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. Author manuscript; available in PMC: 2010 Feb 10.
Published in final edited form as: Cancer Biol Ther. 2008 Oct 22;7(10):1694. doi: 10.4161/cbt.7.10.6665

TGFβ modulates PTEN expression independently of SMAD signaling for growth proliferation in colon cancer cells

Jimmy YC Chow 1,†, Jennifer A Cabral 1,†, Jessica Chang 1, John M Carethers 1,2,3,*
PMCID: PMC2820113  NIHMSID: NIHMS125134  PMID: 18769113

Abstract

Signaling pathways enabling transforming growth factor-beta (TGFβ)’s conversion from a tumor suppressor to a tumor promoter are not well characterized. TGFβ utilizes intracellular SMADs to mediate growth suppression; however, TGFβ-induced proliferative pathways may become more apparent when SMAD signaling is abrogated. Here, we determined regulation of the tumor suppressor PTEN by TGFβ utilizing SMAD4-null colon cancer cells. TGFβ downregulated PTEN mRNA and simultaneously induced growth proliferation. TGFβ also induced both SMAD2 and SMAD3 nuclear translocation, but only triggered SMAD2-specific transcriptional activity in the absence of SMAD4. Interference of SMAD2 with DN-SMAD2 enhanced TGFβ-induced cell proliferation, but downregulation of PTEN expression by TGFβ was unaffected. TGFβ increased PI3K tyrosine phosphorylation, and inhibition of PI3K pharmacologically or by DN-p85 transfection reversed both TGFβ-induced PTEN suppression and TGFβ-induced cell proliferation. Thus, TGFβ activates PI3K to downregulate PTEN for enhancement of cell proliferation that is independent of SMAD proteins.

Keywords: transforming growth factor, PTEN, PI3K, colonic cancer, SMAD

Introduction

TGFβ signals through a heterotetrameric receptor complex comprising type II receptors and type I receptors, both serine/threonine kinases,1 followed by a canonical SMAD-dependent signaling cascade.2 The TGFβ-SMAD cascade in epithelial cells is growth suppressive. However, this signaling pathway is disrupted in the majority of colon and pancreatic cancers, usually by frameshift mutation of the type II receptor in microsatellite unstable tumors, by mutation of the kinase domain of TGFBR2,3,4 or by mutation or loss of SMAD4,3,5 or SMAD2.6,7 Also, interference with activated SMAD translocation from the cytoplasm to the nucleus is accomplished by oncogenic K-RAS, mutated in most colon and pancreatic cancers. The disruption of TGFβ-SMAD suppressive signaling is advantageous for the cancer cells to grow.

Growth suppression is supplanted by growth proliferation with loss of TGFβ-SMAD signaling; however, the proliferation seems to be driven by TGFβ, indicating a switch from its normal cellular function. TGFβ ligand is overexpressed in most colon and pancreatic cancers, and may be a consequence of loss of negative feedback signaling through the SMAD pathway. TGFβ1 overexpression is associated with poor survival in patients with CRC.8 The ligand enhances tumor progression in advanced cancers and metastasis.1,9–11 Additionally, conditional ablation of SMAD4 in mouse mammary epithelium confirmed the importance of signaling through the TGFβ/SMAD pathway during tumor initiation and progression12 and there is enhanced colon tumorigenesis in SMAD4-null mouse models.13,14 These findings suggest the existence of a TGFβ-driven proliferative pathway that may be unmasked with loss of suppressive SMAD signaling. Indeed, patient survival with microsatellite unstable colon cancers is improved when TGFBR2 is mutated compared to patients with wild type TGFBR2, suggesting importance in removing any remaining downstream signaling from TGFBR2 activation.15

PTEN (phosphatase and tensin homolog deleted on chromosome ten, also known as MMAC1/TEP1) is localized to chromosome 10q23.16,17 It is a dual-specificity phosphatase that antagonizes the phosphoinositide-3-kinase (PI3K)/ATP-dependent tyrosine kinases (Akt) signaling pathway,18 and thus plays a functional role in cell cycle arrest and apoptosis.19,20 Description of germline mutations and deletions of PTEN in two hereditary cancer predisposition diseases, Cowden Disease and the Bannayan-Riley-Ruvalcaba syndrome,21–24 point to a role of PTEN as a tumor suppressor gene in the pathogenesis of both benign and malignant growth. Mutations in PTEN are found in a variety of cancers.16,17,25 PTEN expression has also been shown to be downregulated by TGFβ1 in keratinocytes,26 and PTEN mRNA levels were also reduced in a model of TGFβ1 overexpressing transgenic mice that develop pancreatic fibrosis.27,28 Reduction of PTEN mRNA levels in pancreatic cancer cells following TGFβ1 treatment has also been reported.28 Loss of PTEN protein expression appears to be common in colon cancer, although detailed mechanisms for reduced expression are not clear, but do include hypermethylation of the PTEN promoter in some cases.29–31

Our present study focused on the regulation of PTEN by TGFβ in colon cancer cells, and whether this modulation is dependent or independent of the TGFβ-SMAD pathway.

