Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl

We have generated transgenic mice over-expressing human apolipoprotein CI (apo CI) using the native gene joined to the downstream 154-bp liver-specific enhancer that we defined for apo E. Human apo CI (HuCI)-transgenic mice showed elevation of plasma triglycerides (mg/dl) compared to controls in both the fasted (211 +/- 81 vs 123 +/- 52, P = 0.0001) and fed (265 +/- 105 vs 146 +/- 68, P < 0.0001) states. Unlike the human apo CII (HuCII)- and apo CIII (HuCIII)-transgenic mouse models of hypertriglyceridemia, plasma cholesterol was disproportionately elevated (95 +/- 23 vs 73 +/- 23, P = 0.002, fasted and 90 +/- 24 vs 61 +/- 14, P < 0.0001, fed). Lipoprotein fractionation showed increased VLDL and IDL + LDL with an increased cholesterol/triglyceride ratio (0.114 vs 0.065, P = 0.02, in VLDL). The VLDL apo E/apo B ratio was decreased 3.4-fold (P = 0.05) and apo CII and apo CIII decreased in proportion to apo E. Triglyceride and apo B production rates were normal, but clearance rates of VLDL triglycerides and postlipolysis lipoprotein "remnants" were significantly slowed. Plasma apo B was significantly elevated. Unlike HuCII- and HuCIII-transgenic mice, VLDL from HuCI transgenic mice bound heparin-Sepharose, a model for cell-surface glycosaminoglycans, normally. In summary, apo CI overexpression is associated with decreased particulate uptake of apo B-containing lipoproteins, leading to increased levels of several potentially atherogenic species, including cholesterol-enriched VLDL, IDL, and LDL.     

Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.     

Pyridoxal 5'-phosphate and the regulation of ornithine decarboxylase activity and stability

There are two forms of ornithine decarboxylase with respect to pyridoxal 5'-phosphate (pyridoxal-P) affinity in exponentially-growing Swiss 3T3 mouse fibroblasts: form I (Km approximately 10 muM) accounts for 30% of the total activity, and form II (Km approximately 0.4 muM) the remainder. Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal-P; neither form recognizes the Schiff base between ornithine and pyridoxal-P as a substrate. Total pyridoxal-P concentrations indicate that both forms may normally be at least partially active in vivo. Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10-fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme. The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association. The half-lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half-life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal-P serving a major protective function for the enzyme in vivo.     

Neuroprotective role of ornithine decarboxylase activation in transient focal cerebral ischaemia: a study using ornithine decarboxylase-overexpressing transgenic rats

Nuclear magnetic resonance imaging (MRI) was used to study dynamics of maturation and the size of ischaemic stroke lesions in rats with greatly increased activity of ornithine decarboxylase (ODC). Syngenic rats, either with or without chronic pre-ischaemic treatment with an ODC inhibitor, alpha-difluoromethylornithine (DFMO), as well as ODC-overexpressing transgenic rats were subjected either to transient middle cerebral artery (MCA) occlusion or permanent occlusion of the cortical branch of MCA. The two models were chosen to assess the role of ODC activity in damage caused by ischaemia and reperfusion, respectively. Diffusion of water was quantified by means of the trace of the diffusion tensor (D(av) = 1/3 Trace D) to assess the extent of energy failure and cytotoxic oedema, whereas the spin-spin relaxation time (T2) was used as a quantitative indicator of irreversible damage by MRI. Exposure to transient MCA occlusion resulted in significantly smaller stroke lesions in the ODC-overexpressing transgenic (246+/-14 mm3) than in syngenic (320+/-9 mm3) or DFMO-treated (442+/-63 mm3) rats as determined 48 h after the occlusion. The differences in sizes were due to smaller lesions in the cortical tissue (transgenic vs. syngenic) or both in cortical and striatal regions (transgenic vs. DFMO-treated animals). The degree of irreversible oedema was greater in DFMO-treated rats than in syngenic or transgenic animals indicating accelerated development of a permanent damage in the absence of ODC induction. Cortical infarct following permanent MCA occlusion developed faster in the DFMO-treated than in syngenic or transgenic rats as the lesion sizes at 10 h were 26.2+/-4.3 mm3, 14.2+/-2.3 mm3 and 12.3+/-1.9 mm3, respectively. However, the stroke volumes by 48 h were not statistically different in the three animal groups. The present data demonstrate that ODC activation is an endogenous neuroprotective measure in transient cerebral ischaemia.     

