). The phosphotyrosine
signal associated with these proteins rapidly disappeared between 2.5 and 5 min of culture.
|
Peroxovanadate induces extensive tyrosine
phosphorylation of intestinal proteins. The reduction
in phosphotyrosine coupled with our previous demonstration that the
ileal cell suspensions underwent massive programmed cell death led us
to hypothesize that inhibition of PTPases would reverse not only the
reduction in tyrosine phosphorylation but also the initiation of
programmed cell death. However, initial studies with a weak and
reversible phosphatase inhibitor, sodium orthovanadate, at a
concentration as high as 1 mM caused only small increases in tyrosine
phosphorylation and did not prevent DNA fragmentation (data not shown).
Additional studies were performed using a stronger and irreversible
PTPase inhibitor, peroxovanadate (16). Intestinal epithelial mucosal suspensions were exposed to DMEM containing sodium vanadate,
H2O2, or a mixture of sodium vanadate plus
H2O2
(peroxovanadate). After 150 min, the intestinal cells were harvested
and frozen in liquid nitrogen. Extracts were prepared and subjected to
SDS-PAGE and transblot. The blots were exposed to anti-phosphotyrosine
antibodies (RC20H). Preliminary experiments established that
peroxovanadate but not sodium vanadate or
H2O2
appreciably increased tyrosine phosphorylation of many proteins. A
dose-response curve demonstrated the effect of varying concentrations
of peroxovanadate on tyrosine phosphorylation (Fig.
2). Peroxovanadate even at the lowest
concentration (60 µM) resulted in increased tyrosine phosphorylation
(Fig. 2); however, the greatest increase occurred between 500 µM and
1 mM.
|
Peroxovanadate induces a rapid and sustained increase
in tyrosine phosphorylation. To further characterize
the action of PTPase inhibition, we carried out a time course for
peroxovanadate (500 µM sodium vanadate and 8 mM
H2O2).
Maximum tyrosine phosphorylation occurred as early as 15 min of culture
(Fig.
3A), but
a pronounced increase was seen as early as 5 min and extended through
the entire culture period. The time course of phosphorylation varied
for individual proteins. For some proteins, maximal phosphorylation occurred at 15 min. For others, it occurred at later times between 90 and 150 min.
|
Peroxovanadate increases MAP kinase activation. Activation of MAP kinase requires phosphorylation of nearby threonine and tyrosine residues. Using an antibody that recognizes the dually phosphorylated (threonine/tyrosine) MAP kinase (1), we evaluated the effect of peroxovanadate on MAP kinase activation (Fig. 3B). We found that peroxovanadate not only reversed the decline of this enzyme normally seen in dying cells, but it also rapidly induced a sustained increase in MAP kinase activation.
The intestinal epithelial cell crypts show the most
pronounced increase in tyrosine phosphorylation. The
mucosal cell suspensions consist of islands of cells derived from
either villi (70-80%) or crypts (<20%). Preliminary
immunohistochemical experiments using mucosal scrapings indicated that
peroxovanadate disproportionately increased crypt cell phosphotyrosine
staining. To better show this, we exposed a 1-cm piece of intact ileum
(18) as well as mucosal suspensions to a peroxovanadate-containing (500 µM) medium for 150 min and then analyzed the cellular distribution of
phosphotyrosine by immunohistochemistry using a rabbit
anti-phosphotyrosine antibody (Fig.
4). These results confirmed
that peroxovanadate caused a pronounced increase in tyrosine
phosphorylation. They also showed a rather low level of phosphorylation
in normal mouse small intestine. Notably, the crypts showed the most
remarkable increase in tyrosine phosphorylation compared with the
villi. Analysis of digital data on these slides using a Bioquant TCW-95
program (R & M Biometrics, Nashville, TN) and a Sony DXC-151A
charge-coupled device camera indicated that the average
signal in the peroxovanadate-treated crypts was ~100 times that in
the villus.
|
Peroxovanadate treatment reduces the amount of DNA
fragmentation in ex vivo intestinal epithelial cell
cultures. We hypothesized that a reduction in PTPase
activity would not only increase protein tyrosine phosphorylation in
our cell cultures but would also decrease apoptosis. Indeed, the
epithelium of the peroxovanadate-treated ileum had a more orderly
appearance, with deeper crypts and longer villi compared with the
disintegrated dying intestinal epithelium (Fig. 4 and data not shown).
To further evaluate the influence of PTPase inhibition on apoptosis, we
analyzed DNA laddering in treated mucosal suspensions. We have
previously shown that the DNA from dying enterocytes on electrophoretic
gels displays the laddered appearance characteristic of
internucleosomal degradation of DNA, a hallmark feature of most forms
of apoptosis (18). As shown in Fig. 5, DNA
was purified from cells exposed to different concentrations of
peroxovanadate and then resolved by electrophoresis on agarose gels.
