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Am J Physiol Cell Physiol 290: C1310-C1320, 2006. First published December 14, 2005; doi:10.1152/ajpcell.00450.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-{kappa}B signaling in intestinal epithelial cells

Huifang M. Zhang,1,3,* Kaspar M. Keledjian,1,3,* Jaladanki N. Rao,1,3 Tongtong Zou,1,3 Lan Liu,1,3 Bernard S. Marasa,2,3 Shelley R. Wang,3 Lisa Ru,3 Eric D. Strauch,1 and Jian-Ying Wang1,2,3

Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine, Baltimore; and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland

Submitted 6 September 2005 ; accepted in final form 10 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-{kappa}B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-{kappa}B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-{alpha} plus cycloheximide (TNF-{alpha}/CHX). Specific inhibition of NF-{kappa}B by the recombinant adenoviral vector containing the I{kappa}B{alpha} superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-{kappa}B activity and increased the sensitivity to TNF-{alpha}/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-{kappa}B activity and resulted in increased resistance to TNF-{alpha}/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-{alpha}/CHX-induced apoptosis, at least partially, through the activation of NF-{kappa}B signaling in IECs.

polyamines; {alpha}-difluoromethylornithine; X-linked inhibitor of apoptosis protein; I{kappa}B

THE HUMAN INTESTINAL EPITHELIUM has the most rapid turnover rate of any tissue in the body, and its integrity depends on a dynamic balance between cell proliferation, growth arrest, and apoptosis (20, 27, 33). Undifferentiated intestinal epithelial cells (IECs) continuously replicate in the proliferative zone with crypts and differentiate as they migrate up the luminal surface of intestine to replace lost cells (32, 34, 38). To maintain mucosal homeostasis, the rate of IEC proliferation must be counterbalanced by apoptosis, a process that is highly regulated under physiological conditions. It is well established that apoptosis rather than simple exfoliation of enterocytes accounts for the majority of cell loss at the luminal surface of the colon and villous tips in the small intestine (20, 32, 34). Apoptosis also occurs in the crypt area, where this process serves to remove defective or injured undifferentiated progeny cells and maintains the balance in cell number between newly divided and surviving cells (32, 33). Although both crypt and villous cells are susceptible to apoptosis, IEC survival and death are subject to differentiation state-specific control mechanisms (8, 45). Because IECs express differential integrin profiles and interact with specific basement membrane components along the crypt-villous axis, depending on their state of differentiation, distinct cellular signaling pathways are implicated in the modulation of cell survival and apoptosis in undifferentiated/crypt and differentiated/villous cells in the intestinal mucosa. However, the exact mechanisms involved in this process remain elusive.

Focal adhesion kinase (FAK) is a nonreceptor PTK that plays a critical role in a variety of biological functions, including cell proliferation, spreading, motility, and survival or apoptosis (6, 11, 14, 38, 40, 43, 46). FAK was initially isolated and characterized as a tyrosine-phosphorylated protein in v-Src-transformed chicken embryo fibroblast (38). The molecular structure of the FAK protein consists of an NH2-terminal domain with a primary autophosphorylation site, Tyr397; a central catalytic domain with two major sites of phosphorylation, Tyr576/Tyr577; and a COOH-terminal domain containing two proline-rich segments and a FAK-targeting subdomain (12, 14, 38). The activity of FAK is regulated not only by integrin signaling (39, 40) but also by various soluble growth factors, including PDGF, VEGF, and growth hormones (36, 42). FAK is thus involved in signaling pathways initiated by integrins and also plays an essential role in the regulation of cell survival and apoptosis. For example, forced expression of FAK protects human leukemia HL-60 cells from apoptosis caused by oxidative stress, etoposide, and ionizing radiation (21, 43, 44) and protects Madin-Darby canine kidney cells from UV-induced apoptosis (6). To determine the mechanism by which FAK regulates apoptosis, it was demonstrated previously that overexpression of FAK induces the constitutive activation of NF-{kappa}B in several types of cells (43, 48). A recent study further showed that TNF-{alpha}-induced NF-{kappa}B activation is impaired in mouse FAK-deficient fibroblasts (10). To our knowledge, no studies published to date have investigated the potential role of FAK in the regulation of apoptosis and its relationship to NF-{kappa}B in normal IECs.

Because the regulation of apoptosis in IECs involves distinct mechanisms according to the state of differentiation (8, 45) and because FAK has an antiapoptotic role in many types of cells (6, 37, 43, 47), in the current study, we tested the hypotheses that FAK is a cell survival factor in intestinal undifferentiated/crypt cells and that induced FAK expression inhibits apoptosis by activating NF-{kappa}B signaling. To do so, we first developed and characterized stable wild-type (WT)- or dominant-negative mutant (DNM)-FAK-transfected IECs with the undifferentiated phenotype and further determined whether manipulating FAK activity, by either increasing or decreasing its concentration, affected the susceptibility to apoptosis. Second, we determined whether FAK regulates apoptosis by altering NF-{kappa}B signaling in undifferentiated crypt cells. Third, we examined whether increased expression of endogenous FAK by depletion of cellular polyamines by treatment with {alpha}-difluoromethylornithine ({alpha}-DFMO), a specific inhibitor of polyamine biosynthesis (28, 29), alters NF-{kappa}B activity and susceptibility to apoptosis in parental IEC-6 cells. Some of these data have been published previously in abstract form (51).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and supplies. Disposable cultureware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed FBS were purchased from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma Chemical (St. Louis, MO). {alpha}-DFMO was purchased from Ilex Oncology (San Antonio, TX). Antibody against phosphorylated FAK (pFAK) at Tyr397, pFAK at Tyr925, or total FAK (tFAK), and antibody against NF-{kappa}B p65 subunit or c-Rel were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [{gamma}-32P]ATP (3,000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).

