Jerrold M. Ward

Targeted Disruption of the Nuclear Receptor FXR/BAR Impairs Bile Acid and Lipid Homeostasis

Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-/- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice were reduced in size by 50 percent and showed bile duct fibrosis Ahr-/- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system.

Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.

ABSTRACT The numerous functions of the liver are controlled primarily at the transcriptional level by the concerted actions of a limited number of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 1α [HNF1α], -1β, -3α, -3β, -3γ, -4α, and -6 and members of the c/ebp family). Of these, only HNF4α (nuclear receptor 2A1) and HNF1α appear to be correlated with the differentiated phenotype of cultured hepatoma cells. HNF1α-null mice are viable, indicating that this factor is not an absolute requirement for the formation of an active hepatic parenchyma. In contrast, HNF4α-null mice die during embryogenesis. Moreover, recent in vitro experiments using tetraploid aggregation suggest that HNF4α is indispensable for hepatocyte differentiation. However, the function of HNF4α in the maintenance of hepatocyte differentiation and function is less well understood. To address the function of HNF4α in the mature hepatocyte, a conditional gene knockout was produced using the Cre-loxP system. Mice lacking hepatic HNF4α expression accumulated lipid in the liver and exhibited greatly reduced serum cholesterol and triglyceride levels and increased serum bile acid concentrations. The observed phenotypes may be explained by (i) a selective disruption of very-low-density lipoprotein secretion due to decreased expression of genes encoding apolipoprotein B and microsomal triglyceride transfer protein, (ii) an increase in hepatic cholesterol uptake due to increased expression of the major high-density lipoprotein receptor, scavenger receptor BI, and (iii) a decrease in bile acid uptake to the liver due to down-regulation of the major basolateral bile acid transporters sodium taurocholate cotransporter protein and organic anion transporter protein 1. These data indicate that HNF4α is central to the maintenance of hepatocyte differentiation and is a major in vivo regulator of genes involved in the control of lipid homeostasis.

Growth, Adipose, Brain, and Skin Alterations Resulting from Targeted Disruption of the Mouse Peroxisome Proliferator-Activated Receptor β(δ)

ABSTRACT To determine the physiological roles of peroxisome proliferator-activated receptor β (PPARβ), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARβ gene. Homozygous PPARβ-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARβ-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARβ-null mice. PPARβ was not required for induction of mRNAs involved in epidermal differentiation induced byO-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARβ-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARβ-null mice. These results are the first to provide in vivo evidence of significant roles for PPARβ in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.

Prostate Pathology of Genetically Engineered Mice: Definitions and Classification. The Consensus Report from the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee

The Pathological Classification of Prostate Lesions in Genetically Engineered Mice (GEM) is the result of a directive from the National Cancer Institute Mouse Models of Human Cancer Consortium Prostate Steering Committee to provide a hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and the translation to human prostate pathology. The proposed Bar Harbor Classification system is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the Mouse Models of Human Cancer Consortium. A 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, in which study sets of 93 slides from 22 GEM models were provided to individual panel members. The comparison of mouse and human prostate anatomy and disease demonstrates significant differences and considerable similarities that bear on the interpretation of the origin and natural history of their diseases. The recommended classification of mouse prostate pathology is hierarchical, and includes developmental, inflammatory, benign proliferative, and neoplastic disorders. Among the neoplastic disorders, preinvasive, microinvasive, and poorly differentiated neoplasms received the most attention. Specific criteria were recommended and will be discussed. Transitions between neoplastic states were of particular concern. Preinvasive neoplasias of the mouse prostate were recognized as focal, atypical, and progressive lesions. These lesions were designated as mouse prostatic intraepithelial neoplasia (mPIN). Some atypical lesions were identified in mouse models without evidence of progression to malignancy. The panel recommended that mPIN lesions not be given histological grades, but that mPIN be further classified as to the absence or presence of documented associated progression to invasive carcinoma. Criteria for recognizing microinvasion, for classification of invasive gland-forming adenocarcinomas, and for characterizing poorly differentiated tumors, including neuroendocrine carcinomas, were developed and are discussed. The uniform application of defined terminology is essential for correlating results between different laboratories and models. It is recommended that investigators use the Bar Harbor Classification system when characterizing new GEM models or when conducting experimental interventions that may alter the phenotype or natural history of lesion progression in existing models.

Neoplastic and nonneoplastic lesions in aging F344 rats

Abstract Neoplastic and nonneoplastic lesions in untreated F344 rats used as controls in carcinogenesis tests were tabulated and evaluated. The most common neoplasms in 1794 male rats were testicular interstitial cell tumors, leukemias, pituitary adenomas, and adrenal pheochromocytomas. In 1754 female rats, common tumors were pituitary adenomas, mammamary fibroadenomas, uterine endometrial stromal polyps, and leukemias. A variety of less common and rare tumors were seen in almost every tissue. A few malignant neoplasms were metastatic. Nonneoplastic lesions included nephropathy, cardiomypathy, focal hyperplasias in a variety of tissues, and inflammatory lesions of the uterus. The focal hyperplasias were suggestive of the early stages of neoplasia in lung, liver, pituitary, adrenal, thyroid, and testis.

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting{

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Liver-specific disruption of PPARγ in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes

To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.

Lesions of Aryl-hydrocarbon Receptor–deficient Mice

We have analyzed the possible role of the aryl-hydrocarbon receptor (AHR) in the aging process of mice using a homozygous null mouse (Ahr-/-) line as a model. We studied 52 male and female Ahr-/- mice aged from 6-13 months. Forty-six percent died or were ill by 13 months of age. Ahr-/- mice developed age-related lesions in several organs, some of which were apparent after only 9 months of age. Cardiovascular alterations included cardiomyopathy (100%) with hypertrophy and focal fibrosis. Vascular hypertrophy and mild fibrosis were found in the portal areas of the liver (81%), and vascular hypertrophy and mineralization were common in the uterus (70%). Gastric hyperplasia that progressed with age into polyps was evident in the pylorus of 71% of the mice over 9 months of age. Ahr-/- mice had T-cell deficiency in their spleens but not in other lymphoid organs. The immune system deficiency described previously could be the origin for the rectal prolapse found in 48% of the null mice, associated with Helicobacter hepaticus infection. In the dorsal skin (53% incidence), severe, localized, interfollicular and follicular epidermal hyperplasia, with hyperkeratosis and acanthosis, and marked dermal fibrosis, associated with the presence of anagenic hair follicles, were also evident. None of these lesions were found in 42 control (Ahr +/+ or +/-) mice of similar ages. These observations suggest that the AHR protein, in the absence of an apparent exogenous (xenobiotic) ligand, plays an important role in physiology and homeostasis in major organs in mice, and further supports an evolutionary conserved role for this transcription factor.