Functional expression of the human angiotensinogen gene in transgenic mice

The renin-angiotensin system is a major determinant of arterial pressure and volume homeostasis in mammals through the actions of angiotensin II, the proteolytic digestion product of angiotensinogen. Molecular genetic studies in several human populations have revealed genetic linkage between the angiotensinogen gene and both hypertension and increased plasma angiotensinogen. Transgenic mice were generated with a human angiotensinogen genomic clone to develop an animal model to examine tissue- and cell-specific expression of the gene and to determine if overexpression of angiotensinogen results in hypertension. Human angiotensinogen mRNA was expressed in transgenic mouse liver, kidney, heart, adrenal gland, ovary, brain, and white and brown adipose tissue and, in kidney, was exclusively localized to epithelial cells of the proximal convoluted tubules. Plasma levels of human angiotensinogen were approximately 150-fold higher in transgenic mice than that found normally in human plasma. The blood pressure of mice bearing the human angiotensinogen gene was normal but infusion of a single bolus dose of purified human renin resulted in a transient increase in blood pressure of approximately 30 mm Hg within 2 min. These results suggest that abnormalities in the angiotensinogen gene resulting in increased circulating levels of angiotensinogen could potentially contribute in part to the pathogenesis of essential hypertension.     

Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin

Ornithine decarboxylase (ODC) is aberrantly regulated in tumor cells and results in high basal levels of ODC and polyamines in many epithelial tumors. To determine if elevated ODC/polyamine levels can co-operate with a mutant Ha-ras gene in mouse skin tumorigenesis, double transgenic mice were generated by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice. A K6 keratin promoter drives the ODC transgene in K6/ ODC transgenic mice, which results in elevated ODC/ polyamine levels directed to the outer root sheath cells of hair follicles. TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles. Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene did not develop tumors unless treated with either a carcinogen or a tumor promoter, respectively. However, a high percentage of double transgenic mice possessing both the K6/ODC and v-Ha-ras transgenes developed spontaneous tumors. All tumors were well-differentiated keratoacanthomas, some of which progressed to carcinomas within 2 months. The development and the maintenance of these ODC/ras tumors was ODC-dependent since alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the formation and caused the regression of these tumors. These findings indicate that ODC overexpression and an activated Ha-ras are sufficient to produce a high rate of malignant transformation in an animal model. The ODC/ras double transgenic mouse provides a simple in vivo model without the use of chemical carcinogens or tumor promoters in which to test downstream effectors that play a key role in mediating the development of epithelial tumors resulting from the cooperation between ODC and v-Ha-ras.     

Action of prolactin on ornithine decarboxylase activity in mammary gland explants of mice

Prolactin stimulated ornithine decarboxylase activity in mammary gland explants from midpregnant mice. The enhanced enzyme activity occurred in explants which were preincubated for 1 day in medium containing insulin, hydrocortisone, insulin plus hydrocortisone, or in medium containing no hormones. The largest prolactin effect was observed in tissues which were pretreated with insulin plus hydrocortisone; a greater than ten-fold increase in ornithine decarboxylase activity was observed when these tissues were incubated with prolactin for 2 hours. An effect of prolactin on ornithine decarboxylase activity was also observed in explants prepared from lactating mouse mammary glands.     

Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein

We studied the role of the amyloid precursor protein (APP) in ischemic brain damage using transgenic mice overexpressing APP. The middle cerebral artery (MCA) was occluded in FVB/N mice expressing APP695.SWE (Swedish mutation) and in nontransgenic littermates. Infarct volume (cubic millimeters) was assessed 24 hr later in thionin-stained brain sections. The infarct produced by MCA occlusion was enlarged in the transgenics (+32 +/- 6%; n = 12; p < 0. 05; t test). Measurement of APP by ELISA revealed that, although relatively high levels of Abeta were present in the brain of the transgenics (Abeta1-40 = 80 +/- 19 pmol/g; n = 6), there were no differences between ischemic and nonischemic hemispheres (p > 0.05). The reduction in cerebral blood flow produced by MCA occlusion at the periphery of the ischemic territory was more pronounced in APP transgenics (-42 +/- 8%; n = 9) than in controls (-20 +/- 8%; n = 9). Furthermore, the vasodilatation produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (10 microM) was reduced by 82 +/- 5% (n = 8; p < 0.05) in APP transgenics. The data demonstrate that APP overexpression increases the susceptibility of the brain to ischemic injury. The effect is likely to involve the Abeta-induced disturbance in endothelium-dependent vascular reactivity that leads to more severe ischemia in regions at risk for infarction. The cerebral vascular actions of peptides deriving from APP metabolism may play a role in the pathogenic effects of APP.     

Prognostic influence on survival of increased ornithine decarboxylase activity in human breast cancer

Although considerable experimental evidence suggests an important role of polyamines in breast cancer biology, compelling supportive data in patients are lacking. To address this issue, we measured ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase, and spermidine/spermine acetyltransferase (the three key polyamine metabolic enzymes) in a cohort of 50 primary human breast cancers and related their levels of activity to disease-free survival and overall survival. The major finding of our study was that ODC activity level was a negative independent prognostic factor for both end points. With regard to overall survival, the adverse influence of ODC expression was superior even to that provided by the number of positive nodes. Furthermore, the statistical significance of the ODC effect on survival was enhanced when breast cancer-specific mortality was included in the analysis as opposed to death from any cause. In addition, high tumor ODC activity may predict a shorter time from recurrence to death, although this effect was of only borderline statistical significance. In summary, these results provide the first concrete evidence supporting the prognostic role of ODC in human breast cancer.     

Neurodegeneration and gliosis in transgenic mice overexpressing a carboxy-terminal fragment of Alzheimer amyloid-beta protein precursor

In order to study the properties of beta-amyloid in vivo, we generated a total of 28 transgenic founder mice that harbored the gene for the 17-amino acid signal sequence and the 99-amino acid carboxy-terminal fragment (CTF) of the human amyloid-beta protein precursor (beta APP) linked to the cytomegalovirus enhancer and chicken beta-actin promoter. Two of these founders, termed 0304 and 0809, exhibited decreased behavioral activity with gliosis and neurodegeneration in the hippocampus at 2.5 and 9 months of age. Both mice had also decreased levels of synaptophysin, a presynaptic marker, but no evidence for beta-amyloid deposition in their brains. Neurodegeneration in the hippocampus was transmitted to the offspring of mouse 0304, although the frequency was low (5 of 44 mice examined) and the time of onset of the disorder was rather later than that in the founder mouse. This is probably due to reduced levels of the transgene-derived products in the offspring of mouse 0304. The 0809 line failed to produce its offspring. The other remaining transgenic founders appeared normal and had lesser amounts of the CTF mRNA and protein in their brains than did 0304 and 0809 founders, though some mice died in earlier stages or exhibited hydrocephalus. These findings suggest that overexpression of the CTF of human beta APP has the potential to elicit neurodegeneration in vivo without appreciable production of beta-amyloid fibrils.     

Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice

Human erythropoietin (Epo) gene expression is inducible by hypoxia or anaemia in the kidney and liver. Previous transgenic mouse experiments have demonstrated that sequences required for Epo gene induction in the kidney reside in a 7 8 kb Barn HI fragment located 6 kb upstream of the gene. To sublocalize these sequences, we performed Desoxyribonuclease I (DNAse I) mapping studies using transgenic mice which carried this DNA fragment. These studies revealed a DNAse I hypersensitive site (DNAse I HS) located 4 6 kb from the upstream end of the 7.8 kb fragment in anaemic kidney and liver samples. Sequence analysis of the region encompassing the DNAse I HS revealed an element with remarkable homology to the 3' Epo gene hypoxia-inducible enhancer. This suggested the presence of an additional regulatory element that contributes to the control of hypoxia-inducible Epo gene expression in kidney and liver. We constructed transgenic mice containing the human Epo gene linked to either the 5 kb upstream or 2.5 kb downstream portion of the 7.8kb fragment. Inducible expression was limited to the liver. Thus, neither fragment was alone sufficient to confer kidney inducible expression. These findings indicate that sequences more than 8.5 kb upstream of the Epo gene are required for kidney-specific induction. They suggest that either those sequences reside in an 0.3 kb Hind III fragment located between the 5 kb and the 2.5 kb fragments or that sequences in the 5 kb or 0.3 kb fragments must interact with sequences in the 2.5 kb fragment to allow Epo gene induction in the kidney.     