Peroxovanadate treatment decreased DNA fragmentation in a
dose-dependent manner, correlating with the dose-response curve of
protein tyrosine phosphorylation. In contrast, sodium vanadate (1 mM)
or
H2O2
(8 mM) had no appreciable effect on DNA fragmentation at the
concentrations tested (data not shown).
|
Peroxovanadate treatment is required within the
initial 5 min of cell culture at 37°C.
Peroxovanadate caused a sustained increase in tyrosine phosphorylation
in many proteins through the entire culture period. To determine
whether the inhibitory effect of peroxovanadate on DNA fragmentation
occurred at an early, intermediate, or late stage in the death pathway,
cells were treated with peroxovanadate at varying times after the start
of cell culture at 37°C (Fig. 6). This
experiment showed that peroxovanadate effectively prevented DNA
fragmentation when added at 0 or 5 min of culture but became ineffective at later times. Thus the essential PTPase inhibitory action
in this model occurs within minutes of cell culture, coincident with
the initial rapid decrease in protein tyrosine phosphorylation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein tyrosine phosphorylation by transmembrane and cytoplasmic tyrosine kinases and dephosphorylation by PTPases have been shown to play prominent roles in cell function, particularly in the control of growth and differentiation. Less attention has been paid to the potential regulatory role of protein tyrosine phosphorylation and dephosphorylation in the regulation of programmed cell death. This may be partly attributable to the difficulty of synchronizing death in cell lines. We have developed a new and highly synchronous model of intestinal programmed cell death that uses mucosal cell suspension cultures in a serum-free medium (18). Using this model, we found that the overall level of protein tyrosine phosphorylation rapidly decreased within minutes of incubation in DMEM at 37° C (Fig. 1).
Whereas many membrane and cytosolic proteins showed a significant decrease in their phosphotyrosine content during the first 10-15 min of culture, two prominently phosphorylated membrane proteins showed an extremely rapid apparent loss of phosphotyrosine between 2.5 and 5 min. The identity of these proteins, which are estimated to be 87 and 90 kDa, is unknown. We originally speculated that the 87-kDa protein was the regulatory subunit of phosphatidylinositol 3-kinase, which has been implicated in the regulation of programmed cell death (6, 23, 26); however, the position of this protein did not precisely correlate with phosphatidylinositol 3-kinase by immunoblot (data not shown). Whether the loss of phosphotyrosine for the 87- and 90-kDa proteins is due to an abrupt decrease in a specific PTKase activity or an increase in PTPase activity or both is not known. Other explanations for the disappearance of signal are also possible. The apparent loss in phosphotyrosine could be mediated by proteolytic degradation or by translocation from the membrane to the cytosol. The latter possibility seems unlikely, however, because the loss of signal at the 87- and 90-kDa positions in the membrane fraction was not accompanied by a corresponding increase of signal in the cytoplasmic fraction.
Our experiments underscore the potential importance of PTPase activity in either the regulation or possible therapeutic prevention of programmed cell death. Exposure of intestinal epithelial cells to a vanadate/H2O2 mixture resulted in the rapid and sustained appearance of many tyrosine-phosphorylated proteins (Figs. 2 and 3A), including the 87- and 90-kDa proteins noted above. It also increased the dually phosphorylated and active form of the important intracellular regulatory protein MAP kinase (Fig. 3B), which in some cell systems has an anti-apoptotic role (10, 27) and which also became dephosphorylated during cell death. Although an immunohistochemical analysis revealed increased tyrosine phosphorylation in both crypt and villus compartments, it was more pronounced in the crypt (Fig. 4). This may reflect a higher basal level of PTKase activity or a lower basal level of PTPase activity in the crypt than the villus.
Although peroxovanadate inhibited programmed cell death, sodium vanadate and H2O2 had little or no independent action on death at the concentrations tested. Yet, in related recent work, other investigators have presented convincing histochemical evidence that 1 mM vanadate inhibits programmed cell death of isolated rat colonic crypt cells (7). The requirement for a more potent PTPase inhibitor to inhibit programmed cell death in the small intestine suggests that it may have a higher level of endogenous PTPase activity than the colon. If so, this may contribute to the resistance of the ileum compared with the colon to neoplastic transformation.
Peroxovanadate preserved the histological appearance of the cultured intestine, which more closely resembled the normal intestine (Fig. 4 and data not shown). Peroxovanadate also decreased DNA fragmentation in cell cultures, provided it was added within the initial 5 min of cell culture at 37°C (Figs. 5 and 6). When peroxovanadate was added at later times, DNA fragmentation could not be prevented. Because significant DNA fragmentation does not manifest until 60-90 min of cell culture, this experiment suggests that the antiapoptotic action of PTPase inhibition by peroxovanadate occurred shortly after exposure to culture medium and that the rapid tyrosine dephosphorylation seen within the first 5 min of culture may be a critical step in the cellular progression to death.
