In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase–extracellular signal–regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.

We null mutated the mouse angiotensin type 1B (AT1B) receptor gene (Agtr1b) by gene targeting. To identify the specific cell types carrying high Agtr1b gene transcriptional activities, the AT1B coding exon was replaced with a reporter gene, lacZ. In 6- to 8-wk-old Agtr1b -/- mice, high AT1B transcriptional activity was observed in adrenal zona glomerulosa cells and the testis, including mature and immature spermatic cells, whereas low activity was detected homogeneously in anterior pituitary cells and choroidal plexus vessel walls. A similar pattern was observed in Agtr1b +/- mice with less intensity. Microscopically, the anterior pituitary, heart, adrenal, zona glomerulosa, kidney, and the testis of Agtr1b -/- mice were intact and were indistinguishable from those of Agtr1b +/+ mice. Systemic blood pressure was comparable in Agtr1b -/- and Agtr1b +/+ mice. Moreover, plasma aldosterone level was comparable between the two mouse groups. No compensatory enhancement of AT1A mRNA was found in the kidney and adrenal gland of Agtr1b -/- mice. The observed absence of the abnormal phenotypes in Agtr1b -/- mice, which have been described for homozygous angiotensinogen null mutant mice, indicates that 1) AT1A receptors can take over the role of AT1B receptors in Agtr1b -/- mice or 2) functionally significant non-AT1, non-AT2 receptor(s) may exist for the action of angiotensin.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK −/− mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. Highfrequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after lowfrequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory. cholecystokinin | NMDA receptor | long-term potentiation | memory | entorhinal cortex M emory is stored in neural networks through changes in synaptic strength (1). Long-term potentiation (LTP) and long-term depression (LTD) are two forms of synaptic plasticity that are believed to represent a neural basis of memory in different brain regions (2-5). The major form of LTP in the hippocampus and neocortex is induced through theta burst stimulation or highfrequency stimulation (HFS) (2, 3). Previous studies have shown that NMDA receptors (NMDARs) play a crucial role in HFSinduced LTP in the hippocampus (6-9) and neocortex (2, 10), and in the formation and consolidation of associative memory (11,12).Serving as the gateway from the hippocampus to the neocortex, the entorhinal cortex forms strong reciprocal connections with the neocortex (13, 14) and shows extensive cholecystokinin (CCK) labeling (15-17) with projections to neocortical areas, including the auditory cortex (13,14,18). CCK is the most abundant cortical neuropeptide (19), and mice lacking the CCK gene exhibit poor performance in a passive avoidance task and display impaired spatial memory (20). Although many studies have focused on GABAergic CCK neurons (21-24), many glutamatergic neurons in the neocortex express CCK (25, 26). We previously found that local infusion of CCK into the auditory cortex of anesthetized rats induces plastic changes that enable auditory cortical neurons to start responding to a light stimulus after its pairing with an auditory stimulus (18). Activation of the entorhinal cortex potentiates neuronal responses in the auditory cortex, and this effect is suppressed by infusion of a CCK-B receptor (CCKBR) antagonist (18), suggesting that the entorhinal cortex enables neocortical plasticity via CCK-containing neurons projecting to the neocortex.If CCK enables cortical neuroplasticity and associative memory formation, then we would expect CCK-induced neuroplasticity to affect LTP. The release of neuropeptides occurs slowly in response to repetitive firing (27,28)....

Patients with damage to the medial temporal lobe show deficits in forming new declarative memories but can still recall older memories, suggesting that the medial temporal lobe is necessary for encoding memories in the neocortex. Here, we found that cortical projection neurons in the perirhinal and entorhinal cortices were mostly immunopositive for cholecystokinin (CCK). Local infusion of CCK in the auditory cortex of anesthetized rats induced plastic changes that enabled cortical neurons to potentiate their responses or to start responding to an auditory stimulus that was paired with a tone that robustly triggered action potentials. CCK infusion also enabled auditory neurons to start responding to a light stimulus that was paired with a noise burst. In vivo intracellular recordings in the auditory cortex showed that synaptic strength was potentiated after two pairings of presynaptic and postsynaptic activity in the presence of CCK. Infusion of a CCKB antagonist in the auditory cortex prevented the formation of a visuo-auditory association in awake rats. Finally, activation of the entorhinal cortex potentiated neuronal responses in the auditory cortex, which was suppressed by infusion of a CCKB antagonist. Together, these findings suggest that the medial temporal lobe influences neocortical plasticity via CCK-positive cortical projection neurons in the entorhinal cortex.