Plasmid construction and trasnfection. The Myc-tagged cDNA encoding full-length murine WT-FAK or DNM-FAK, the F397-F576-F577 triple mutation, was subcloned into the NotI (blunt ended with Klenow polymerase)-to-XbaI restriction enzyme site of the pRc/cytomegalovirus (CMV) promoter-driven eukaryotic expression vector (Invitrogen) (4, 48). The IEC-6 cells were transfected with the pRc WT-FAK, pRc DNM-FAK, or pRc vector containing no FAK cDNA using the Lipofectamine kit as recommended by the manufacturer (GIBCO-BRL, Gaithersburg, MD). After 3-h incubation, the transfection medium was replaced with standard growth medium containing 5% FBS for 2 days before exposure to the selection medium. These transfected cells were selected for FAK integration by incubation with the selection medium containing 0.6 mg/ml G418, and clones resistant to the selection medium were isolated, cultured, and screened for FAK expression using Western blot analysis with the specific anti-Myc tag antibody. The recombinant adenoviral vector expressing I{kappa}B{alpha} superrepressor (Ad-I{kappa}BSR) was constructed using the Adeno-X expression system (Clontech, Palo Alto, CA) as described in our previous publication (52). The adenoviral particles were propagated in human embryonic kidney HEK-293 cells and purified using CsCl ultracentrifugation. Cells were infected using various concentrations of the Ad-I{kappa}BSR or control vector, and cell samples were collected for various measurements 48 h after infection.

Cell culture and general experimental protocols. The IEC-6 cell line was purchased from the American Type Culture Collection (Manassas, VA) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (35). The IEC-6 cells originated from intestinal crypt cells judged on the basis of morphological and immunological criteria. They were nontumorigenic and retained the undifferentiated character of intestinal crypt cells. Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin and 50 µg/ml gentamicin. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2, and passages 15–20 were used in experiments. There were no significant changes in biological function or characterization of IEC-6 cells at passages 15–20 (24, 25).

In the first series of studies, we developed stable IEC-6 cells highly expressing either WT- or DNM-FAK and further determined whether altered kinase activity of FAK, either increased or decreased, affected the sensitivity of IECs to apoptosis. After transfection and selection, FAK protein levels in stable WT- and DNM-FAK-transfected cells were examined using Western blot analysis, and the functional activities of induced FAK were elucidated by measurement of levels of pFAK at Tyr397 and at Tyr925 using the specific anti-pFAK antibodies. To determine the role of FAK in the regulation of apoptosis in normal IECs, stable WT- and DNM-FAK-transfected cells were initially grown in standard DMEM for 4 days and then exposed to TNF-{alpha} (20 ng/ml) in combination with cycloheximide (CHX; 25 µg/ml). Apoptotic cell death was measured 4 h after administration of TNF-{alpha}/CHX.

In the second series of studies, we determined whether FAK regulated apoptosis of IECs by altering NF-{kappa}B activity. After stable WT- and DNM-FAK-transfected IEC-6 cells were grown in DMEM for 4 days, levels of NF-{kappa}B proteins, sequence-specific DNA binding activity, and NF-{kappa}B transcriptional activity were examined using Western blot analysis, EMSA, and luciferase reporter gene assays, respectively. The observed increase in NF-{kappa}B activity in WT-FAK-transfected cells was specifically prevented by ectopic expression of I{kappa}BSR through infection with the Ad-I{kappa}BSR vector. Apoptosis was induced after cells were infected with Ad-I{kappa}BSR or control adenoviral vector for 48 h.

In the third series of studies, we examined whether increased endogenous FAK by depletion of cellular polyamines altered NF-{kappa}B activity and regulated the sensitivity to TNF-{alpha}/CHX-induced apoptosis in IEC-6 cells. After parental IEC-6 cells were grown in control cultures and cultures containing either 5 mM {alpha}-DFMO alone or {alpha}-DFMO plus 5 µM spermidine (SPD) for 8 days, changes in the levels of tFAK, pFAK, NF-{kappa}B activity, and the apoptotic response to TNF-{alpha}/CHX were examined. In studies dealing with FAK-transfected cells, stable WT- and DNM-FAK-transfected cells were treated with 5 mM {alpha}-DFMO for the same time, and the levels of NF-{kappa}B activity and the apoptotic response to treatment with TNF-{alpha}/CHX were determined.

Western blot analysis. Cell samples dissolved in ice-cold RIPA buffer (49 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM PMSF, 20 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 mM sodium orthovanadate) were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentration of the supernatant was measured using the methods described by Bradford (3), and each lane was loaded with 20 µg of protein equivalent. The supernatant was boiled for 5 min and then subjected to electrophoresis on 7.5% acrylamide gels according to the method of Laemmli (23). Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x Tris-buffered saline (TBS), pH 7.4, with 0.1% Tween 20 (TBST). Overnight immunological evaluation was then performed at 4°C in 5% nonfat dry milk-TBST buffer containing different specific antibodies. The filters were subsequently washed with 1x TBST and incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on the filters were reacted for 1 min with chemiluminiscence reagent (NEL-100; DuPont NEN, Boston, MA).

Preparation of nuclear protein and EMSA. Nuclear proteins were prepared via a procedure described previously (25, 52), and the protein contents in nuclear preparations were measured using the Bradford method (3). The double-stranded oligonucleotides used in these experiments included 5'-AGTTGAGGGGACTTTCCCAGGC-3', which contains a consensus NF-{kappa}B binding site (underlined). These oligonucleotides were end labeled radioactively with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega, Madison, WI). For EMSA, 0.035 pmol/l 32P-labeled oligonucleotides (~30,000 cpm) and 10 µg of nuclear protein were incubated in a total volume of 20 µl in the presence of 10 mM Tris·HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% glycerol, and 1 µl of poly(dI-dC). The binding reactions were allowed to proceed at room temperature for 20 min. Thereafter, 2 µl of bromphenol blue (0.1% in water) were added, and protein-DNA complexes were resolved by performing electrophoresis on nondenaturing 5% polyacrylamide gels and were visualized using autoradiography.