Results

TGFβ treatment suppresses PTEN expression in SMAD4-null colonic epithelial cells

We examined the effects of TGFβ upon expression of PTEN in SMAD4-null colon cancer cells. SW480 cells expressed abundant PTEN mRNA as determined by semi-quantitative RT-PCR (Fig. 1A). TGFβ treatment did not affect PTEN expression immediately after treatment but reduced PTEN mRNA by 48 hours. To further assess transcriptional activity, we transfected PTEN-luc into SW480 cells. TGFβ treatment blocked the nascent transcriptional activity of PTEN-luc after 24 hours of treatment and extending to 48 hours (Fig. 1B), compared with our semiquantitative PCR experiments showing loss of amplifiable PTEN mRNA. Thus, TGFβ downregulates PTEN mRNA gene expression by affecting its promoter in SMAD4-null colon cancer cells.

Figure 1.

Figure 1

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TGFβ suppresses PTEN transcription through promoter activity in SMAD4-null colonic epithelial cells. (A) SW480 cells were treated with TGFβ (10 ng/mL) and observed through different time points as indicated. PTEN mRNA expression was measured using RT-PCR. PTEN expression was suppressed 48 hours after TGFβ treatment. GAPDH is a loading control. The agarose gel is representative of 3 separate experiments. (B) SW480 cells were transfected with a plasmid that encoded a PTEN promoter sequence (PTEN-luc). The cells were then treated with TGFβ, which blocks PTEN promoter activity in SW480 colonic cancer cells. ***p < 0.05 vs. control. Data are from at least 3 independent experiments.

TGFβ induces TGFβ-responsive cell proliferation via activation of SMAD2 but not SMAD3, without affecting PTEN transcription

We investigated whether the TGFβ-induced downregulation of PTEN expression was due to activation of canonical intracellular SMAD2 and/or SMAD3, in the absence of SMAD4, as there are reports of TGFβ-SMAD transcriptional activity in SMAD4-null cells.36 To address this, we characterized if both SMAD2 and SMAD3 were activated in SMAD4-null SW480 cells and determined if disruption had any effect on cell proliferation and PTEN expression. Cells transiently transfected with p3TP-luc, containing a binding region for the entire SMAD2/3/4 complex, were transactivated by TGFβ treatment, indicating TGFβ-SMAD activity in these SMAD4-null cells (Fig. 2). However, cells transfected with pCAGA-luc, a specific SMAD3-sensitive promoter, was not activated by TGFβ treatment. This suggests that in SMAD4-null SW480 cells, TGFβ induces a predominant SMAD2-specific transcriptional response and no SMAD3-specific response, even without the presence of SMAD4. To further study the role of SMAD2, we utilized a dominant negative (DN)-SMAD2 plasmid to inhibit native SMAD2 function. Cells were transfected with an empty vector or DN-SMAD2-plasmid containing an upstream FLAG sequence to verify transfection at Western blotting. We demonstrated that DN-SMAD2 expression was persistent through 72 hrs after transfection (Fig. 3A). As shown in Figure 3B, TGFβ treatment for 48 hours induces proliferation of SMAD4-null SW480 cells consistent with and coinciding with the observed reduction of PTEN tumor suppressor expression. Inhibition of TGFβ-activated SMAD2 with DN-SMAD2 enhances the TGFβ-induced cell proliferation (Fig. 3B), indicating that SMAD2 is an important downstream mediator of growth suppression. To determine if TGFβ-induced PTEN suppression was dependent on SMAD2, we assessed PTEN expression while native SMAD2 was inhibited by DN-SMAD2. DN-SMAD2 transfection failed to prevent the TGFβ-induced reduction of PTEN expression, suggesting that TGFβ-induced PTEN suppression is neither dependent on activation of SMAD2 nor the presence of SMAD4 (Fig. 3C). These findings indicate that the regulation of PTEN by TGFβ is SMAD-independent, and TGFβ is mediating its effects on PTEN by another signaling pathway.30,31

Figure 2.

