Regulation of mTORC1 Signalling and Amino Acid Uptake by Iron Availability in Caco-2 Intestinal Epithelial Cells

  • Ailsa Downie Watson

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Iron is an essential micronutrient in the human diet that plays a vital role in the control of cell growth and multiple other cellular processes. Iron deficiency is the most prevalent nutritional deficiency in the UK (WHO). The Scottish Government (RESAS division) has funded the current study in an attempt to gain a better understanding of how iron deficiency specifically impacts upon the functional properties of the gut and how these changes may alter health as a whole.

The iron chelator deferoxamine (DFO) was used to induce iron deficiency in Caco-2 cells (a cell line derived from a human colorectal carcinoma which has functional properties typical of absorptive enterocytes of the small intestine) in order to assess the effects of iron depletion on various aspects of the physiology of intestinal epithelium.

Sustained exposure (16 h) of Caco-2 cells to DFO effectively suppressed protein synthesis and cell proliferation, consistent with known effects of iron depletion. A key metabolic pathway which has been implicated strongly as a regulator of cell growth and proliferation is that centred on the mammalian or mechanistic target of rapamycin (mTOR). Consistent with previous studies using other cell lines and tissues, iron depletion induced in human intestinal Caco-2 cells results in decreased mTORC1 signalling. A key discovery from the present work is that REDD1 (Regulated in DNA Damage and Development 1), a gene that exerts a potent inhibitory effect on mTORC1 signalling when expressed, is induced by iron deficiency at both the mRNA and protein level in Caco-2 cells. The induction of REDD1 was associated with a striking reduction in Thr389 phosphorylation of S6K1, Ser240/244 phosphorylation of ribosomal S6 protein and that of 4E-BP1 phosphoryation on Ser65, all of which serve as positive downstream readouts of mTORC1 signalling. The loss in phosphorylation of these mTORC1 targets was reversed upon cellular resupplementation of iron, treatment with Actinomycin D (which prevented the transcription of REDD1) and REDD1 silencing using siRNA. Interestingly, there appears to be a link between REDD1 expression and activity of the protein phosphatase 2A (PP2A). Treatment of iron depleted cells with the PP2A inhibitor okadaic acid prevented both the induction of REDD1 and the inhibitory effect it has on mTORC1 signalling. Conversely, treating cells with MG132 (a proteasome inhibitor) enhanced REDD1 protein levels in Caco-2 cells and suppressed mTORC1 signalling.

The mTORC1 pathway is regulated by nutrients (e.g. amino acids) as well as iron. Thus, it was hypothesised that modification of mTORC1 signalling under iron depleted conditions might result from repression or inhibition of amino acid transport. However, DFO-induced iron depletion was found to stimulate upregulation in the expression and function of the amino acid transporters studied, (i.e. B0AT1, y+LAT1, y+LAT2, SNAT1, SNAT2 and PAT1) with the single exception of LAT2 which was downregulated. The intracellular amino acid concentrations of Caco-2 cells were maintained when cells were rendered iron deficient, perhaps as a result of the reciprocal changes in amino acid transporter expression.

The research presented in this thesis indicates that impaired mTORC1 signalling may contribute to atrophy and resultant dysfunction of the intestinal epithelium in individuals who are chronically iron deficient and therefore helps underscore the potential health benefits of dietary iron supplementation. The research also identifies REDD1 and PP2A as potential therapeutic targets whose modulation may help mitigate loss in mTORC1 activity and thereby potentially limit intestinal mucosal atrophy associated with chronic or recurrent iron deficiency disorders.
Date of Award2015
Original languageEnglish
SupervisorHari Hundal (Supervisor) & Peter Taylor (Supervisor)

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