Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.     

Lessons from TGF-beta transgenic mice

Aberrant expression of TGF-beta and/or receptor/signaling function is present in a wide variety of disease processes. Overexpression of TGF-beta isoforms in transgenic mice using tissue-specific promoters has provided model systems to study the effects of increased activity of TGF-beta in the intact organism. We will review the pertinent features of some of these models, and discuss new insights provided by these studies into regulation and role of TGF-beta in health and disease.     

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Expression of a dominant-negative mutant human insulin receptor in the muscle of transgenic mice

To examine the in vivo effects of a kinase-deficient mutant human insulin receptor, we used the muscle creatine kinase promoter to express a putative dominant-negative receptor: Ala1134-->Thr (Moller, D. E., Yokota, A., White, M. F., Pazianos, A. G., and Flier, J. S. (1990) J. Biol. Chem. 265, 14979-14985) in transgenic mice. Two lines were generated, where receptor expression was restricted to striated muscle and was increased by 5-12-fold in skeletal muscle. Transgenic gluteal muscle insulin receptor kinase activity was reduced by approximately 80% after maximal in vitro insulin stimulation. Glycogen content in this muscle was reduced by 45% in transgenic mice. Insulin levels were approximately 2-fold higher, and glucose concentrations were 12% higher in transgenics fed ad libitum. Transgenic mice exhibited reduced in vivo sensitivity to low dose (0.1 milliunits/g) intravenous insulin. In isolated soleus muscles from transgenics, where mutant receptors were expressed at lower levels, insulin-stimulated receptor kinase activity was reduced by 42%, but insulin-stimulated 2-deoxyglucose uptake was unaffected. These results indicate that (i) overexpression of a kinase-deficient human insulin receptor in muscle causes dominant-negative effects at the level of receptor kinase activation, (ii) impairment of insulin-stimulated muscle receptor tyrosine kinase activity can cause decreased insulin sensitivity in vivo, (iii) kinase-defective receptor mutants may be used to create novel animal models of tissue-specific insulin resistance.     

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen alpha1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.     

Using knockout and transgenic mice to study neurophysiology and behavior

Reverse genetics, in which detailed knowledge of a gene of interest permits in vivo modification of its expression or function, provides a powerful method for examining the physiological relevance of any protein. Transgenic and knockout mouse models are particularly useful for studies of complex neurobiological problems. The primary aims of this review are to familiarize the nonspecialist with the techniques and limitations of mouse mutagenesis, to describe new technologies that may overcome these limitations, and to illustrate, using representative examples from the literature, some of the ways in which genetically altered mice have been used to analyze central nervous system function. The goal is to provide the information necessary to evaluate critically studies in which mutant mice have been used to study neurobiological problems.     

p53-dependent impairment of T-cell proliferation in FADD dominant-negative transgenic mice

Members of the tumour necrosis factor (TNF) receptor family exert pleiotropic effects and can trigger both apoptosis and proliferation [1]. In their cytoplasmic region, some of these receptors share a conserved sequence motif - the 'death domain' - which is required for transduction of the apoptotic signal by recruiting other death-domain-containing adaptor molecules like the Fas-associated protein FADD/MORT1 or the TNF receptor-associated protein TRADD [2-4]. FADD links the receptor signal to the activation of the caspase family of cysteine proteases [5,6]. Functional inactivation of individual receptor family members often fails to exhibit a distinctive phenotype, probably because of redundancy [7-9]. To circumvent this problem, we used a dominant-negative mutant of FADD (FADD-DN) which should block all TNF receptor family members that use FADD as an adaptor. We established transgenic mice expressing FADD-DN under the influence of the lck promoter and investigated the consequences of its expression in T cells. As expected, FADD-DN thymocytes were protected from death induced by CD95 (Fas/Apo1), whereas apoptosis induced by ultraviolet (UV) irradiation, anti-CD3 antibody treatment or dexamethasone was unaffected, as was spontaneous cell death. Surprisingly, however, we also observed profound inhibition of thymocyte proliferation in vivo and of activation-induced proliferation of thymocytes and mature T cells in vitro. This inhibition of proliferation was not due to increased cell death and appeared to be p53 dependent.     

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.     

Amyloidogenic role of cytokine TGF-beta1 in transgenic mice and in Alzheimer's disease

Deposition of amyoid-beta peptide in the central nervous system is a hallmark of Alzheimer's disease and a possible cause of neurodegeneration. The factors that initiate or promote deposition of amyloid-beta peptide are not known. The transforming growth factor TGF-beta1 plays a central role in the response of the brain to injury, and increased TGF-beta1 has been found in the central nervous system of patients with Alzheimer's disease. Here we report that TGF-beta1 induces amyloid-beta deposition in cerebral blood vessels and meninges of aged transgenic mice overexpressing this cytokine from astrocytes. Co-expression of TGF-beta1 in transgenic mice overexpressing amyloid-precursor protein, which develop Alzheimer's like pathology, accelerated the deposition of amyloid-beta peptide. More TGF-beta1 messenger RNA was present in post-mortem brain tissue of Alzheimer's patients than in controls, the levels correlating strongly with amyloid-beta deposition in the damaged cerebral blood vessels of patients with cerebral amyloid angiopathy. These results indicate that overexpression of TGF-beta1 may initiate or promote amyloidogenesis in Alzheimer's disease and in experimental models and so may be a risk factor for developing Alzheimer's disease.     

Behavioral differences among 129 substrains: Implications for knockout and transgenic mice.