Figure 2

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TGFβ stimulates SMAD2 but not SMAD3 promoter activity in SW480 cells. Cells were transfected with p3TP-luc (binding region for the SMAD complex) or (B) pCAGA-luc (binding region for SMAD3). The cells were then treated with TGFβ and promoter activities of p3TP-luc and pCAGA-luc were studied. TGFβ is able to induce p3TP promoter activity without significantly affecting pCAGA-luc promoter activity. This demonstrates that SMAD2 is a functional and transcriptionally-active factor in SMAD4-null SW480 cells. *p < 0.05 vs. control. Data are from at least 3 independent experiments.

Figure 3.

Figure 3

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Inhibition of SMAD2 by DN-SMAD2 transfection neither reverses TGFβ-induced cell proliferation nor suppression of PTEN expression. (A) SW480 cells were transfected with empty vector or DN-SMAD2 plasmid for 24, 48 and 72 hours. Cells were lysed and the proteins were analyzed by Western blotting. The FLAG protein gene has been subcloned into the DN-SMAD2 plasmid (DN-SMAD2-FLAG) that is expressed in the cells up to 72 hrs after transfection. (B) The proliferation of SW480 cells transfected with DN-SMAD2 followed by TGFβ treatment was ascertained. DN-SMAD2 transfection does not reverse TGFβ-induced cell proliferation. Instead, it potentiates TGFβ-induced cell proliferation (*p < 0.05). Data are from at least 3 independent experiments. (C) PTEN mRNA expression n SW480 cells was suppressed by TGFβ 48 hours after treatment in the control as well as in DN-SMAD2 transfected cells. GAPDH is a loading control. Agarose gel is representative of 3 independent experiments.

TGFβ induces PI3K activity independent of SMAD activation

It has been previously demonstrated that TGFβ can stimulate the PI3K pathway in mammary tumor epithelial cells.37 To examine if TGFβ cold do so in colon tumor cells, we immunoprecipitated the p85 subunit of PI3K from SW480 cells and determined if it was activated by phosphorylation at tyrosine residues. TGFβ induced PI3K activation 24 hours after treatment (Fig. 4), approximating the time for PTEN suppression by TGFβ. To confirm if TGFβ is able to downregulate PTEN protein expression, Western blot analysis demonstrated that PTEN expression is reduced by TGFβ 72 hours after treatment (subsequent to PTEN mRNA reduction) and accompanied by increased Akt activating starting at 24 hours but more prominently activated 72 hours after treatment, using two different SMAD4-null colon cell lines (Fig. 5). To evaluate if PI3K activation was dependent on SMAD2 activation, we assessed if the phosphorylated p85 subunit of PI3K was affected by inhibition of native SMAD2 by DN-SMAD2 after TGFβ treatment. Although DN-SMAD2 significantly reduced TGFβ-induced pSMAD2, DN-SMAD2 did not alter PI3K activation, as determined by expression of phosphorylated p85 (Fig. 6). This finding suggests that TGFβ induces PI3K activation is independent of SMAD activation.

Figure 4.

Figure 4

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TGFβ induces PI3K activation in SW480 cells. Cells were treated with TGFβ and cell lysates were extracted. The equilibrated proteins were then immunoprecipitated with an anti-p85 antibody, and the antibody protein complex were then loaded to a SDS-PAGE and transferred to PVDF membrane and probed with an anti-phospho-tyrosine antibody. TGFβ induced PI3K activity at 24 and 48 hours. Blot is representative of 3 independent experiments.

Figure 5.

Figure 5

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Downregulation of PTEN protein is accompanied by upregulation of Akt activity by TGFβ. (A) SW480 cells and (B) HT29 cells were treated with TGFβ at various time points as shown. Cell lysates were loaded to SDS-PAGE and transferred to PVDF membrane. The proteins were then probed with an anti-PTEN, pAkt and Akt antibodies. TGFβ treatment is able to downregulate PTEN expression at 72 hours. Simultaneously, TGFβ activates Akt beginning at 24 hours, prior to PTEN protein downregulation. Blots are representative of 3 independent experiments.

Figure 6.

Figure 6

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TGFβ no longer activates SMAD2 but still induces PI3K activity in DN-SMAD2 transfected cells. SW480 cells were transfected with a DN-SMAD2 plasmid and treated with TGFβ for 24 hours. Cells were then lysed, the lysates were extracted and proteins equilibrated. For the phosphorylated SMAD2 experiment, total lysates were separated by SDS-PAGE and transferred to PVDF. For the phosphorylated p85 experiment, total lysates were immunoprecipitated with an anti-p85 antibody, and the antibody protein complex were then loaded to a SDS-PAGE and transferred to PVDF membrane. The membranes were then probed with either anti-p-SMAD2 antibody, or anti-phospho-tyrosine antibody. The results indicate that DN-SMAD2 is able to reduce SMAD2 phosphorylation induced by TGFβ, but does not affect TGFβ-induced PI3K activity. Blot is representative of 3 independent experiments.

TGFβ-induced PTEN suppression is dependent on growth proliferative PI3K signaling

To examine the role of PI3K on both PTEN regulation and cell growth by TGFβ, we used a specific PI3K inhibitor, LY290042. This inhibitor reversed TGFβ-induced cell proliferation (Fig. 7A). To confirm this finding, we transfected cells with a DN-p85 plasmid that is identical to the regulatory subunit of PI3K except that it has a p110 binding site mutated,35 disabling the lipid kinase’s function. Like pharmacological inhibition, transfection of DN-p85 reversed TGFβ-induced cell proliferation, but to a greater extent than LY294002 (Fig. 7A). To elucidate if TGFβ-induced activation of PI3K is mediating PTEN suppression, we assessed PTEN expression while simultaneously inhibiting PI3K activation with DN-p85. Inhibition of PI3K activation reversed TGFβ-induced PTEN suppression in two different SMAD4-null colon cancer cell lines, indicating that the PI3K pathway is the mediator of TGFβ’s effects on PTEN (Fig. 7B).

Figure 7.

Figure 7

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Inhibition of PI3K reverses TGFβ-induced cell proliferation. (A) SW480 cells were treated with LY294002 (a specific PI3K inhibitor) or transfected with DN-p85 plasmid followed by TGFβ treatment. Cell numbers were counted after 48 hours. Both pharmacological treatment and DN-p85 transfection showed a dramatic reduction of TGFβ-induced cell proliferation. *p < 0.05 vs. control. This results represents the average of 6 experiments. Baseline represents untreated growth. (B and C) Inhibition of PI3K prevents TGFβ-induced PTEN suppression in SMAD4-null (B) SW480 cells and (C) HT29 cells. Cells were transfected with DN-p85 and treated with TGFβ for 24 hours, and total RNAs were extracted and collected another 48 hours later. PTEN expression was suppressed by TGFβ treatment in the control, but its suppression was blocked by DN-p85 transfection. GAPDH is used as a loading control. Gels are representative of 3 independent experiments.

Discussion

TGFβ is known to function in two divergent roles in epithelial cells: growth suppression in normal and early transformed cells, and growth proliferative in mature or metastatic cancers. It is well demonstrated that TGFβ-SMAD signaling is the key pathway that mediates growth suppression. However, it is not clear how TGFβ converts from its suppressor function to that of a growth promoter, nor is it clear if simply loss of TGFβ-SMAD signaling is the key event for this transformation, or if there is active participation by TGFβ with proliferative pathways. Here, we show (a) TGFβ activates PI3K signaling in colon cancer cells, (b) TGFβ suppresses PTEN expression in a SMAD-independent but PI3K-dependent fashion, (c) TGFβ-induced cell proliferation is in part dependent on PI3K signaling and simultaneous suppression of PTEN, while TGFβ-SMAD signaling is growth suppressive, (d) TGFβ-SMAD2 transactivation can occur in the absence of SMAD4. Our findings suggest that the TGFβ-PI3K-PTEN proliferative pathway is unmasked with loss of the suppressive TGFβ-SMAD pathway in colon cancer cells, converting TGFβ’s dominant role as a tumor suppressor into a tumor promoter.

PTEN is a tumor suppressor and expression of which in cancer cells are frequently altered. Removing PTEN function promotes cell proliferation for cancer cells. Downregulation of PTEN mRNA by TGFβ was first described in keratinocytes when PTEN was originally cloned,26 but the underlying mechanisms for TGFβ-induced PTEN regulation had not been fully examined. PTEN is commonly affected in different cancers, often inactivated by genetic or epigenetic events during the pathogenesis of colon cancers.30,31 In our current study, we find that PTEN expression is negatively regulated by TGFβ, possibly via its inhibition of the PTEN promoter.

