We have identified a novel T/EBP/NKX2.1 target gene, claudin-18
, which is a member of the claudin multigene family. Unlike other claudin gene family members, claudin-18
encodes two types of lung- and stomach-specific polypeptides by alternative splicing. An alternative 12-bp insertion at the 3′ end of exon 4 produces an isoform that lacks the C-terminal cytoplasmic domain. Most claudins have tyrosine-valine (Y-V) amino acid sequences at their C termini, which directly bind to the membrane-associated guanylate kinase family of proteins, ZO-1, ZO-2, and ZO-3, through their first PDZ (postsynaptic protein-95 [PSD-95]/discs large [DLG]/zonula occludens-1 [ZO-1]) domains (18
). These proteins are believed to function as scaffolds of the TJ plaque to cross-link TJ strands to the actin-based cytoskeleton, and they are involved in the regulation of the barrier function of TJs by modulating actin filament-TJ strand association (18
). Considering these hypotheses and the fact that claudins can be polymerized to form TJ strands without interacting with ZO-1 (13
), the C-terminally truncated isoform may have a dominant-negative-type function in regulating the barrier function of TJs. Determination of whether this is the case or not must await further studies.
Alternative usage of two promoters and their own first exons leads to production of lung- and stomach-specific claudins. Although detailed descriptions of most claudins have not yet been reported, most claudins have wide tissue distribution patterns, except claudins-11 and -16 (27
; our unpublished observation). Claudin-11 is the only claudin expressed in the myelin sheaths of oligodendrocytes in the central nervous system and Sertoli cells in the testis (14
). Claudin-16 expression is mainly restricted to the thick ascending limb of Henle in the kidney, and it is assumed that claudin-16 creates a magnesium-selective channel through the TJs (38
). In our analyses by RT-PCR, more than 10 claudins are expressed in mouse lung (this study) and stomach (unpublished observation). However, lung- and stomach-restricted expression of claudin-18 suggests that claudin-18 may have an important role such as a channel-like activity as seen in claudin-16.
The results of transient-transfection studies indicate that the homeodomain-containing transcription factor T/EBP/NKX2.1 is required to trans
activate expression of the mouse claudin-18a1
promoter even though the level of induction is low (two to threefold). To our knowledge, this is the first report that describes functional analysis of the claudin
gene promoter. Cotransfection experiments using a plasmid expressing T/EBP/NKX2.1 with claudin-18a1
–luciferase reporter constructs delineated the minimal region of the mouse claudin-18a1
gene promoter that is sufficient to activate transcription of the gene. This region contains two consensus T/EBP/NKX2.1 binding elements, 5′-CAAG-3′ (19
). Mutation of each motif interfered equally with T/EBP/NKX2.1 binding to the site and reduced claudin-18a1
promoter activity by approximately one-half. Mutation of both sites nearly abolished the transcriptional activity of the gene. Data obtained from electrophoretic mobility shift assays were consistent with these results. The dose-dependent increase in reporter activity in response to cotransfected T/EBP/NKX2.1 is also consistent with the hypothesis that the mouse claudin-18a1
gene is a direct target of T/EBP/NKX2.1. Interestingly, claudin-18a1
is the only gene that appears to be activated by T/EBP/NKX2.1, although at least 10 claudins are expressed in mouse embryonic lungs (27
). T/EBP/NKX2.1 not only functions as a transcriptional regulator of surfactant protein (1
) and Clara cell secretory protein (34
) genes in lung but also is proposed to be one of the key regulators of early lung development (26
). Recently, it was demonstrated that morphogenesis and cellular differentiation of the distal lung compartments are strictly dependent on the activity of T/EBP/NKX2.1 (26
). Whether claudin-18a1
has a role in lung morphogenesis remains to be studied.
is a stomach-specific isoform of the claudin-18
gene that has a promoter distinct from that of claudin-18a1
. High levels of expression in adult mouse stomach were demonstrated by immunostaining. Of interest is its immunolocalization, where claudin-18 appears to be located on entire cell membranes and in some cases cytoplasm rather than just TJs. This pattern of expression is very similar to those of caludin-3 and -5 in stomach (33
). Diffuse cytoplasmic staining of claudin-18 may be the result of using thicker paraffin sections (4 to 6 μm) for immunostaining instead of very thin sections such as the 50- to 100-nm sections used for EM. However, IEM demonstrated claudin-18 immunogold spots only at TJs and not within any other cellular organelles. For claudin-3 and -5, IEM has yet to be performed (33
). Thus, it is possible, as proposed for claudin-3 and -5 (33
), that claudin-18 may be localized along the lateral and basolateral intercellular space without forming direct cell-to-cell contacts that can influence ion or solute movement along the lateral or basolateral membranes. It is possible that these claudins are recruited into TJs to quickly establish new paracellular transport properties in response to acute physiological challenge. Our data on claudin-18 argue for the proposal that some, if not all, of the TJ barriers may be regulated by both transcriptional and subcellular localization mechanisms (33
Our results demonstrated that the stomach-specific form of the transcript is present in both mouse and human. In preliminary experiments, there was no cis-regulatory element for stomach-specific expression within 1.8 kb upstream of the mouse claudin-18a2 gene when a human stomach adenocarcinoma-derived AGS cell line was used in transfection assays. Initially, the expression of the human claudin-18A2 gene in this cell line was confirmed by using RT-PCR. To our knowledge, no stomach-specific transcription factor has been reported. Further sequences upstream of 1.8 kb of the mouse claudin-18a2 gene may contain a cis-regulatory element for stomach-specific expression. It is not yet known whether the two different tissue-specific forms of claudin-18 possess distinct functions at their specific expression sites. In this regard, it is interesting that claudin-18al is the only claudin that is missing in T/ebp/Nkx2.1-null mouse lungs. Determination of whether claudin-18A1 plays a role in lung development requires further experiments.