Epidermal ornithine decarboxylase and polyamines in mice exposed to 50 Hz magnetic fields and UV radiation

We studied the influence of magnetic fields (MFs) and simulated solar radiation (SSR) on ornithine decarboxylase (ODC) and polyamines in mouse epidermis. Chronic exposure to combined MF and SSR did not cause persistent effects on ODC activity or polyamines compared to the animals exposed only to UV, although the same MF treatment was previously found to accelerate skin tumor development. In an acute 24-h experiment, an elevation of putrescine and down-regulation of ODC activity was observed in the animals exposed to a 100-microT MF. No effect was seen 24 h after a single 2-MED (minimal erythemal dose) exposure to SSR. The results indicate that acute exposure to 50 Hz MF does exert distinctive biological effects on epidermal polyamine synthesis.     

Alteration of hormone levels in transgenic tobacco plants overexpressing the rice homeobox gene OSH1

In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.     

Association between high levels of ornithine decarboxylase activity and favorable prognosis in human colorectal carcinoma

Several studies have documented increased expression of ornithine decarboxylase (ODC) in neoplastic colorectal tissue versus normal-appearing colonic mucosa. The present study was undertaken to determine whether there is an association between the degree of overexpression of ODC in colorectal carcinomas and survival in a series of 74 patients. A high level of tumor ODC expression was found to be significantly associated with greater survival in our patient series. Patients with tumor ODC activities greater than the median and especially in the highest quartile experienced a more favorable outcome than those patients with ODC values below the median or in the lowest quartile (P = 0.03 and 0.02, respectively). The presence of a GTP-activatable isoform of ODC was also significantly associated with a favorable prognosis but only in tumors of the right colon (P = 0.01). There was no association found between ODC activity and tumor grade, tumor size, or patient age, sex, or race. Our results demonstrate that high levels of ODC expression (and presence of a GTP-activatable isoform for right-sided colon tumors) predict a favorable prognosis in human colorectal carcinoma. Knowledge of a patient's ODC status at the time of surgery may be useful in decisions regarding adjuvant therapy. Understanding the mechanism(s) involved should lead to new therapeutic approaches for advanced colorectal carcinoma.     

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.     

Mast cell-/basophil-specific transcriptional regulation of human L-histidine decarboxylase gene by CpG methylation in the promoter region

L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine, and in hematopoietic cell lineages the gene is expressed only in mast cells and basophils. We attempted here to discover how HDC gene expression is restricted in these cells. In the cultured cell lines tested, only the mast cells and basophils strongly transcribed the HDC gene. However, in transient transfection analysis, the reporter constructs with the HDC promoter were active not only in expressing cells but also in nonexpressing cells. Detailed analyses of the HDC promoter region revealed that the GC box is essential for transactivation. Also, the promoter region of the HDC gene proved to be sensitive to DNase I and restriction endonucleases exclusively in HDC-expressing cells, suggesting that the promoter region is readily accessible to trans-acting factor(s). Furthermore, the promoter region in HDC-expressing cell lines was found to be selectively unmethylated. The correlation between HDC expression and hypomethylation was also found in primary human mast cells. Methylation of the HDC promoter in vitro reduced the luciferase reporter activity in transient expression analysis, suggesting that methylation of the promoter region is functionally important for HDC gene expression. These results imply that alteration of DNA methylation is one of the mechanisms regulating cell-specific expression of the HDC gene.