Regulation of intestinal glutamine absorption by transforming growth factor-beta

Abstract

Introduction: Transforming growth factor-beta (TGF-β) plays a central role in regulating intestinal epithelial cell proliferation, migration and wound repair but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulation mechanisms and intracellular signaling pathways involved in the regulation of TGF-β on glutamine transport in cultured intestinal cells. Methods: [3H]-L-glutamine (50 μM) transport activity and mRNA levels for the intestinal glutamine transporter ATB0 were measured in cultured intestinal Caco-2 cells. Cells were treated with TGF-β (0 – 50 ng/ml), inhibitors of the mitogen-activated protein kinase (MAPK) MEK1 (PD 98059, 0 – 50 μM), the MAPK p38 (SB 203580, 0–10 μM), as well as actinomycin-D (0 – 1 μM) and cycloheximide (0 – 20 μM). Data were analyzed by ANOVA. Results: Continuous incubation with IGF-2 stimulated glutamine transport activity in Caco-2 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 12 hours) resulted in a 50% increase in transport activity (0.93 ± 0.20 nmole/mg protein/minute in TGF-β vs. 0.61 ± 0.17 nmole/mg protein/minute in control) and a 1.8-fold increase of glutamine transporter ATB0 mRNA levels. TGF-β stimulated transport activity by increasing transport maximal capacity (Vmax 9.21 ± 0.51 nmole/mg protein/minute in TGF-β vs. 5.91 ± 0.34 nmole/mg protein/minute in control) without affecting the transport affinity (Km 290 ± 40 μM glutamine in TGF-β vs 305 ± 34 μM glutamine in controls, p = NS). This TGF-β induced glutamine transport activity was individually attenuated by inhibitors of transcription (actinomycin-D), translation (cycloheximide), and MAPK MEK1 (PD 98059). Mitogen-activated protein kinase p38 inhibitor SB 203580 had no effect on this TGF-β stimulation of glutamine transport activity. Conclusion: TGF-β stimulates intestinal glutamine uptake in cultured human intestinal epithelial cells via a mechanism that involves activation of transcription of the transporter gene and translation of the transporter protein. Activation of mitogen-activated protein kinase ERK1 cascade is involved in the regulation. This increase in glutamine uptake may occur to support intestinal cell homeostasis and wound repair.