Most knockout (KO) mice are produced with embryonic stem cells derived from a 129 strain. Because most KO strains are backcrossed to B6 yet retain a portion of their genome from 129, especially around the ablated target locus, phenotypes previously attributed to the ablated locus may be due to passenger 129 genes. Thus, the authors decided to test several 129 substrains for their behavioral characteristics. Seven 129 substrains were put through a battery of tasks to determine their behavioral profiles. Differences were found in anxiety-related behaviors in the zero-maze, habituation to the open field, and cued fear conditioning. All strains successfully performed the rotorod task. The behavioral differences observed may have important implications for the interpretation of data and show divergence of behavioral performance in these 129 substrains. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit

Amino acid changes S180A (S-->A at site 180), H197Y, Y277F, T285A, and A308S are known to shift the maximum wavelength of absorption (lambda max) of red and green visual pigments toward blue, essentially in an additive fashion. To test the generality of this "five-sites" rule, we have determined the partial amino acid sequences of red and green pigments from five mammalian orders (Artiodactyla, Carnivora, Lagomorpha, Perissodactyla, and Rodentia). The result suggests that cat (Felis catus), dog (Canis familiaris), and goat (Capra hircus) pigments all with AHYTA at the five critical sites have lambda max values of approximately 530 nm, whereas rat (Rattus norvegicus) pigment with AYYTS has a lambda max value of approximately 510 nm, which is accurately predicted by the five-sites rule. However, the observed lambda max values of the orthologous pigments of European rabbit (Oryctolagus cuniculus), white-tailed deer (Odocoileus virginianus), gray squirrel (Sciurus carolinensis), and guinea pig (Cavia procellus) are consistently more than 10 nm higher than the predicted values, suggesting the existence of additional molecular mechanisms for red and green color vision. The inferred amino acid sequences of ancestral organisms suggest that the extant mammalian red and green pigments appear to have evolved from a single ancestral green-red hybrid pigment by directed amino acid substitutions.     

Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice

Aberrant expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) has been linked to developmental abnormalities in vertebrate models, and has been implicated in multiple disease states in humans. To identify cis-acting regulatory elements that dictate expression of this receptor, we generated transgenic mice bearing the reporter gene beta-galactosidase (lacZ) under the control of a 6-kb promoter sequence. Expression of lacZ was monitored throughout embryonic development, with special focus on nervous tissue, skeleton, and several organ systems wherein PDGF alpha R expression is thought to play a pivotal role. In several independent transgenic mouse strains, lacZ expression recapitulated predominant features of PDGF alpha R gene expression during mouse development. These results demonstrate that critical tissue-specific regulatory elements for PDGF alpha R expression are located within a 6-kb upstream region of the PDGF alpha R gene.     

mu Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality

Previous studies on the Khat plant (Catha edulis, Celastraceae) illustrated the importance of using freshly harvested young shoots and leaves such that cathinone, the principle active component and Schedule I controlled drug contained within the plant, could be suitably isolated and identified. The purpose of this work was to develop a quantitative analytical technique for the determination of cathinone. The proposed method is based on treating the reductant cathinone with copper(II)-neocuproine reagent in sodium acetate-buffered medium followed by measuring the absorbance of the copper(I)-neocuproine complex at 455 nm. The calibration plot is linear in the range 0.08-25 micrograms ml-1 with a detection limit of 0.08 microgram ml-1. The precision of the method, expressed as the relative standard deviation, is 1.35% for 10 micrograms ml-1 cathinone. Good recoveries have been obtained in applying the method to the analysis of cathinone in Khat leaves.     

Dopamine neurons from transgenic mice with a knockout of the p53 gene resist MPTP neurotoxicity

Haemagglutination activity (HA) was found and characterized in a midgut homogenate of Ixodes ricinus (L.). HA was induced by tick feeding; it was not detected in starved ticks. In a haemagglutination inhibition test, HA showed an affinity for some carbohydrates (N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, rhamnose, and dulcit) and glycoconjugates (especially lipopolysaccharides). Midgut protein components of 37, 60, 65, and 73 kDa were identified by immunoblotting as potential structural subunits of the new agglutinin.     

Ethanol inhibits hepatocyte proliferation in insulin receptor substrate 1 transgenic mice

BACKGROUND & AIMS: Long-term ethanol consumption is known to impair the ability of the liver to regenerate, but the molecular mechanisms are poorly understood. Multiple growth factors promote hepatocyte proliferation, some of which involve the insulin receptor substrate 1 (IRS-1)-mediated signal transduction pathway. To explore effects of ethanol on the IRS-1 signal liver growth in vivo, studies in transgenic mice overexpressing IRS-1 in the liver were performed because these mice show constitutive activation of the downstream signal transduction pathways leading to enhanced hepatocyte proliferation. METHODS: Tyrosyl phosphorylation of IRS-1 and subsequent protein-protein interactions were examined in liver lysates from animals fed ethanol or control diet. Activity of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) was assessed by specific enzymatic assays. Hepatocyte proliferation was measured by incorporation of [3H]thymidine into liver DNA. RESULTS: Tyrosyl phosphorylation of IRS-1, association of IRS-1 with PI3K, and activation of downstream PI3K and MAPK pathways were greatly reduced as a result of long-term ethanol consumption. Ethanol virtually abolished the enhanced hepatocyte DNA synthesis induced by expression of the IRS-1 transgene. CONCLUSIONS: Altered transmission of growth signals through the IRS-1-mediated signal transduction cascade may represent a molecular mechanism of how ethanol inhibits hepatocyte proliferation.