We have previously found that the oncogenic RAS/ERK pathway and calcium-activated PKC-alpha are was involved in TGFβ-induced PTEN downregulation in pancreatic cancer cells.28,38 TGFβ-induced PTEN suppression is mediated by PI3K as shown in the current study, which is linked to non-oncogenic RAS signaling. PI3K is typically induced by activated receptor tyrosine kinases such as the EGF receptor.39 We demonstrate that TGFβ induces PI3K activation 24 hrs after treatment, suggesting activation of intracellular pathways (independent of SMADs) that could even involve gene transcription or protein degradation steps for activation. Inhibition of PI3K by LY294002 or dominant negative construct transfection reversed TGFβ-induced cell proliferation and PTEN suppression. These findings suggest that TGFβ ligand alone is able to promote cell proliferation in SMAD4-null colon cancer cells, including when SMAD2 is inactivated, and this is completely independent of SMAD activation. How TGFβ activates PI3K in colon cancer is not known. An interaction between PI3K and SMAD proteins had been suggested in the literature,40,41 but our data indicating independence from SMAD signaling has also been shown in normal fibroblasts.42 PI3K and PTEN antagonize the production of PIP3 in cells. Here, we suggest a TGFβ-mediated PI3K activation, which in turn reduces transcription of PTEN, indicating a different level of antagonism. It is not clear what mediators downstream of PI3K activation mediate PTEN suppression. However, the consequences of PI3K-activated PTEN suppression mean less PTEN protein available to reverse PIP3 levels allowing the induction of cell proliferation.

Canonical TGFβ-SMAD signaling suggests that SMAD4 is required for the translocation of SMAD2/3 into the nucleus to carry out TGFβ-responsive gene transcription and to promote TGFβ-induced tumor suppression. Our findings corroborate other studies in that there can be SMAD2/3 induced transcription in the absence of SMAD4.36 In SMAD4-null SW480 cells, SMAD2 but not SMAD3 was activated by ligand. Functionally, the TGFβ-SMAD2 pathway in SW480 cells is growth suppressive, as inhibition of SMAD2 increased TGFβ-induced cell proliferation. However, although interruption of this growth suppressive pathway induced increased proliferation by TGFβ, the mechanism does not involve regulation of PTEN, which we show is SMAD-independent and regulated by PI3K signaling.

In summary, we demonstrate in SMAD4-null cells that PTEN is ligand regulated by TGFβ in a SMAD-independent but PI3K-dependent manner. Our findings support that the frequent inactivation of growth suppressive SMAD signaling in colon and pancreatic cancers as well as in cancer-prone hamartomatous polyposis syndrome unmasks an unbalanced and TGFβ-induced growth proliferative PI3K pathway that suppresses PTEN expression and function. The imbalance created by loss of TGFβ-SMAD signaling may be a mechanism for TGFβ’s conversion to a growth promoter.

Materials and Methods

Materials

Human recombinant TGFβ1 was obtained from R&D Systems (Minneapolis, MN). The PI3K inhibitor, LY294002 was obtained from EMD Biosciences Inc., (San Diego, CA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture media and supplements came from Invitrogen (Carlsbad, CA) unless otherwise indicated.

Cell lines and culture

The SW480 and HT29 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained, respectively, in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) and Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) with 10% FBS and penicillin G-streptomycin (Invitrogen).

Immunoprecipitation and western blot analysis

Immunoprecipitation of the p85 subunit of PI3K was performed by overnight incubation by rotary rotation with p85 antibody with 500 µg of cell extract at 4°C. Protein agarose A beads (Upstate; Lake Placid, NY) were added, and the mixture were further rotated for 3 h at 4°C. After the mixture was washed with cold PBS, the antibody-protein complex was denatured at 100°C for 5 min, and the protein was loaded onto a 7.5% polyacrylamide gel. After electrophoresis, proteins were transferred onto a PVDF membrane, blocked for 30 min with 5% skim milk, and probed overnight with primary antibodies at 4°C. Blotting was done with antibodies against phospho-tyrosine, total PTEN (1:250), SMAD2 (1:250), and SMAD4 (1:500) from Santa Cruz Biotechnology (Santa Cruz, CA), pAkt (1:1000), Akt (1:1000), FLAG (1:1000), pSMAD2 (1:1000) (Upstate Biotechnology, Chicago, IL), and GAPDH (1:5000; Ambion, Austin, TX). The next day, four 0.1% PBS-Tween (PBS-T) washes were performed along with the appropriate secondary antibody incubation. Blotted proteins were detected with horseradish peroxidase-linked secondary antibodies (Sigma; St. Louis, MO) for enhance chemi-luminescence detection.

