Mucosal Healing through Trefoil Factors
Explore and order pathway-specific siRNAs, real-time PCR assays, and expression vectors. View pathway information and literature references for your pathway.
  • Click on your proteins of interest in the pathway image or review below
  • Select your genes of interest and click "add selection"
  • When you have finished your gene selection, click "Find Products" to find assays, arrays, or create custom products
Download Image Terms of Use Download PPT
Pathway Navigator
Mucosal Healing through Trefoil Factors

Epithelial continuity depends on a family of small, yet abundant, secreted proteins the TFFs (Trefoil Factors). TFFs are considered as Rapid Response agents to mucosal injury; with up-regulation of expression in the early stages of mucosal repair. These peptides are involved in mucosal maintenance and repair through motogenic and anti-apoptotic activities. They also function as scatter factors, pro-invasive and angiogenic agents. TFFs are connected with multiple oncogenic pathways. As a consequence, the TFF signaling pathways serve as potential targets in the control of chronic inflammation and progression of human solid tumors (Ref.1 & 2). The TFFs constitute a family of small regulatory gastric peptides consisting of three members, TFF1/pS2 (Gastrointestinal Trefoil Protein-pS2), TFF2/SP (Spasmolytic Protein-1) and TFF3/ITF (Intestinal Trefoil Factor). These have seven conserved cysteines residues, six of which form three intra-chain disulfide bonds that result in a characteristic three-loop structure named Trefoil domain or P-domain. This P-domain renders the protein resistant to proteases and acid degradation. TFF response elements in TFF gene promoters allow increase in TFF expression through auto-induction and cross-induction of other TFFs, in addition to Mucin expression and tumor suppression (Ref.3 & 4).

TFFs are expressed differentially, with TFF1 and TFF2 principally in gastric epithelia and TFF3 in goblet cells of intestine and lung. TFF2 is located in the cytoplasm of the gastric mucous neck and catalyze the formation of stable Mucin complexes. TFF3 binds to epithelial cell surface and is regulated by Mucins. It is through association with Mucins that TFFs influence protection and healing of the gastrointestinal mucosa, serving as a barrier to protect the epithelium from mechanical stress, noxious agents, viruses and other pathogens. The roles of TFF2 and TFF3 are linked to ECAD (Epithelial Calcium Dependent Adhesion Protein)/Ctnn (Catenin) complexes that are the common targets for motogenic factors during cell migration. TFF3 down-regulates ECAD and Ctnn-Beta (Catenin-Beta) and decreases cell adhesion (Ref.3 & 4). TFF3-induced cell migration requires perturbation of ECAD functions and tyrosine phosphorylation of Ctnn-Beta. TFF signal transduction pathways in neoplastic progression and tumor cell invasion require COXs (Cyclooxygenases). The metabolites of the COXs enzymes activated by TFFs induce DNA mutations through formation of free radicals or mutagenic agents such as MDA (Malondialdehyde). TFFs are overexpressed in several human solid tumors of breast, prostate, esophagus, stomach, biliary, pancreas and intestine (Ref.5). 

Cell migration is the result of integrated disruption of cell-cell, cell-substratum adhesion and prevention of apoptosis through cell detachment. TFFs are the only peptides essential for restitution and this is achieved without promotion of proliferation or tumorigenesis. Cell-cell adherens junctions are established and maintained by the adhesion molecules ECAD, Ctnn-Beta and calcium-dependent homophilic interactions with adjacent cells. Epithelial movement therefore requires integration of motogenic and cell-survival signals. This is achieved by activation of several intracellular signaling pathways that converge on ERKs (Extracellular Signal-Regulated Kinases). Serine phosphorylation of the ERKs is central to TFF-mediated signaling, lying downstream of ErbB (v-ErbB Avian Erythroblastic Leukemia Viral Oncogene Homolog) activation and FAK1 (Focal Adhesion Kinase-1) activation (through recruitment of GRB2 (Growth Factor Receptor-Bound Protein-2), SOS1 (Son of Sevenless-1) and SHC1 ((SHC (Src Homology-2 Domain Containing) Transforming Protein))) (Ref.1 & 2). Disruption of focal adhesions between a cell and its substratum requires dissociation of the focal adhesion complex. This, in turn, requires autophosphorylation of FAK1 on tyrosine 397, which forms a docking site for Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2) Viral Oncogene), the p85 subunit of PI3K (Phosphatidylinositde-3 Kinase) and SH2 domain of the adaptor protein SHC1. FAK1 phosphorylation and the binding of either SHC1 or Src transduce signals through HRas (v-Ha-Ras Harvey Rat Sarcoma Viral Oncogene Homolog), Raf to ERKs, which links Itg (Integrin) signaling to mitogenic signaling. TFF induces FAK1 phosphorylation and activation. Cell migration thus results due to interaction between ERKs, RhoA and ROCK (Rho-Associated Coiled-Coil-Containing Protein Kinase) proteins along with FAK1 activation, Itg-Beta1 (Integrin-Beta-1) clustering and Ctnn-Beta activation. Signaling elements downstream TFF signaling also comprise MYL (Myosin Light Chain) phosphorylations which are involved in cytoskeletal activation during cell growth, differentiation and invasion. Translocation of phosphorylated ERKs to the nucleus leads to amplification and derestriction of TFF expression to ensure sustained action (Ref.6).

