TTR exon-humanized mouse optimal for verifying new therapies for FAP.

Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.

Cell-type-specific roles for COX-2 in UVB-induced skin cancer.

In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2(flox/flox) mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2(flox/flox);K14Cre(+) mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2(flox/flox);K14Cre(+) papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2(flox/flox); LysMCre(+) myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer.

Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

RUNX3 is a tumor suppressor for a variety of cancers. RUNX3 suppresses the canonical Wnt signaling pathway by binding to the TCF4/ß-catenin complex, resulting in the inhibition of binding of the complex to the Wnt target gene promoter. Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells. Notably, RUNX3 expression increased the ratio of the Wnt signaling-high population in the KatoIII cells. although the maximum Wnt activation level of individual cells was similar to that in the control. As found previously, RUNX3 also binds to TCF4 and ß-catenin in KatoIII cells, suggesting that these molecules form a ternary complex. Moreover, the ChIP analyses revealed that TCF4, ß-catenin and RUNX3 bind the promoter region of the Wnt target genes, Axin2 and c-Myc, and the occupancy of TCF4 and ß-catenin in these promoter regions is increased by the RUNX3 expression. These results suggest that RUNX3 stabilizes the TCF4/ß-catenin complex on the Wnt target gene promoter in KatoIII cells, leading to activation of Wnt signaling. Although RUNX3 increased the Wnt signaling activity, its expression resulted in suppression of tumorigenesis of KatoIII cells, indicating that RUNX3 plays a tumor-suppressing role in KatoIII cells through a Wnt-independent mechanism. These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/ß-catenin complex by cell context-dependent mechanisms.

Candidate chromosome 1 disease susceptibility genes for Sjogren's syndrome xerostomia are narrowed by novel NOD.B10 congenic mice.

Sjogren's syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.

Requirement for deoxycytidine kinase in T and B lymphocyte development.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, "fine-tuning" role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.

Prostaglandin E2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors.

BACKGROUND & AIMS: Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS: We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS: Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS: Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Cox-2 deletion in myeloid and endothelial cells, but not in epithelial cells, exacerbates murine colitis.

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer. Pharmacological inhibition of cyclooxygenase (COX) function exacerbates symptoms in colitis patients. Animal models of colitis using Cox-2-knockout mice and COX inhibitors also indicate that COX-2 has a protective role against colon inflammation. However, because conventional Cox-2 deletion and COX-2 inhibitors eliminate COX-2 function in all cells, it has not been possible to analyze the role(s) of COX-2 in different cell types. Here, we use a Cox-2(flox) conditional knockout mouse to analyze the role of COX-2 expression in distinct cell types in the colon in response to dextran sulfate sodium (DSS)-induced colitis. We generated Cox-2 conditional knockouts in myeloid cells with LysMCre knock-in mice, in endothelial cells with VECadCreERT2 transgenic mice and in epithelial cells with VillinCre transgenic mice. When treated with DSS to induce colitis, both myeloid cell-specific and endothelial cell-specific Cox-2-knockout mice exhibited greater weight loss, increased clinical scores and decreased epithelial cell proliferation after DSS injury when compared with littermate controls. In contrast, epithelial-specific Cox-2 knockouts and control littermates did not differ in response to DSS. These results suggest that COX-2 expression in myeloid cells and endothelial cells, but not epithelial cells, is important for protection of epithelial cells in this murine colitis model.

Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Absence of myeloid COX-2 attenuates acute inflammation but does not influence development of atherosclerosis in apolipoprotein E null mice.

OBJECTIVE: The role of myeloid cell cyclooxygenase-2 (COX-2) in the progression of atherosclerosis has not been clearly defined. METHODS AND RESULTS: We investigated the role of COX-2 expressed in the myeloid lineage in the development of atherosclerosis using a myeloid-specific COX-2(-/-) (COX-2(-M/-M)) mouse on a hyperlipidemic apolipoprotein (apo) E(-/-) background (COX-2(-M/-M)/apoE(-/-)). Myeloid COX-2 depletion resulted in significant attenuation of acute inflammation corresponding with decreased PGE(2) levels in an air pouch model. COX-2 depletion in myeloid cells did not influence development of atherosclerosis in COX-2(-M/-M)/apoE(-/-) when compared to apoE(-/-) littermates fed either chow or western diets. The unanticipated lack of contribution of myeloid COX-2 to the development atherosclerosis is not attributable to altered maintenance, differentiation, or mobilization of myeloid and lymphoid populations. Moreover, myeloid COX-2 depletion resulted in unaltered serum prostanoid levels and cellular composition of atherosclerotic lesions of COX-2(-M/-M)/apoE(-/-) mice. CONCLUSIONS: Our results suggest that COX-2 expression in myeloid cells, including macrophages, does not influence the development of atherosclerosis in mice.