Measurement of caspase-3 activity. Caspase-3 activity was measured using the caspase-3 colorimetric assay kit (R&D Systems, Minneapolis, MN) and performed according to the protocol recommended by the manufacturer. Briefly, cells were treated with TNF-{alpha}/CHX for 4 h, washed with ice-cold Dulbecco's PBS (DPBS), and scraped from the dishes. The collected cells were washed with DPBS and then lysed in ice-cold cell lysis buffer. The assay for caspase-3 activity was performed in a 96-well plate. Each well contained 50 µl of cell lysate (~150 µg of total proteins), 50 µl of reaction buffer {50 mM HEPES, pH 7.4, 0.1% 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, 100 mM NaCl, 10 mM DTT, and 1 mM EDTA}, 5 µl of caspase-3 colorimetric substrate, and a caspase-specific peptide conjugated to a chromogen, p-nitroanilide (p-NA). The 96-well plate was incubated at 37°C for 90 min, during which the caspase-3 in the sample presumably cleaved the chromophore p-NA from the substrate molecule. Absorbency readings at 405 nm were obtained after the incubation, with caspase-3 activity found to be directly proportional to the color reaction. Protein levels of each sample were determined using the Bradford method (3).

Assessment of morphology and annexin V staining. After various experimental treatments, cells were photographed with a Nikon inverted microscope before fixation. Annexin V staining of apoptosis was performed using a commercial apoptosis kit (Clontech) and performed according to the protocol recommended by the manufacturer. Briefly, cells were rinsed with 1x binding buffer and resuspended in 200 µl of 1x binding buffer. Five microliters of annexin V and propidium iodide were added onto the slide and incubated at room temperature for 10 min in the dark. Annexin-stained cells were visualized and photographed under a fluorescence microscope using a dual filter set for FITC and rhodamine, and the percentage of apoptotic cells was determined.

Luciferase assay and transient transfection. The NF-{kappa}B-dependent luciferase reporter gene construct containing the synthetic sequence with four copies of connective NF-{kappa}B-binding elements was obtained from Clontech. Transient transfection was performed using the Lipofectamine kit and performed as recommended by the manufacturer (Invitrogen). The cells were collected at 48 h after transfection, and the luciferase activity from individual transfections was normalized by beta-galactosidase activity from cotransfected plasmid pCMV beta-galactosidase. The experiments were conducted in triplicate and are reported as the means of relative light units/beta-galactosidase.

Statistics. Values are means ± SE of three to six samples. Autoradiographic results were repeated three times. The significance of differences between means was determined using ANOVA. The level of significance was determined using Duncan's multiple-range test (15).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Changes in apoptotic response in stable FAK-transfected IECs. To determine the involvement of FAK in the regulation of apoptosis in intestinal undifferentiated crypt cells, stable DNM-FAK- and WT-FAK-transfected IEC-6 cells were developed and characterized. The expression vectors encoding either DNM-FAK or WT-FAK cDNA with a Myc tag under the control of the CMV promoter were constructed (Fig. 1A). As shown in Fig. 1B, four DNM-FAK and two WT-FAK clones were resistant to the selection medium containing 0.6 mg/ml G418 and highly expressed DNM-FAK or WT-FAK proteins as indicated by an increase in Myc tag levels. Consistently, these stable DNM-FAK-transfected IECs were associated with a significant decrease in levels of pFAK at Tyr397 and Tyr925 (Fig. 1B, left), whereas stable WT-FAK-transfected IECs were paralleled by a dramatic increase in levels of pFAK proteins (Fig. 1B, right). Cells transfected with the pRc-null vector containing no FAK cDNA exhibited no significant change in levels of total and pFAK proteins compared with those observed in parental IEC-6 cells without transfection. We also examined the effects of increased or decreased expression of FAK on cell differentiation by measuring the expression of brush-border enzymes such as sucrase-isomaltase, which demonstrated that both stable DNM-FAK- and WT-FAK-transfected IECs retained the undifferentiated characteristic (data not shown).


Figure 1
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  		Fig. 1. Characterization of stable dominant-negative mutant focal adhesion kinase (DNM-FAK)- and wild-type (WT)-FAK-transfected intestinal epithelial cells (IECs). A: structure of FAK expression vectors. a, Control vector (pRc-null); b, DNM-FAK; and c, WT-FAK. Complete open reading frame (ORF) of DNM-FAK or WT-FAK cDNA containing a Myc tag at the 3' end of the FAK ORF was cloned to an expression pRc vector. PCMV, human cytomegalovirus immediate-early promoter. B: representative immunoblots for phosphorylated FAK (pFAK) and Myc tag proteins. IEC-6 cells were transfected with expression vector containing either DNM-FAK cDNA or WT-FAK cDNA using Lipofectamine, and clones resistant to selection medium containing 0.6 mg/ml G418 were isolated and screened for DNM-FAK or WT-FAK expression using Western blot analysis. Whole cell lysates from each clone (C) and cells transfected with control vector were harvested, applied to each lane equally (20 µg), and subjected to electrophoresis on 7.5% acrylamide gel. pFAK and Myc tag proteins were identified by probing nitrocellulose with the specific antibody that recognizes either pFAK at Tyr397, pFAK at Tyr925, or Myc tag. After the membrane was stripped, it was reprobed for actin, which served as an internal control for equal loading. Three experiments with similar results were performed.

 
Using these stable FAK-transfected IECs, the following two experiments were performed. First, we determined changes in spontaneous apoptotic cell death without any challenge of apoptotic stimulators and demonstrated that forced expression of DNM-FAK or WT-FAK protein failed to induce apoptosis directly in IEC-6 cells. No typical morphological features of apoptosis were identified in cells that highly expressed DNM-FAK or WT-FAK protein as measured by annexin V staining (data not shown). No differences in cell viability were observed between parental IEC-6 cells or in cells transfected with either DNM-FAK or WT-FAK cDNA (Fig. 2A, a vs. c and e).