If so, then the tyrosine phosphorylation/dephosphorylation steps required for cell survival may occur in the initiation phase of programmed cell death as opposed to the later commitment and execution phases. The later phases involve alterations in mitochondria, leakage of cytochrome c, and activation of caspases and DNA fragmentation factors. Much less is known about the initiation phase, which is associated with cell surface proteins and membrane events such as membrane blebbing. Because the disruption of cell-cell and cell-basement membrane contacts normally occurs as intestinal epithelial cells die and exfoliate, the phosphatase-mediated dephosphorylation of proteins involved in cell-cell or cell-basement membrane contacts may initiate a death program in our suspension cultures.
Focal adhesion kinase (FAK) and EGFR are two candidate PTPase targets that play an important role in the maintenance of tissue integrity and may be involved in the progression of intestinal apoptosis. Both proteins have been shown to be regulated by PTPases (12, 15). The interaction of extracellular matrix proteins and integrins can activate FAK, which is a cytoplasmic tyrosine kinase implicated in integrin-mediated signal transduction pathways. During integrin-mediated cell adhesion, FAK in surviving cells becomes activated, tyrosine phosphorylated, and colocalized to focal contacts with integrins and other cytoskeletal proteins. During endothelial cell apoptosis, FAK became dephosphorylated on tyrosine residues (2, 8, 15, 27). Moreover, constitutively activated forms of FAK rescued two established epithelial cell lines from anoikis-type apoptotic death (2).
A potential role for the EGFR and other Erb-related proteins in
enterocyte adherence, migration, and death is of particular interest,
since these proteins have no clear function in postmitotic intestinal
epithelial cells but are expressed at a relatively high level (13, 19).
Recently, integrins have been shown to physically associate with EGFR
and to cause it to autophosphorylate in a ligand-independent manner as
part of a novel signaling pathway to promote adhesion-dependent cell
survival (10). Moreover, in some tumor cells, EGFR kinase blockade by
monoclonal antibodies or pharmacological inhibitors caused apoptosis
(9). In other tumor cells, an early cell surface event in the death
pathway induced by tumor necrosis factor- was EGFR dephosphorylation by PTPase 1B (12).
By inhibiting PTPases, peroxovanadate globally increases tyrosine phosphorylation of cell surface receptors, cytoplasmic and nuclear proteins, even in the absence of receptor tyrosine kinase-activating ligands (16). The present report suggests that PTPases may play an important role in the positive regulation of programmed cell death of intestinal epithelial cells. Although the role of PTPases in this process has not received much attention, the forced overexpression of the leukocyte common antigen-related (LAR) phosphatase has recently been shown to cause apoptosis in 3T3 cells (24). We have found a high expression level of this transmembrane tyrosine phosphatase in the ileum (data not shown). Thus it will be of interest to determine whether LAR or other membrane-bound or cytosolic PTPases are activated or stabilized during the initiation phase of cell death and to determine to what extent the death pathways in the replicating crypt and postmitotic villus cells differ from each other and from various apoptotic pathways currently being defined in other models.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by Ross Products Division-Abbott Laboratories (BE97) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45925.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. A. Scheving, Dept. of Pediatrics, Div. of Pediatric Gastroenterology, Vanderbilt Univ. School of Medicine, 21st and Garland, Nashville, TN 37232-2561 (E-mail: larry.scheving{at}mcmail.vanderbilt.edu).
Received 10 February 1999; accepted in final form 27 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Boylan, J. M.,
and
P. A. Gruppuso.
Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat.
J. Biol. Chem.
273:
3784-3790,
1998
2.
Frisch, S. M.,
K. Vuori,
E. Ruoslahti,
and
P. Y. Chan-Hui.
Control of adhesion-dependent cell survival by focal adhesion kinase.
J. Cell Biol.
134:
793-799,
1996[Abstract].
3.
Grossmann, J.,
J. M. Maxson,
C. M. Whitacre,
D. E. Orosz,
N. A. Berger,
C. Fiocchi,
and
A. D. Levine.
New isolation technique to study apoptosis in human intestinal epithelial cells.
Am. J. Pathol.
153:
53-62,
1998
4.
Grossmann, J.,
S. Mohr,
E. G. Lapetina,
C. Fiocchi,
and
A. D. Levine.
Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G1117-G1124,
1998
5.
Ikeda, H.,
Y. Suzuki,
M. Suzuki,
M. Koike,
J. Tamura,
J. Tong,
M. Nomura,
and
G. Itoh.
Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium.
Gut
42:
530-537,
1998
6.
Kulik, G.,
A. Klippel,
and
M. J. Weber.
Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.
Mol. Cell. Biol.
17:
1595-1606,
1997[Abstract].
7.