Preserved heart function and maintained response to cardiac stresses in a genetic model of cardiomyocyte-targeted deficiency of cyclooxygenase-2.

Cyclooxygenase-1 and -2 are rate-limiting enzymes in the formation of a wide array of bioactive lipid mediators collectively known as prostanoids (prostaglandins, prostacyclins, and thromboxanes). Evidence from clinical trials shows that selective inhibition of the second isoenzyme (cyclooxygenase-2, or Cox-2) is associated with increased risk for serious cardiovascular events and findings from animal-based studies have suggested protective roles of Cox-2 for the heart. To further characterize the function of Cox-2 in the heart, mice with loxP sites flanking exons 4 and 5 of Cox-2 were rendered knockout specifically in cardiac myocytes (Cox-2 CKO mice) via cre-mediated recombination. Baseline cardiac performance of CKO mice remained unchanged and closely resembled that of control mice. Furthermore, myocardial infarct size induced after in vivo ischemia/reperfusion (I/R) injury was comparable between CKO and control mice. In addition, cardiac hypertrophy and function four weeks after transverse aortic constriction (TAC) was found to be similar between the two groups. Assessment of Cox-2 expression in purified adult cardiac cells isolated after I/R and TAC suggests that the dominant source of Cox-2 is found in the non-myocyte fraction. In conclusion, our animal-based analyses together with the cell-based observations portray a limited role of cardiomyocyte-produced Cox-2 at baseline and in the context of ischemic or hemodynamic challenge.

Compensatory hypertrophy induced by ventricular cardiomyocyte-specific COX-2 expression in mice.

Cyclooxygenase-2 (COX-2) is an important mediator of inflammation in stress and disease states. Recent attention has focused on the role of COX-2 in human heart failure and diseases owing to the finding that highly specific COX-2 inhibitors (i.e., Vioxx) increased the risk of myocardial infarction and stroke in chronic users. However, the specific impact of COX-2 expression in the intact heart remains to be determined. We report here the development of a transgenic mouse model, using a loxP-Cre approach, which displays robust COX-2 overexpression and subsequent prostaglandin synthesis specifically in ventricular myocytes. Histological, functional, and molecular analyses showed that ventricular myocyte specific COX-2 overexpression led to cardiac hypertrophy and fetal gene marker activation, but with preserved cardiac function. Therefore, specific induction of COX-2 and prostaglandin in vivo is sufficient to induce compensated hypertrophy and molecular remodeling.

Tumor formation in a mouse model of colitis-associated colon cancer does not require COX-1 or COX-2 expression.

Cyclooxygenase-2 (COX-2), a key enzyme of prostanoid biosynthesis, plays an important role in both hereditary and spontaneous colon cancer. Individuals with ulcerative colitis are also at high risk for colorectal cancer. To investigate the role of Cox-2 in colitis-associated colon cancer, we subjected Cox-2 luciferase-knock-in mice and Cox-2-knockout mice to a well-known mouse model of colitis-associated cancer in which animals are treated with a single-azoxymethane (AOM) injection followed by dextran sulfate sodium (DSS) administration. Tumors induced by AOM and DSS expressed significantly higher Cox-2 levels when compared with surrounding areas of colon, as detected both by luciferase reporter gene expression driven from the endogenous Cox-2 promoter and by western blotting of COX-2 protein in Cox-2 luciferase heterozygous knock-in mice. Immunofluorescence revealed that tumor stromal fibroblasts, macrophages and endothelial cells express COX-2 protein. In contrast, little COX-2 expression was observed in myofibroblasts or epithelial cells. Despite a significant elevation of COX-2 expression in AOM/DSS-induced colon tumors in wild-type mice, similar tumors developed in AOM/DSS-treated Cox-2(-/-)- and Cox-1(-/-)-knockout mice. These results indicate that cyclooxygenase-derived prostanoids are not major players in colitis-associated cancer. In contrast, tumor formation induced by multiple injections of AOM (with no DSS-induced colitis) did not occur in Cox-2(-/-)-knockout mice. Our data suggest that the mechanism of colorectal tumor promotion in colitis-associated cancer differs from the mechanism of tumor promotion for hereditary and sporadic colorectal cancer.