Figure 2
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  		Fig. 2. Apoptotic response to TNF-{alpha} in combination with cycloheximide (CHX) in parental IEC-6 cells and stable DNM-FAK- and WT-FAK-transfected IECs. A: changes in TNF-{alpha}/CHX-induced apoptosis after various treatments. Parental IEC-6 cells and stable DNM-FAK- and WT-FAK-transfected cells were grown in standard DMEM for 6 days and then exposed to TNF-{alpha}/CHX. Apoptosis was measured using morphological analysis (middle) and ApoAlert annexin V staining (left) 4 h after treatment with TNF-{alpha}/CHX. a, control; b, control cells exposed to TNF-{alpha}/CHX; c, DNM-FAK cells (C1); d, DNM-FAK cells exposed to TNF-{alpha}/CHX; e, WT-FAK cells (C1); and f, WT-FAK cells exposed to TNF-{alpha}/CHX. Original magnification, x150. B: percentage of apoptotic cells after different treatments as described in A. Values are means ± SE of data from 3 experiments. *P < 0.05 vs. cells treated without TNF-{alpha}/CHX; +P < 0.05 vs. control cells treated with TNF-{alpha}/CHX; #P < 0.05 vs. DNM-FAK cells treated with TNF-{alpha}/CHX. C: changes in levels of caspase-3 protein and its enzymatic activity in cells described in A,a, levels of caspase-3 protein, and A,b, caspase-3 activity. After cells were exposed to TNF-{alpha}/CHX for 4 h, levels of procaspase-3 (PCas3), active caspase-3 (Cas3), and caspase-3 activity were identified. Values are means ± SE of data from 6 samples. *P < 0.05 vs. cells treated without TNF-{alpha}/CHX; +P < 0.05 vs. control cells treated with TNF-{alpha}/CHX; #P < 0.05 vs. DNM-FAK cells treated with TNF-{alpha}/CHX.

 
Second, we examined changes in the susceptibility of stable DNM-FAK- and WT-FAK-transfected IECs to TNF-{alpha}/CHX-induced apoptosis. Inhibition of FAK activation by ectopic expression of DNM-FAK significantly enhanced IECs to TNF-{alpha}/CHX-induced apoptosis (Fig. 2). Typical morphological features of apoptosis increased markedly when stable DNM-FAK-transfected IECs were exposed to TNF-{alpha}/CHX (Fig. 2A,b vs. d). The percentages of apoptotic cells were increased from ~50% in cells transfected with the control vector to ~80% in clone 1 (C1) of DNM-FAK-transfected cells after exposure to TNF-{alpha}/CHX (Fig. 2B). Consistently, stable DNM-FAK-transfected cells exhibited increased activation of caspase-3 as indicated by increases in active caspase-3 protein levels (Fig. 2C,a) and enzymatic activity (Fig. 2C,b) compared with those observed in control cells after treatment with TNF-{alpha}/CHX. Increased susceptibility to TNF-{alpha}/CHX-induced apoptosis in C1 of DNM-FAK-transfected cells did not result simply from clonal variation, because identical results were obtained when the other three clones, C2, C3, and C4, of stable DNM-FAK-transfected cells were treated with TNF-{alpha}/CHX (data not shown). On the other hand, induced activation of FAK by ectopic expression of WT-FAK protected IECs against TNF-{alpha}/CHX-induced apoptosis (Fig. 2A,b vs. f, and B, right). The percentages of TNF-{alpha}/CHX-induced apoptosis were decreased from ~50% in control cells to ~34% in C1 of stable WT-FAK-transfected IECs. The activation of caspase-3 in C1 of stable WT-FAK-transfected IECs was lower than that of control cells and cells transfected with DNM-FAK (Fig. 2C). We made similar observations when C2 of stable WT-FAK-transfected IECs was examined (data not shown). These results indicate that FAK plays a critical role in the regulation of apoptosis in IECs and that increased FAK activation promotes cell survival, but that decreased FAK enhances apoptotic cell death after administration of TNF-{alpha}/CHX.

Effect of FAK activation on NF-{kappa}B activity. To determine the possibility that FAK activation regulates apoptosis by altering the NF-{kappa}B signaling pathway, we examined changes in NF-{kappa}B sequence-specific DNA binding, NF-{kappa}B-dependent transcriptional activity, and a NF-{kappa}B downstream target protein, X-linked inhibitor of apoptosis protein (XIAP), in stable DNM-FAK- and WT-FAK-transfected IECs. Stable DNM-FAK-transfected IECs exhibited dramatically decreased levels of NF-{kappa}B binding activity (Fig. 3A, left) and NF-{kappa}B transcriptional activity as measured using the luciferase reporter of NF-{kappa}B-dependent promoter assay (Fig. 3B). Levels of NF-{kappa}B sequence-specific DNA biding activity and NF-{kappa}B-dependent transcriptional activity were decreased by ~85% compared with those measured in cells transfected with control vector containing no FAK cDNA. Inhibition of NF-{kappa}B-dependent transcriptional activity after inactivated FAK in DNM-FAK-transfected cells is specific, because there were no significant differences in luciferase reporter activity of Smad-dependent promoter constructs between parental IEC-6 cells (12 ± 0.6; n = 6), DNM-FAK-transfected cells (11.2 ± 0.7; n = 6), or WT-FAK-transfected cells (13.3 ± 0.6; n = 6) as measured using the methods described in our previous publication (26). Consistent with a decrease in NF-{kappa}B activity, levels of XIAP also significantly decreased in stable DNM-FAK-transfected IECs and were ~35% of the control value (Fig. 3C). Because NF-{kappa}B and NF-{kappa}B-mediated XIAP expression play a critical role in the protection of IECs against apoptosis (25, 52), these results suggest that increased susceptibility of DNM-FAK-transfected IECs to apoptosis results at least partially from the inactivation of NF-{kappa}B signaling pathway.


Figure 3
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  		Fig. 3. Changes in NF-{kappa}B activity and its downstream target X-linked inhibitor of apoptosis protein (XIAP) in parental IEC-6 cells and stable DNM-FAK- and WT-FAK-transfected IECs. A: representative autoradiograms of NF-{kappa}B sequence-specific DNA binding. Nuclear extracts were prepared, and EMSA was performed using 10 µg of nuclear proteins and 0.035 pmol/l of 32P end-labeled oligonucleotides containing a single NF-{kappa}B binding site. Position of the specifically bound DNA-protein complex is indicated. Three separate experiments were performed with similar results. B: changes in NF-{kappa}B transcriptional activity measured using NF-{kappa}B-dependent promoter activity in cells described in A. Cells were transfected with NF-{kappa}B-dependent promoter luciferase reporter plasmid, and luciferase activity was measured 48 h after transfection. NF-{kappa}B-dependent promoter activity data was normalized to beta-galactosidase activity from cotransfection of pRSV beta-galactosidase. Values are means ± SE of data gathered from 6 dishes. *P < 0.05 vs. control groups. C: levels of NF-{kappa}B downstream target XIAP in cells described in A. Levels of XIAP were measured using Western blot analysis, and actin served as an internal control for equal loading.

 
In contrast, stable WT-FAK-transfected IECs exhibited a marked increase in NF-{kappa}B binding activity (Fig. 3A, right) and NF-{kappa}B-dependent transcriptional activity (Fig. 3B). Levels of NF-{kappa}B-dependent transcriptional activity in stable WT-FAK-transfected IECs were ~3 times control values and >10 times the values observed in stable DNM-FAK-transfected cells. Expression of XIAP was also increased in stable WT-FAK-transfected IECs (Fig. 3C, right). Levels of XIAP in stable WT-FAK-transfected cells were more than twice the control value. These results indicate that increased FAK activity activates NF-{kappa}B signaling pathway, suggesting that FAK-mediated NF-{kappa}B activation results in resistance to apoptosis in stable WT-FAK-transfected IECs.

Effect of inactivation of NF-{kappa}B on apoptosis in WT-FAK-transfected IECs. To further determine the relationship between activated NF-{kappa}B and increased resistance to apoptosis, we examined the effect of inactivation of NF-{kappa}B by ectopic expression of the I{kappa}B{alpha}SR on the susceptibility to TNF-{alpha}/CHX-induced apoptosis in stable WT-FAK-transfected IECs. The adenoviral vector encoding nondegradable I{kappa}B{alpha} mutant (I{kappa}B{alpha}SR) cDNA under the control of the CMV promoter (Ad-I{kappa}BSR) was constructed (19, 31). Consistent with our previous study (25, 52), I{kappa}B{alpha}SR protein was expressed in amounts that increased in tandem with Ad-I{kappa}BSR load in WT-FAK-transfected IECs (data not shown), whereas an adenovirus that lacked exogenous I{kappa}B{alpha} mutant cDNA (Ad-null) did not induce I{kappa}B{alpha}SR protein levels. Increased levels of I{kappa}B{alpha}SR protein by infection with the Ad-I{kappa}BSR at a concentration of 25 plaque-forming units (PFU)/cell completely blocked NF-{kappa}B sequence-specific DNA binding activity in stable WT-FAK-transfected IECs (Fig. 4A) and also totally decreased expression of XIAP and cellular inhibitor of apoptosis protein (data not shown). We reported similar results in our previous publication (52).


Figure 4
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  		Fig. 4. Effect of inhibition of the NF-{kappa}B activity by ectopic expression of the I{kappa}B superrepressor (I{kappa}B{alpha}SR) gene on apoptosis in stable WT-FAK-transfected IECs after exposure to TNF-{alpha}/CHX. A: NF-{kappa}B sequence-specific DNA binding activity. Stable WT-FAK-transfected IECs were infected with the recombinant adenoviral vector encoding human I{kappa}B{alpha}SR cDNA (Ad-I{kappa}BSR) or an adenoviral vector lacking I{kappa}B{alpha}SR cDNA (Ad-null) at a multiplicity of infection of 25 plaque-forming units (PFU)/cell for 48 h. NF-{kappa}B binding activity was measured using EMSA. Three separate experiments were performed with similar results. B: changes in TNF-{alpha}/CHX-induced apoptosis after various treatments. B,a, cells infected with an Ad-null alone; B,b, cells infected with the Ad-null and then exposed to TNF-{alpha}/CHX; B,c, cells infected with Ad-I{kappa}BSR alone; and B,d, cells infected with the Ad-I{kappa}BSR and then exposed to TNF-{alpha}/CHX. After 48-h infection, cells were exposed to TNF-{alpha}/CHX, and apoptotic cell death was examined 4 h thereafter. Original magnification, x150. C: %apoptotic cells among cells described in B. Values are means ± SE from 3 separate experiments. *P < 0.05 vs. no-TNF-{alpha}. +P < 0.05 vs. cells infected with the Ad-null and then treated with TNF-{alpha}/CHX.

 
The results presented in Fig. 4, B and C, show that inhibition of NF-{kappa}B activity by infection with Ad-I{kappa}BSR prevented FAK-mediated resistance to TNF-{alpha}/CHX-induced apoptosis in IECs. In the present study, stable WT-FAK-transfected IECs were initially grown in control culture dishes for 4 days and then infected with Ad-I{kappa}BSR or Ad-null at the same concentration. Apoptosis was induced 48 h after infection. Inactivation of NF-{kappa}B by the infection with the Ad-I{kappa}BSR significantly increased the sensitivity of stable WT-FAK-transfected cells to TNF-{alpha}/CHX-induced apoptosis. At 4 h after exposure to TNF-{alpha}/CHX, the percentages of apoptotic cell death were increased from ~37% in WT-FAK-transfected IECs infected with Ad-null vector to ~72% in cells infected with Ad-I{kappa}BSR at the concentration of 25 PFU/cell. In addition, infection with the Ad-null vector did not affect the sensitivity to apoptosis in stable WT-FAK-transfected IECs. There were no significant changes in percentages of apoptosis in WT-FAK-transfected IECs infected with and without the Ad-null vector after treatment with TNF-{alpha}/CHX. These findings suggest that induced activation of NF-{kappa}B in stable WT-FAK-transfected cells plays an important role in the increased resistance to TNF-{alpha}/CHX-induced apoptosis.

Increased FAK and susceptibility to apoptosis in polyamine-deficient cells. To determine the physiological role of induced FAK in the regulation of apoptosis in IECs, the effect of the observed increase in endogenous FAK activity after polyamine depletion on TNF-{alpha}/CHX-induced apoptosis was examined in parental IEC-6 cells. Our previous studies (25, 50, 52) and studies performed by other researchers (7, 31) have shown that polyamines are multiple functional cellular regulators and that depletion of cellular polyamines promotes the resistance to apoptosis in normal IECs. In the present study, we first elucidated the effect of polyamine depletion on FAK activity and then determined the potential role of the observed increase in FAK activity in the regulation of apoptosis in polyamine-deficient cells. Exposure of IEC-6 cells to 5 mM {alpha}-DFMO completely inhibited ornithine decarboxylase (a key enzyme required for polyamine biosynthesis) activity and almost totally depleted cellular polyamines. The levels of putrescine and SPD were undetectable on day 8 after treatment with {alpha}-DFMO, and spermine was decreased by ~65% (data not shown). We reported similar results in our previous publications (25, 53).