Lifshitz, S.,
B. Schwartz,
S. Polak-Charcon,
D. Benharroch,
I. Prinsloo,
and
S. A. Lamprecht.
Extensive apoptotic death of rat colonic cells deprived of crypt habitat.
J. Cell. Physiol.
177:
377-386,
1998[Medline].
8.
Marushige, Y.,
and
K. Marushige.
Alterations in focal adhesion and cytoskeletal proteins during apoptosis.
Anticancer Res.
18:
301-307,
1998[Medline].
9.
Modjtahedi, H.,
K. Affleck,
C. Stubberfield,
and
C. Dean.
EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5.
Int. J. Oncol.
13:
335-342,
1998[Medline].
10.
Moro, L.,
M. Venturino,
C. Bozzo,
L. Silengo,
F. Altruda,
L. Beguinot,
G. Tarone,
and
P. Defilippi.
Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival.
EMBO J.
17:
6622-6632,
1998
11.
Noda, T.,
R. Iwakiri,
K. Fujimoto,
S. Matsuo,
and
T. Y. Aw.
Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G270-G276,
1998
12.
Perez, M.,
B. Haschke,
and
N. J. Donato.
Differential expression and translocation of protein tyrosine phosphatase 1B-related proteins in ME-180 tumor cells expressing apoptotic sensitivity and resistance to tumor necrosis factor: potential interaction with epidermal growth factor receptor.
Oncogene
18:
967-978,
1999[Medline].
13.
Polk, D. B.
Epidermal growth factor receptor-stimulated intestinal epithelial cell migration requires phospholipase C activity.
Gastroenterology
114:
493-502,
1998[Medline].
14.
Potten, C. S.,
and
T. D. Allen.
Ultrastructure of cell loss in intestinal mucosa.
J. Ultrastruct. Res.
60:
272-277,
1977[Medline].
15.
Retta, S. F.,
S. T. Barry,
D. R. Critchley,
P. Defilippi,
L. Silengo,
and
G. Tarone.
Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity.
Exp. Cell Res.
229:
307-317,
1996[Medline].
16.
Ruff, S. J.,
K. Chen,
and
S. Cohen.
Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney.
J. Biol. Chem.
272:
1263-1267,
1997
17.
Scheving, L. A.,
and
W. Gardner.
Circadian regulation of CREB transcription factor in mouse esophagus.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1011-C1016,
1998
18.
Scheving, L. A.,
W. H. Jin,
K. M. Chong,
W. Gardner,
and
F. O. Cope.
Dying enterocytes downregulate signaling pathways converging on Ras: rescue by protease inhibition.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1363-C1372,
1998
19.
Scheving, L. A.,
R. A. Shiurba,
T. D. Nguyen,
and
G. M. Gray.
Epidermal growth factor receptor of the intestinal enterocyte. Localization to laterobasal but not brush border membrane.
J. Biol. Chem.
264:
1735-1741,
1989
20.
Scheving, L. E.,
T. H. Tsai,
and
L. A. Scheving.
Chronobiology of the intestinal tract of the mouse.
Am. J. Anat.
168:
433-465,
1983[Medline].
21.
Su, Y.,
Y. Shi,
M. A. Stolow,
and
Y. B. Shi.
Thyroid hormone induces apoptosis in primary cell cultures of tadpole intestine: cell type specificity and effects of extracellular matrix.
J. Cell Biol.
139:
1533-1543,
1997
22.
Sun, Z.,
X. Wang,
R. Wallen,
X. Deng,
X. Du,
E. Hallberg,
and
R. Andersson.
The influence of apoptosis on intestinal barrier integrity in rats.
Scand. J. Gastroenterol.
33:
415-422,
1998[Medline].
23.
Ueno, H.,
H. Honda,
T. Nakamoto,
T. Yamagata,
K. Sasaki,
K. Miyagawa,
K. Mitani,
Y. Yazaki,
and
H. Hirai.
The phosphatidylinositol 3' kinase pathway is required for the survival signal of leukocyte tyrosine kinase.
Oncogene
14:
3067-3072,
1997[Medline].
24.
Weng, L. P.,
J. Yuan,
and
Q. Yu.
Overexpression of the transmembrane tyrosine phosphatase LAR activates the caspase pathway and induces apoptosis.
Curr. Biol.
8:
247-256,
1998[Medline].
25.
Yamada, M.,
Y. Enokido,
T. Ikeuchi,
and
H. Hatanaka.
Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons.
Eur. J. Neurosci.
7:
2130-2138,
1995[Medline].
26.
Yao, R.,
and
G. M. Cooper.
Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor.
Science
267:
2003-2006,
1995[Medline].
27.
Yang, C.,
J. Chang,
M. Gorospe,
and
A. Passaniti.
Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.
Cell Growth Differ.
7:
161-171,
1996[Abstract].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |