Kazuaki Takaku

Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc Δ716 knockout mice

Arachidonic acid is metabolized to prostaglandin H2 (PGH2) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH2, PGE2 is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE2, EP2, causes decreases in number and size of intestinal polyps in ApcΔ716 mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE2 through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE2 receptors, EP1 or EP3, did not affect intestinal polyp formation in ApcΔ716 mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

INTESTINAL TUMORIGENESIS IN COMPOUND MUTANT MICE OF BOTH DPC4 (SMAD4) AND APC GENES

The DPC4 (SMAD4) gene plays a key role in the TGFbeta signaling pathway. We inactivated its mouse homolog Dpc4 (Smad4). The homozygous mutants were embryonic lethal, whereas the heterozygotes showed no abnormality. We then introduced the Dpc4 mutation into the Apc(delta716) knockout mice, a model for human familial adenomatous polyposis. Because both Apc and Dpc4 are located on chromosome 18, we constructed compound heterozygotes carrying both mutations on the same chromosome by meiotic recombination. In such mice, intestinal polyps developed into more malignant tumors than those in the simple Apc(delta716) heterozygotes, showing an extensive stromal cell proliferation, submucosal invasion, cell type heterogeneity, and in vivo transplantability. These results indicate that mutations in DPC4 (SMAD4) play a significant role in the malignant progression of colorectal tumors.

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase‐1 gene

Heme oxygenase‐1 (HO‐1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho‐1. Here we show that a heme‐binding factor, Bach1, is a critical physiological repressor of ho‐1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho‐1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho‐1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho‐1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound‐deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho‐1. Thus, regulation of ho‐1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor–activator antagonism.

Suppression of intestinal polyposis in Apc(Δ716) knockout mice by an additional mutation in the cytosolic phospholipase A2 gene

Arachidonic acid is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Cytosolic phospholipase A2 (cPLA2) plays a key role in the release of arachidonic acid as the substrate of cyclooxygenase-1 (COX-1) or COX-2. We found that the level of cPLA2 mRNA was markedly elevated in the polyps and correlated with the polyp size in the small intestine of theApc Δ 716 knockout mouse, a model for human familial adenomatous polyposis. To determine the role of cPLA2 in intestinal tumorigenesis, we then introduced a cPLA2 gene mutation intoApc Δ 716 mice. In the compound mutant mice, the size of the small intestinal polyps was reduced significantly, although the numbers remained unchanged. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of polyps in the small intestine rather than in the initiation process. In contrast, colonic polyps were not affected in either size or number. Interestingly, group X sPLA2 was constitutively expressed in the colon at much higher levels than in the small intestine. These results suggest that in the colon, group X sPLA2 supplies arachidonic acid in both the normal epithelium and the polyps even in the absence of cPLA2.

Involvement of caspase 3 in apoptotic death of cortical neurons evoked by DNA damage

Previous reports have shown that DNA-damage-evoked death of embryonic cortical neurons is delayed by general caspase inhibitors and is accompanied by an increase in DEVD-AFC cleavage activity. We show here that this cleavage activity is lacking in camptothecin-treated caspase 3-deficient neurons. Moreover, we report that death of camptothecin-treated caspase 3-deficient neurons cultured from E16 embryos is delayed and that no significant increase in survival is observed with cotreatment with the general caspase inhibitor BAF. These results indicate that caspase-dependent death of camptothecin-treated cortical neurons requires caspase 3 activity. The delay in death is accompanied by impairment of DNA fragmentation. However, Bax-dependent cytochrome c release still occurs in camptothecin-treated caspase 3-deficient cortical neurons. Accordingly, we hypothesize that the delayed death which occurs in the absence of caspase 3 activity may be due to mitochondrial dysfunction. Finally, we show that the delay in death observed with E16 caspase 3-deficient neurons does not occur in neurons cultured from E19 embryos. This suggests that the requirement for caspase 3 in death of neurons evoked by DNA damage may differ depending upon the developmental state of the cell.

Gastro-intestinal tumorigenesis in Smad4 mutant mice

The SMAD4 gene plays a key role in the TGF-beta signaling pathway. We inactivated its mouse homolog Smad4. The homozygous mutants were embryonically lethal, whereas the heterozygotes were viable and fertile. Although young heterozygotes appeared normal, old mice developed gastric and duodenal polyps similar to human juvenile polyps characterized by abundant stroma and eosinophilic infiltrations. These data are consistent with the reports that a subset of human juvenile polyposis kindreds carry germline mutations in the SMAD4 gene. We then introduced the Smad4 mutation into the Apc(Delta716) knockout mice, a model for human familial adenomatous polyposis. Because both Apc and Smad4 are located on mouse chromosome 18, we constructed by meiotic recombination compound heterozygotes carrying both mutations on the same chromosome. In such mice, intestinal polyps developed into more malignant tumors than those in the simple Apc(Delta716) heterozygotes, showing an extensive stromal cell proliferation and strong submucosal invasion. These results indicate that mutations in SMAD4 play a significant role in the malignant progression of colorectal tumors.