As shown in Fig. 5A, depletion of cellular polyamines by {alpha}-DFMO significantly increased levels of pFAK at Tyr397 and Tyr925, although it had no effect on the expression of tFAK protein. Levels of pFAK at Tyr397 and Tyr925 in the {alpha}-DFMO-treated IEC-6 cells were ~2.5 and ~2.6 times the control values (without {alpha}-DFMO), respectively. The addition of exogenous polyamine (5 µM SPD) together with {alpha}-DFMO completely prevented the increased levels of pFAK. The addition of putrescine (10 µM) in place of SPD had an identical effect on levels of pFAKs when it was added to cultures that contained {alpha}-DFMO (data not shown). Consistent with observations in stable WT-FAK-transfected IECs (Fig. 3), increased levels of pFAKs were associated with activation of NF-{kappa}B as indicated by increases in NF-{kappa}B protein (Fig. 5B,a) and its sequence-specific DNA binding activity (Fig. 5B,b). Levels of p65 protein and NF-{kappa}B binding activity in polyamine-deficient cells were more than twice the control values, which were completely prevented by adding exogenous SPD together with {alpha}-DFMO. As reported in our previous publications (25, 50, 52), polyamine depletion by treatment with {alpha}-DFMO protected IEC-6 cells from TNF-{alpha}/CHX-induced apoptosis. In polyamine-depleted cells, treatment with TNF-{alpha}/CHX caused no significant apoptosis, although it induced typical apoptotic cell death in control cells (Fig. 5C). When SPD was added concomitantly with {alpha}-DFMO, the protective effect of polyamine depletion on TNF-{alpha}/CHX-induced apoptosis was completely prevented.


Figure 5
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  		Fig. 5. Effect of increased levels of endogenous pFAK by depletion of cellular polyamines on NF-{kappa}B activity and TNF-{alpha}/CHX-induced apoptosis in IEC-6 cells. Cells were grown in the control culture or cultures containing either 5 mM {alpha}-difluoromethylornithine ({alpha}-DFMO; specific inhibitor of polyamine biosynthesis) or {alpha}-DFMO plus 5 µM spermidine (SPD) for 8 days. A: representative immunoblots for pFAK and total FAK (tFAK) in presence or absence of cellular polyamines. Whole cell lysates were harvested, and levels of pFAK at Tyr397 and Tyr927 and tFAK were assessed using Western blot analysis with different specific antibodies. Actin immunoblot analysis was performed as an internal control for equal loading. Three experiments were performed with similar results. B: changes in NF-{kappa}B protein (a) and its DNA binding activity (b) in cells described in A. Total and nuclear proteins were isolated, and levels of NF-{kappa}B subunit p65 and specific-sequence DNA binding activity were measured using Western blot analysis and EMSA, respectively. C: apoptotic response of control and polyamine-deficient IEC-6 cells to TNF-{alpha}/CHX. Apoptosis was measured after treatment with TNF-{alpha}/CHX for 4 h. Values are means ± SE of data from 3 experiments. *P < 0.05 vs. cells treated without TNF-{alpha}/CHX.

 
On the other hand, inactivation of FAK by ectopic expression of DNM-FAK not only prevented the induced NF-{kappa}B activation but also blocked increased resistance to TNF-{alpha}/CHX-induced apoptosis in polyamine-deficient cells. As shown in Fig. 6, treatment of stable DNM-FAK-transfected IECs with {alpha}-DFMO induced neither NF-{kappa}B activation nor a protective effect on TNF-{alpha}/CHX-induced apoptosis. In fact, there was no significant induction of NF-{kappa}B binding activity (Fig. 6A,a) or NF-{kappa}B-dependent transcriptional activity (Fig. 6A,b) in stable DNM-FAK-transfected cells, regardless of treatment with or without {alpha}-DFMO. Consistently, exposure to TNF-{alpha}/CHX caused typical apoptotic cell death in stable DNM-FAK-transfected IECs treated with or without {alpha}-DFMO. The percentages of apoptotic cells in DNM-FAK-transfected IECs were ~75% after being cultured in control medium and ~70% after being cultured in medium containing 5 mM {alpha}-DFMO. In contrast, levels of NF-{kappa}B binding activity and NF-{kappa}B-dependent transcriptional activity increased dramatically in stable WT-FAK-transfected IECs in the presence or absence of {alpha}-DFMO. NF-{kappa}B binding activity levels and luciferase reporter activities of the NF-{kappa}B-dependent promoter led to additional increases in stable WT-FAK-transfected IECs after polyamine depletion (Fig. 6A, right). Furthermore, the percentages of TNF-{alpha}/CHX-induced apoptosis in {alpha}-DFMO-treated WT-FAK-transfected IECs were lower than those of stable WT-FAK-transfected cells cultured in medium containing no {alpha}-DFMO (Fig. 6B, right). These results indicate that induced expression of endogenous FAK after polyamine depletion also activates NF-{kappa}B, thus contributing to the resistance to TNF-{alpha}/CHX-induced apoptosis in IECs.


