Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells.

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.

Persistent BDNF exon I-IX mRNA expression following the withdrawal of neuronal activity in neurons.

It is still unclear whether an active state of transcription once established in chromatin persists in neurons. Here, we focused on BDNF exon I-IX mRNA expression because of its marked induction upon the treatment of rat cortical neurons with trichostatin A, suggesting strong repression of the expression through histone deacetylase activity. Acetylation of histones H3 and H4 in promoter-I of the BDNF gene (BDNF-PI) was induced by membrane depolarization time- and dose-dependently, corresponding with the increase in mRNA expression. Following withdrawal of the depolarization, the mRNA level remained elevated for at least 6h, the persistence of which depended upon the strength of depolarization, whereas the BDNF exon IV-IX expression did not. The acetylation of histones was also maintained with BDNF-PI. Thus, BDNF exon I-IX mRNA expression remained increased after depolarization was withdrawn, suggesting that once activated, the BDNF-PI transcription persists due to chromatin remodeling.

Remote control of activity-dependent BDNF gene promoter-I transcription mediated by REST/NRSF.

To know the role of repressor element-1 (RE-1)-silencing transcription factor (REST) in activity-dependent gene transcription in neurons, we investigated whether the Ca2+ signal-induced transcription of brain-derived neurotrophic factor promoter-I (BDNF-PI) is repressed by RE-1 located in exon II from far downstream of BDNF promoter-II (BDNF-PII). By constructing plasmids in which the location between BDNF-PI, -PII, and -RE-1 is maintained, we found, by conducting promoter assays with cortical neurons, that the promoter activity was constitutively repressed through the actions of BDNF-RE-1 but activated by Ca2+ signals evoked via membrane depolarization, which was due to BDNF-PI but not to BDNF-PII. The over-expression of REST reduced the level of transcriptional activation through the N- and C-terminals, suggesting the recruitment of a histone deacetylase. On over-expression of REST, an increased depolarization did not allow the activation. Thus, REST remotely represses activity-dependent gene transcription, the level of which controls the magnitude of the repression.

Interference with activity-dependent transcriptional activation of BDNF gene depending upon the expanded polyglutamines in neurons.

Expanded polyglutamines (polyQ) have been demonstrated to impair the CREB-dependent transcription in established cell lines. Since activity-dependent transcription in neurons, which plays an important role in forming neuronal plasticity, is largely controlled by CREB, it is important to study whether polyQ interferes with the activity-dependent transcriptional activation of genes in neurons. In cultured rat cortical neurons, over-expression of truncated dentatorubral-pallidoluysian atrophy proteins containing expanded polyQ, which form aggregation bodies in nucleus, reduced the calcium (Ca(2+)) signal-mediated transcriptional activation of brain-derived neurotrophic factor, c-fos, and pituitary adenylate cyclase-activating polypeptide gene promoters in a dose-dependent manner. The interference with the transcriptional activation was dependent upon the presence of polyQ, the strength of which was increased as the length of polyQ stretches was expanded. Thus, polyQ interferes with the activity-dependent transcription in a polyQ-length-dependent manner, which may correspond to the severity of polyglutamine diseases.