Background: Phorbol esters activate an array of biological activity, including amino acid transport activity, via intracellular protein kinase C (PKC) activation. We previously observed that phorbol ester activates glutamine transport activity and transporter ATB0/ASCT2 levels. B0AT1, the gene responsible for impaired amino acid absorption in Hartnup disorder, was identified as the gene encoding the predominant intestinal glutamine transporter B0. The purpose of this study was to investigate the effect of phorbol ester and PKC on this B0AT1 gene.
Methods: Sub-confluent human intestinal epithelial Caco-2 cells were treated with DMSO as a control, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 0 - 0.5 uM) and protein kinase C (PKC) inhibitor chelerythrine chloride (CHE, 0-0.5 uM). B0AT1 mRNA and B0AT1 protein levels were measured using real-time PCR and western blot techniques, respectively. Data were mean ± SE and analyzed with ANOVA with p<0.05.
Results: TPA treatment significantly alters the steady-state glutamine transporter B0AT1 mRNA levels in a time-dependent manner. TPA treatment resulted in a 4.5-fold increase in B0AT1 mRNA levels at 2 hours, a 15-fold increase at 4 hours and a 6-fold increase at 6 hours compared with the control group. Prolonged TPA treatment (12, 18, 24 and 48 hours) resulted in no significant change in B0AT1 mRNA levels, compared with the control group. These findings were in contrast to the TPA effect on another prominent intestinal glutamine transporter ATB0/ASC2, which starts to increase at 6 hours of incubation with TPA and reaches maximal stimulation at 24 hours. Additionally, the stimulatory effect of TPA on ATB0/ASC2 is via PKC activation, whereas the specific PKC inhibitor CHE had no effect on the TPA-induced increase in B0AT1 mRNA levels, suggesting that PKC activation was not involved. TPA treatment resulted in no change in B0AT1 protein levels at 2 hours, a 2.5-fold increase of protein levels at 4 hours and 50 % increase at 6 hours, respectfully.
Conclusion: Phorbol ester TPA regulates glutamine transporters B0AT1 and ATB0/ASC2 via different intracellular mechanisms. These findings enhance our understanding of the regulation mechanism of intestinal glutamine absorption.