Figure 6
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  		Fig. 6. Effect of inhibition of FAK activation by ectopic expression of DNM-FAK on NF-{kappa}B binding activity and TNF-{alpha}/CHX-induced apoptosis in the presence or absence of cellular polyamines. A: changes in NF-{kappa}B activity. A,a: representative autoradiograms of sequence-specific NF-{kappa}B binding. Parental IEC-6 cells and stable DNM-FAK- or WT-FAK-transfected cells were grown in control cultures and in cultures containing 5 mM {alpha}-DFMO for 8 days. Nuclear proteins were prepared, and NF-{kappa}B binding activity was measured using EMSA. Position of specifically bound DNA-protein complex is indicated. Three experiments were performed with similar results. A,b: changes in NF-{kappa}B transcriptional activity measured on basis of NF-{kappa}B dependent promoter activity in cells described in A,a. Cells were transfected with NF-{kappa}B-dependent promoter luciferase reporter plasmid, and luciferase activity was measured 48 h after transfection. Values are means ± SE of data gathered from 6 dishes. *P < 0.05 compared with cells treated without {alpha}-DFMO. B: apoptotic response to TNF-{alpha}/CHX in cells described in A. After control IEC-6 cells and stable DNM-FAK- or WT-FAK-transfected cells were grown in presence or absence of {alpha}-DFMO for 8 days, apoptosis was measured after exposure to TNF-{alpha}/CHX for 4 h. Values are means ± SE of data from 3 experiments. *P < 0.05 compared with cells treated without TNF-{alpha}/CHX.

 
Finally, we examined the effect of treatment with the apoptotic stimulator TNF-{alpha}/CHX on FAK phosphorylation and NF-{kappa}B-dependent transcriptional activity in parental IEC-6 cells. The results presented in Fig. 7A showed that exposure to TNF-{alpha}/CHX significantly inhibited FAK phosphorylation as indicated by a decrease in levels of pFAK at Tyr925, although TNF-{alpha}/CHX did not alter levels of tFAK protein. Treatment with TNF-{alpha}/CHX also suppressed NF-{kappa}B-dependent transactivation as shown by an inhibition of luciferase reporter activities of NF-{kappa}B-dependent promoter constructs. The level of NF-{kappa}B-dependent transcriptional activity was inhibited by ~55% after exposure to TNF-{alpha}/CHX for 3 h. These results further support our conclusion and provide additional evidence suggesting that FAK is an important cell survival factor in IECs.


Figure 7
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  		Fig. 7. Effect of treatment with TNF-{alpha}/CHX on FAK phosphorylation and NF-{kappa}B dependent transcriptional activity in IEC-6 cells. A: representative immunoblots for phosphorylated FAK (pFAK) and total FAK (tFAK). Cells were treated with TNF-{alpha}/CHX for 3 h, and then levels of pFAK and tFAK were measured using Western blot analysis. Actin immunoblot analysis was performed as an internal control for equal loading. Three experiments were performed with similar results. B: changes in NF-{kappa}B transcriptional activity measured on basis of NF-{kappa}B dependent promoter activity in cells described in A. After cells were transfected with NF-{kappa}B-dependent promoter luciferase reporter plasmid for 45 h, they were exposed to TNF-{alpha}/CHX. Luciferase activity was measured 3 h after treatment with TNF-{alpha}/CHX. Data regarding NF-{kappa}B dependent promoter activity were normalized to beta-galactosidase activity from cotransfection of pRSV beta-galactosidase. Values are means ± SE of data collected from 6 dishes. *P < 0.05 vs. control groups.

 

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Apoptosis undoubtedly plays a central role in the control of the intestinal mucosal homeostasis, and an imbalance between IEC proliferation and apoptosis has significant pathological consequence; however, the exact mechanisms involved in this process at the cellular and molecular levels are still unclear. Herein we describe our investigation of whether FAK is implicated in the regulation of cell survival and apoptosis in IECs. Induced FAK expression by stable transfection with the FAK gene protected undifferentiated IECs against TNF-{alpha}/CHX-induced apoptosis, whereas overexpression of DNM-FAK promoted susceptibility to apoptosis (Fig. 2). Induced FAK activation was also associated with a significant increase in NF-{kappa}B activity (Fig. 3), and specific inhibition of NF-{kappa}B by infection with the recombinant adenoviral vector containing I{kappa}B{alpha}SR prevented the protective effect of FAK on TNF-{alpha}/CHX-induced apoptosis in stable WT-FAK-transfected IECs (Fig. 4), suggesting that induced FAK suppresses apoptosis by activating NF-{kappa}B signaling. Furthermore, increased levels of endogenous FAK by depletion of cellular polyamines also increased NF-{kappa}B activity and promoted resistance to TNF-{alpha}/CHX-induced apoptosis (Fig. 5), which were completely prevented by overexpression of DNM-FAK (Fig. 6). These findings strongly suggest that FAK has an antiapoptotic role in undifferentiated crypt cells and is important to the biological regulation of intestinal epithelial integrity.

It has been shown that FAK functions as an important cell survival signal in several apoptosis-inducing systems (6, 9, 10, 17, 18, 22, 43, 44). For example, microinjection with the specific anti-FAK antibody or a peptide corresponding to the portion of the beta1-integrin cytoplasmic domain presumed to be required for the beta-integrin-FAK interaction leads to apoptosis in anchorage-dependent cells (17). Constitutively activated FAK protects Madin-Darby canine kidney cells from apoptosis as a result of the loss of matrix contact (6), whereas attenuation of FAK expression results in apoptosis in some tumor cells (9, 11). In another study, ECM survival signals transduced by FAK were shown to inhibit p53-regulated apoptosis by serum withdrawal (12, 18). However, whether FAK plays a role in the regulation of cell survival and apoptosis in normal IECs has not been explored to date. In this study, we have provided evidence demonstrating that induced FAK expression protects undifferentiated crypt cells against TNF-{alpha}/CHX-induced apoptosis and that inactivation of FAK by overexpression of DNM-FAK increases sensitivity to apoptosis. In this study, the TNF-{alpha}/CHX-induced apoptosis model was used for the following reasons: 1) TNF-{alpha} is a proinflammatory cytokine that has a potent cytotoxic effect on IECs and is widely used as a biological apoptosis inducer (5, 25), 2) the cytotoxic effects of TNF-{alpha} are evident only if protein synthesis is inhibited (41), and 3) increased release of TNF-{alpha} in the gut mucosa commonly occurs together with downregulation of protein synthesis under various physiological and pathological conditions (20, 27). Therefore, this model is more applicable than most of the apoptotic models to in vivo conditions and was suitable for the current study.