Luciferase reporter assays

The TGFβ-responsive element luciferase plasmid PAI-1 promoter (p3TP-luc) was utilized to assess the effects of TGFβ on combined SMAD2 and SMAD3 transactivation. The pCAGA-luc plasmid contains a TGFβ-response element between −271 and −255 bp, which overlaps the CAGACA sequence recognized only by SMAD3,32 and used to assess the effects of TGFβ on SMAD3 transactivation. The PTEN-luc reporter gene (a generous gift of Dr. Adamson, Burnham Institute, La Jolla, CA) contains the PTEN promoter cloned in pGL3 vector.33 The above reporter vectors (0.75 µg/ml) and the pRL-TK vector (20 ng/ml) were transiently delivered to the colon cancer cells by Transfectin (Promega; Madison, WI) in 12-well plates with a ratio of 3:1 of vector to transfection reagent in OPTI-MEM reduced serum-free media (GIBCO; Carlsbad, CA). Two hours posttransfection, 1 ml of complete media was added per well, and 12–16 h posttransfection cells were treated with 10 ng/ml TGFβ. Firefly luciferase activity was measured by a Dual-Luciferase kit (Promega, Madison, WI) 6, 24 and 48 h after treatment, and normalization was performed using the Renilla luciferase activity by co-transfecting pRL-TK vector.

Dominant negative SMAD2 and p85 transfections

Dominant negative (DN)-SMAD2 (generous gifts from Dr. Rik Derynck at University of California, San Francisco34), or DN-p85 (a gift from Dr. Cullen Taniguchi at Harvard University35) were transiently delivered by Transfectin (Promega) at a ratio of 3:1 of vector to transfection reagent using 1 µg/ml DN-SMAD2 or 3 µg/ml DN-p85 plasmids dissolved in OPTI-MEM reduced serum-free media (GIBCO/Invitrogen, Carlsbad, CA). After 2–3 h, IMDM with FBS and penicillin G-streptomycin was added to the transfected cells. At 2 h posttransfection, complete media were added and later used in the experiments.

Cell growth assay

Cells were seeded at a density of 10,000 cells/well and, 24 hours later, were treated with 10 ng/ml TGFβ in the absence of FBS. After 48 hours, cells were treated in 0.5 ml of 0.05% trypsin and counted using a hemacytometer.

Total RNA extraction and semi-quantitative reverse transcription-PCR

Total RNA extraction was performed using TRIzol reagent (Invitrogen, Carlsbad, CA). Cells grown on six-well plates were lysed with TRIzol (1 ml/well), combined with chloroform, and mixed. Supernatants were then precipitated with isopropanol, and RNA pellets were washed with 75% ethanol, air dried, and then resuspended in water. Two micrograms of total RNA were converted into cDNA by reverse transcriptase followed by amplification of PTEN (SuperScript II, Invitrogen). Briefly, after inactivation at 65°C for 10 min, 1 µl of the reaction mixture was incubated in buffer containing 0.2 mM each of dATP, dCTP, dGTP and dTTP, 0.2 µM each of the oligonucleotide primers, 3 mM MgCl2, and 10× buffer consisting of 200 mM Tris·HCl (pH 8.0), 500 mM KCl, and 1 unit Taq polymerase. The following primers were designed to amplify PTEN: forward 5′-CCCAGCGTGAAAAGAGAGAC-3′ and reverse 5′-GAGACCGCAGTCCGTCTAAG-3′; and GAPDH served as a loading control: forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. PCR was performed as follows: denaturation at 95°C for 3 min and 35 cycles of 94°C for 30 s, 55°C for 30 s, and 74°C for 4 min; denaturation at 95°C for 3 min.

Statistical analysis

Statistical significance was determined using either Student’s t-test or one-way ANOVA by GraphPad Instat 3 (San Diego, CA). p values less than 0.05 were considered to be significant.

Acknowledgements

Supported by the United States Public Health Service (DK073090 to J.Y.C.C., and DK067287 to J.M.C.), the UCSD Digestive Diseases Research Development Center (DK080506), and the VA Research Service.

Abbreviations

TGFβ

transforming growth factor-β

DN

dominant negative

PI3K

phosphatidylinositol 3 phosphate

PTEN

phosphate and tensin homolog deleted on chromosome ten

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