Novel anti-inflammatory functions for endothelial and myeloid cyclooxygenase-2 in a new mouse model of Crohn's disease.

Cyclooxygenase-2 (COX-2) is an important regulator of inflammation implicated in the development of a variety of diseases, including inflammatory bowel disease (IBD). However, the regulation of intestinal inflammation by COX-2 is poorly understood. We previously reported that COX-2(-/-) mice fed a cholate-containing high-fat (CCHF) diet had high mortality of unknown mechanisms attributable to severe intestinal inflammation in the ileo-ceco-colic junction that presented characteristics similar to Crohn's disease (CD). To further characterize the role of COX-2 in intestinal inflammation, we established cell-specific conditional COX-2(-/-) mice. Endothelial cell-specific (COX-2(-E/-E)) and myeloid cell-specific (COX-2(-M/-M)) COX-2(-/-) mice, but not wild-type mice, on the CCHF diet developed localized CD-like pathology at the ileo-ceco-colic junction that was associated with cellular infiltration, increased expression of myeloperoxidase and IL-5, and decreased IL-10 expression. The CD-like pathology in COX-2(-E/-E) mice was also accompanied by increased expression of cytokines (IL-6, TNF-alpha, and INF-gamma), compared with wild-type mice and COX-2(-M/-M) mice. In contrast, the ileo-ceco-colic inflammation in COX-2(-M/-M) mice was associated with more pronounced infiltration of granulocytes and macrophages than COX-2(-E/-E) mice. COX-2(-ME/-ME) (COX-2(-M/-M) x COX-2(-E/-E)) mice on the CCHF diet developed CD-like pathology in the ileo-ceco-colic junction reminiscent of total COX-2(-/-) mice on CCHF diet and wild-type mice on CCHF diet treated with COX-2 inhibitor, celecoxib. The pathology of diet-mediated ileo-ceco-colic inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the protective roles of endothelial and myeloid COX-2 and the molecular pathogenesis of CD.

Feedback regulation of cyclooxygenase-2 transcription ex vivo and in vivo.

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostaglandin biosynthesis. Its inducible expression is regulated by complex pathways. To monitor Cox-2 transcriptional activity in vivo, we generated a knock-in mouse expressing a firefly luciferase reporter. In this study we examined, by comparing luciferase activity of Cox-2(luc/+) and Cox-2(luc/-) cells and mice, effects of prostanoid products on Cox-2 promoter transcriptional activation. In peritoneal macrophages, luciferase induction by LPS in Cox-2(luc/-) cells was less than that of Cox-2(luc/+) cells. However, in the presence of PGE(2), induction was comparable, suggesting positive Cox-2 feedback regulation by PGE(2) occurs for macrophages. In contrast, feedback modulation was not observed in TPA-induced Cox-2(luc/+) and Cox-2(luc/-) mouse embryonic fibroblasts (MEFs). Using non-invasive in vivo imaging, we observed negative feedback regulation of Cox-2 expression during paw inflammation in living mice. Our results suggest Cox-2 expression is regulated by cell type specific feedback mechanisms, both in cultured cells and in living animals.

Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model.

Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS). Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u.) or intraperitoneal (i.p.) HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p.) prior to HK-GBS (i.p.) delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse.

COX-2 expression and function in the hyperalgesic response to paw inflammation in mice.

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.

The threshold level of adenomatous polyposis coli protein for mouse intestinal tumorigenesis.

The adenomatous polyposis coli (APC) gene, whose mutations are responsible for familial adenomatous polyposis, is a major negative controller of the Wnt/beta-catenin pathway. To investigate the dose-dependent effects of APC protein in suppressing intestinal tumorigenesis, we constructed mutant mice carrying hypomorphic Apc alleles Apc(neoR) and Apc(neoF) whose expression levels were reduced to 20% and 10% of the wild type, respectively. Although both hypomorphic heterozygotes developed intestinal polyps, tumor multiplicities were much lower than that in Apc(Delta716) mice, heterozygotes of an Apc null allele. Like in Apc(Delta716) mice, loss of the wild-type Apc allele was confirmed for all polyps examined in the Apc(neoR) and Apc(neoF) mice. In the embryonic stem cells homozygous for these hypomorphic Apc alleles, the level of the APC protein was inversely correlated with both the beta-catenin accumulation and beta-catenin/T-cell factor transcriptional activity. These results suggest that the reduced APC protein level increases intestinal polyp multiplicity through quantitative stimulation of the beta-catenin/T-cell factor transcription. We further estimated the threshold of APC protein level that forms one polyp per mouse as approximately 15% of the wild type. These results also suggest therapeutic implications concerning Wnt signaling inhibitors.