TFF signaling is active at both the paracrine and autocrine levels. For several years, it has been hypothesized that TFF acts by binding to specific receptors. Nevertheless, despite numerous studies, to date no canonical receptors have been identified. However, TFF directly binds to Mucins that constitute the mucus layer lining the gastrointestinal tract. TFF (mostly TFF1) triggers two types of cellular responses (Ref.7). On one hand, it lowers cell proliferation by delaying G1-S cell phase transition. On the other hand, TFF protects cells from chemical-, anchorage free-, or BAD (Bcl2-Antagonist of Cell Death)-induced apoptosis. Thus TFF has dual anti-proliferative and anti-apoptotic role. One function of TFF is to delay cell cycle commitment. TFF leads to an increased level of CDK (Cyclin-Dependent Kinase) inhibitors of both INK4 and CIP subfamilies. Members of the INK4 family (p16(INK4A), p15(INK4B), p18(INK4C) and p19(INK4D)) specifically bind to CDK4 and inhibit Rb (Retinoblastoma) phosphorylation, thereby causing G1 phase arrest. The function of CDK inhibitors of the CIP family (p21(CIP1) (Cyclin Dependent Kinase Inhibitor-p21), p27(KIP1) (Cyclin Dependent Kinase Inhibitor-p27) and p57-KIP2 (Cyclin Dependent Kinase Inhibitor-p57)) is more complex. They display mainly inhibitory activity towards the CcnE (Cyclin-E)/CDK2 complex which is required for correct E2F (E2F Transcription Factor) activity, and thus act to block S phase entry. However, in the absence of INK4 proteins, they can also bind CcnD1 (Cyclin-D1)/CDK4 complexes which effectively sequester them, facilitating CcnE/CDK2 function. Thus, the reduction of cell cycle commitment in the presence of TFF results from the upregulation of both INK4 and CIP CDK inhibitors. TFF represses cell progression towards the S phase of the cell cycle, and therefore proliferation. The transition between the G1 and S phase is tightly regulated. At this checkpoint, a decision is made by the cell whether to proceed or not. Rb and E2F are major players in a regulatory circuit in the late G1 phase. E2F triggers S phase entry by activating promoters of S phase-regulated genes. A prerequisite for the Rb growth-suppressor function is binding of its unphosphorylated form to the E2F transcription factor, thus inhibiting E2F transcriptional activity, and arresting cells in G1. Consistently, the presence of TFF leads to a two-fold increase in Rb activity that is associated with a two-fold decrease in E2F transcriptional activity. The relationship between a member of the TFF family and cell cycle regulation indicates that the effector of the anti-mitotic function of TFF is the Rb/E2F pathway (Ref.7 & 8).

Abrogation of cell death requires both PI3K activation (a consequence of TFF interaction with the GHR (Growth Hormone Receptor) and GH1 (Growth Hormone-1)) and ERK activation; the former operates through Serine/Threonine phosphorylation of Akt1 (v-Akt Murine Thymoma Viral Oncogene Homolog-1), serine phosphorylation of BAD and inhibition of mitochondrial CytoC (Cytochrome-C) release; and formation of the Apoptosome (APAF1 (Apoptotic Protease Activating Factor-1), Caspase9 (Apoptosis Related Cysteine Protease-9) and CytoC) (Ref.2). The phosphorylation of BAD at specific serine residues, results in loss of the ability of BAD to heterodimerize with the survival proteins like Bcl2 (B-Cell CLL/Lymphoma-2). TFFs are used in therapies for the chronic debilitating conditions ulcerative colitis, Crohn’s disease and also IBDs (Inflammatory Bowel Diseases). TFFs are also effective in the treatment of the Mucositis (Ref.4).