The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1-S phase transition and reducing apoptosis

Abstract

Trefoil factor (TFF)1 is synthesized and secreted by the normal stomach mucosa and by the gastrointestinal cells of injured tissues. The link between mouse TFF1 inactivation and the fully penetrant antropyloric tumor phenotype prompted the classification of TFF1 as a gastric tumor suppressor gene. Accordingly, altered expression, deletion, and/or mutations of the TFF1 gene are frequently observed in human gastric carcinomas. The present study was undertaken to address the nature of the cellular and molecular mechanisms targeted by TFF1 signalling. TFF1 effects were investigated in IEC18, HCT116, and AGS gastrointestinal cells treated with recombinant human TFF1, and in stably transfected HCT116 cells synthesizing constitutive or doxycycline-induced human TFF1. We observed that TFF1 triggers two types of cellular responses. On one hand, TFF1 lowers cell proliferation by delaying G1-S cell phase transition. This results from a TFF1-mediated increase in the levels of cyclin-dependent kinase inhibitors of both the INK4 and CIP subfamilies, leading to lower E2F transcriptional activity. On the other hand, TFF1 protects cells from chemical-, anchorage-free-, or Bad-induced apoptosis. In this process, TFF1 signalling targets the active form of caspase-9. Together, these results provide the first evidence of a dual antiproliferative and antiapoptotic role for TFF1. Similar paradoxical functions have been reported for tumor suppressor genes involved in cell differentiation, a function consistent with TFF1.

Figure 1.

Human recombinant TFF1 decreases IEC18, HCT116 and AGS gastrointestinal cell numbers. The number of live cells was estimated by the MTT method. Human recombinant TFF1 addition to the culture medium (0.1–50 μM) induces a dose-dependent reduction in the number of HCT116 (▴), IEC18 (•) and AGS (cross) cells but has no effect on that of hematopoietic Jurkat (▪) and cervix HeLa (□) cells. Standard deviations are indicated. *P < 0.05; **P < 0.01.

Figure 2.

Western blot analysis of constitutive and doxycycline-induced TFF1 synthesis in stably transfected HCT116 cell lines. 10 μl of conditioned culture medium from two clones of each HCT116 cell line were loaded. TFF1 detection was done using the p2802 specific antibody. (Lanes 1 and 2) pCMV-transfected control clones. (Lanes 3 and 4) Clones transfected with the pCMV-hTFF1 constitutive expression vector. (Lanes 5, 6, 9, and 10) pUHD-transfected control clones. (Lanes 7, 8, 11, and 12) Clones transfected with the pUHD-hTFF1 inducible vector. (Lanes 5–8) Absence (Dox. −) of doxycycline treatment. (Lanes 9–12) Presence (Dox. +) of doxycycline treatment. (Lanes 13–15) 5, 10, and 50 ng of human recombinant TFF1. Molecular weight scale is indicated on the left in kD.

Figure 3.

TFF1 induces the accumulation of cells in the G1 phase of the cell cycle. Analysis of the cell cycle patterns was done using propidium iodide. Cell cycle profiles of G1-enriched HCT116 parental cells after a 1 hour release into the cell cycle in the absence (control) or presence (TFF1, 1 μM) of human recombinant TFF1 (1μM). Cells in the G1 (M1), S (M2), and G2/M (M3) phases are indicated. TFF1 delays G1/S transition.

Figure 4.

TFF1 modifies pRb and E2F transcriptional activity. Parental HCT116- and HCT116-expressing TFF1 constitutively or under induction were transfected with vectors containing a specific response element for pRb (pRb-Ta-Luc) or E2F (pE2F-Ta-Luc) upstream from a TATA-like promoter driving a luciferase reporter gene. The pTA-Luc vector was used as a negative control. Luciferase activities were corrected for β-galactosidase activities. pTA-Luc luciferase activity was set at 1. TFF1 treatment (10 μM), constitutive or inducible TFF1 synthesis increased pRb transcriptional activity and decreased E2F transcriptional activity. (Gray bars) Absence of TFF1. (White bars) Presence of TFF1. Standard deviations are indicated. **P < 0.01.

Figure 5.

TFF1 prevents induced apoptosis of IEC18, HCT116, and AGS gastrointestinal cells. Apoptosis was induced by treatment with 5 mM sodium butyrate (But), 50 μM ceramide (Cer), anchorage-free condition (Anc), or Bad expression (Bad), as indicated. The number of live cells was estimated by the MTT method. Independent of the nature of the apoptosis inducer, addition of recombinant TFF1 (0.1 to 10 μM) protected gastrointestinal IEC18 (top), HCT116 (middle) and AGS (bottom) cells from apoptosis in a dose-dependent manner. Standard deviations are indicated. *P < 0.05; **P < 0.01.

Figure 6.

TFF1 reduces caspase activities. Apoptosis of HCT116 cells was induced using either 5 mM sodium butyrate treatment (A), anchorage-free condition (B), or Bad expression (C). Enzymatic activities of caspase-1, -2, -3, -4/5, -6, -8, and -9 (C1 to C9) were investigated by quantification of the cleavage of specific fluorometric substrates. Results were given as fold increase compared with controls. TFF1 treatment (10 μM), constitutive or induced TFF1 expression reduces activity of several caspases. (Gray bars) Absence of TFF1. (White bars) Presence of TFF1. Standard deviations are indicated. *P < 0.05; **P < 0.01.

Figure 7.

TFF1 does not alter procaspase-9 cleavage. Western blot analysis of cell extracts (15 μg protein) from butyrate-treated (But. +) parental (lanes 3 and 4) and stably transfected HCT116 cell lines expressing constitutive (lane 6) or doxycycline-induced (lane 8) TFF1. TFF1 treatment (10 μM, TFF1 +) was as indicated. (Lane 9) Cleaved caspase-9 as positive control. Arrows indicate the inactive procaspase-9 (47 kD) and the active caspase 9 (37 kD). TFF1 does not modify procaspase-9 cleavage. Molecular weight scale is indicated on the left in kD.

Figure 8.

Schematic model of signaling pathways mediating TFF1 antiproliferative and anti-apoptotic function. Cells integrate external mitogenic or antimitogenic stimuli to decide whether or not to proliferate. Although the number and the nature of the molecular mechanisms participating in transduction of the external TFF1 stimulus remain unknown (?), we demonstrate that TFF1 triggers two types of responses. One function of TFF1 is to delay cell cycle commitment. TFF1 signaling results in increased levels of INK4 and CIP cdk inhibitors. Increased amounts of INK4 alter the distribution of cdk4 in favor of INK4/cdk4 and thereby reverse the sequestration of CIP. The release of CIP proteins leads to cyclinE/cdk2 inhibition, and a lower rate of pRb phosphorylation. This event results in the formation of pRb/E2F complexes, and subsequently, to the reduction of E2F transcriptional activity and reduced expression of E2F-responsive genes needed to drive cells through the G1/S transition. On the other hand, TFF1 signaling targets the active caspase-9, possibly via inhibitors of the IAP family, thereby down-regulating apoptosis. Apopt: apoptosome; C9, caspase-9; D and E, cyclins D and E; ext and int: extra- and intracellular.