Transgenic mouse model for imaging of ATF4 translational activation-related cellular stress responses in vivo.

Activating transcription factor 4 (ATF4) is a translationally activated protein that plays a role in cellular adaptation to several stresses. Because these stresses are associated with various diseases, the translational control of ATF4 needs to be evaluated from the physiological and pathological points of view. We have developed a transgenic mouse model to monitor the translational activation of ATF4 in response to cellular stress. By using this mouse model, we were able to detect nutrient starvation response, antivirus response, endoplasmic reticulum (ER) stress response, and oxidative stress in vitro and ex vivo, as well as in vivo. The reporter system introduced into our mouse model was also shown to work in a stress intensity-dependent manner and a stress duration-dependent manner. The mouse model is therefore a useful tool for imaging ATF4 translational activation at various levels, from cell cultures to whole bodies, and it has a range of useful applications in investigations on the physiological and pathological roles of ATF4-related stress and in the development of clinical drugs for treating ATF4-associated diseases.

Imaging cyclooxygenase-2 (Cox-2) gene expression in living animals with a luciferase knock-in reporter gene.

The cyclooxygenase-2 (Cox-2) gene plays a role in a variety of normal and pathophysiological conditions. Expression of the Cox-2 gene is induced in a broad range of cells, in response to many distinct stimuli. The ability to monitor and quantify Cox-2 expression noninvasively in vivo may facilitate a better understanding of the role of Cox-2, both in normal physiology and in different diseases. We generated a "knock-in" mouse in which the firefly luciferase reporter enzyme is expressed at the start site of translation of the endogenous Cox-2 gene. Correlation of luciferase and Cox-2 expression was confirmed in heterozygous Cox-2luc/+ mouse embryonic fibroblasts isolated from the knock-in mouse. In an acute sepsis model, following injection of interferon gamma and endotoxin, ex vivo imaging and Western blotting demonstrated coordinate Cox-2 and luciferase induction in multiple organs. Using both paw and air pouch inflammation models, we can monitor repeatedly localized luciferase expression in the same living mouse. Cox-2luc/+ knock-in mice should provide a valuable tool to analyze Cox-2 expression in many disease models.

Gastrointestinal hamartomatous polyposis in Lkb1 heterozygous knockout mice.

Peutz-Jeghers syndrome (PJS) is a hereditary disorder characterized by gastrointestinal hamartomatous polyposis associated with mucocutaneous pigmentation. Germ-line mutations of the gene encoding LKB1 (STK11), a serine/threonine kinase, are identified in most PJS patients. To investigate the role of LKB1 in the PJS phenotypes, we introduced a germ-line mutation in the mouse Lkb1 gene by homologous recombination in mouse embryonic stem cells. In most Lkb1 (+/-) mice >20 weeks of age, hamartomatous polyps developed in the glandular stomach, often in the pyloric region. Small intestinal hamartomas also developed in approximately one-third of the Lkb1 (+/-) mice >50 weeks of age. A genomic PCR and sequence analysis showed that all hamartomas retained both the wild-type and targeted Lkb1 alleles, indicating that allelic loss of the wild-type Lkb1 was not the cause of polyp formation. Moreover, the LKB1 protein level was not reduced in hamartomatous polyps compared with that in the Lkb1 (+/-) normal gastric mucosa. In addition, the remaining allele showed neither missense mutations in the coding sequence nor produced truncated LKB1 in the hamartoma. Taken together, these data suggest that the wild-type Lkb1 is expressed in the hamartoma at the haploid amount. Accordingly, the gastrointestinal hamartomas appear to develop because of the Lkb1 haploinsufficiency. Although additional genetic events may be critical in hamartoma and adenocarcinoma development, these data strongly suggest that the initiation of polyposis is not the result of loss of heterozygosity in Lkb1.