The results from the present study also suggest that phosphorylation at Tyr397 and Tyr925 is involved in the FAK-protective effect on apoptosis in undifferentiated crypt cells. Levels of pFAK Tyr397 and Tyr925 were increased in stable WT-FAK-transfected IECs, but their levels were significantly decreased in stable DNM-FAK-transfected IECs (Fig. 1) or after exposure to TNF-{alpha}/CHX (Fig. 7). These findings are consistent with results published by other investigators who have demonstrated that Tyr397 and Tyr925 are essential for the antiapoptotic action of FAK as demonstrated by transfection with FAK mutants (Y397F and Y925F) (30, 38). FAK Tyr397 is an autophosphorylation site and a high-affinity binding site for Src homology 2 domains of the Src family of kinases, phosphatidylinositol 3-kinase (PI3-kinase), and PLC-{gamma}. In addition, FAK Tyr925 is a binding site for the Grb2 Src homology 2 domain, and this interaction is implicated in integrin-stimulated activation of Ras. FAK Lys454 is essential for its kinase activity, and transfection with kinase-inactive FAK (K454R) has shown that the catalytic activity of FAK is also necessary for its full antiapoptotic effect (12, 38). These results suggest that the signals through the Src family of kinases, PI3-kinase, and/or Grb2 mediate downstream antiapoptotic signals such as NF-{kappa}B and IAPs in different types of cells.

The results presented in Figs. 3 and 4 provide direct evidence that induced FAK regulates apoptosis by altering NF-{kappa}B signaling in undifferentiated IECs. Stable WT-FAK-transfected IECs showed higher basal NF-{kappa}B activation than that observed in IEC-6 cells transfected with a control vector, but DNM-FAK-transfected IECs exhibited a significant decrease in NF-{kappa}B activity. Specific inhibition of NF-{kappa}B by I{kappa}BSR prevented the increased resistance of WT-FAK-transfected IECs to apoptosis. NF-{kappa}B is an inducible transcription factor and is thought to be the central regulator of the transcription of genes involved in apoptosis (25, 31, 52). Under nonstress conditions, NF-{kappa}B is sequestered in the cytoplasm by binding to inhibitory I{kappa}B proteins. In response to a host of stimuli, I{kappa}B proteins are phosphorylated and then degraded, allowing free NF-{kappa}B to translocate to the nucleus to activate the transcription of specific genes (19). The results presented in Fig. 3C further show that high constitutive NF-{kappa}B activation in stable WT-FAK-transfected IECs was associated with increased expression of XIAP protein, suggesting that FAK-activated NF-{kappa}B suppresses TNF-{alpha}/CHX-induced apoptosis through the stimulation of XIAP expression. This result is not surprising, because our previous studies (52) and of studies conducted by others (16) have demonstrated that IAPs, including XIAP, are downstream targets of NF-{kappa}B in epithelial and endothelial cells. Consistent with our current results, Sonoda et al. (43) reported that overexpression of FAK protects human leukemic HL-60 cells against hydrogen peroxide- or etoposide-induced apoptosis by increasing IAP expression. It has been established that IAPs are potent natural suppressors of apoptosis and function by directly inhibiting the activity of caspases, the principal effectors of apoptotic cell death (16, 19, 52).

The data produced in the present study further suggest that the observation that induced FAK expression suppresses apoptosis in undifferentiated crypt cells is of physiological significance because increased levels of endogenous FAK by depletion of cellular polyamines have an identical effect on NF-{kappa}B and apoptosis in parental IEC-6 cells. The depletion of cellular polyamines by inhibiting their biosynthesis with {alpha}-DFMO-induced levels of pFAKs and increased NF-{kappa}B activity, which were associated with the increased resistance to TNF-{alpha}/CHX-induced apoptosis (Fig. 5). Inactivation of FAK by ectopic expression of DNM-FAK not only prevented the increased NF-{kappa}B but also suppressed resistance to apoptosis in polyamine-deficient cells (Fig. 6), indicating that induced FAK after polyamine depletion inhibits apoptosis at least partially by activating NF-{kappa}B signaling. The natural polyamines SPD and spermine and their precursor, putrescine, are organic cations found in all eukaryotic cells (28, 29). Polyamines have been implicated in a wide variety of biological functions, and the regulation of cellular polyamines has been recognized as the central convergence point for the multiple signaling pathways driving distinct IEC functions. An increasing body of evidence indicates that polyamines are implicated in the regulation of apoptosis in different cell types and that polyamines modulate apoptosis in IECs through multiple signaling pathways. To date, several signaling pathways, including NF-{kappa}B (25, 31, 52), Akt kinase (50), ERK1/2 (1), Bcl-2/Bax (7), and JNK (2), have been shown to be involved in the process by which polyamines modulate apoptosis in IECs. However, the current studies provide new evidence suggesting a role for induced FAK in the regulation of apoptosis in intestinal undifferentiated crypt cells after polyamine depletion.

In summary, these results indicate that FAK is implicated in the regulation of apoptosis in intestinal undifferentiated crypt cells. Our present study further demonstrates that induced FAK expression suppresses apoptosis by stimulating XIAP expression as a result of NF-{kappa}B activation. Increased FAK by stable WT-FAK transfection increases NF-{kappa}B activity and induces XIAP expression, leading to increased resistance to TNF-{alpha}/CHX-induced apoptosis. On the other hand, inactivation of FAK by ectopic expression of DNM-FAK decreases NF-{kappa}B activity, inhibits expression of XIAP, and increases sensitivity to TNF-{alpha}/CHX-induced apoptosis. In addition, increased levels of endogenous pFAK by the depletion of cellular polyamines also activate NF-{kappa}B signaling and induce resistance to TNF-{alpha}/CHX-induced apoptosis. Specific inhibition of FAK activity by overexpression of DNM-FAK not only blocks induced NF-{kappa}B activation but also prevents increased resistance to apoptosis in polyamine-deficient cells. These findings suggest that increased FAK is critical for cell survival in the intestinal mucosa in vivo and contributes to maintenance of IEC integrity under physiological and pathological conditions.


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This work was supported by a Merit Review Grant from the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491. J.-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201 (e-mail: jwang{at}smail.umaryland.edu)

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. Section 1734 solely to indicate this fact.

* H. M. Zhang and K. M. Keledjian contributed equally to this work. Back


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