Functional studies of *Ras and Bcl-2 oncoproteins in keratinocyte homeostasis and multistep skin carcinogenesis

To assess the effect of deregulated Ha-ras and bcl-2, individually and in combination on epidermal keratinocyte homeostasis and during multistep skin carcinogenesis, we generated skin-specific transgenic mice and keratinocyte transfectants constitutively expressing oncogenic Ha-ras and bcl-2 proteins. The deregulated Ha-ras and bcl-2 expression contributing to homeostatic imbalances in the skin had an additive effect on the probability of tumor development. They were also cooperative in incidence, growth, and latency of tumor formation, and they exhibited synergistic cooperation in malignant transformation of benign papillomas. To explain the homeostatic imbalances by Ha-ras and bcl-2 overexpression in the skin, we investigated the three major cellular processes of proliferation, cell death, and differentiation. Epidermal expression of Bcl-2 retarded keratinocyte proliferation in the epidermis of neonatal mice compared with results for control littermates. Constitutive expression of Ha-ras increased keratinocyte proliferation, and co-expression of bcl-2 modestly suppressed the ras-mediated abnormal proliferation of neonatal keratinocytes. Bcl-2 proteins in keratinocytes protected UV-treated cells from apoptotic cell death regardless of oncogenic ras expression in both non-neoplastic neonatal epidermis and human keratinocyte cell lines. The spontaneous apoptotic index (AI) was also lower in papillomas constitutively expressing bcl-2 compared with the ones that developed in control mice. Ras-overexpressing epidermis, including that in ras/bcl-2 double transgenic mice, had abnormal differentiation patterns compared with controls. The oncogenic ras protein had alterations in both epidermal distribution and the extent of cytokeratin 14 and involucrin expression. Abnormal expression of the hyperproliferation marker cytokeratin 6 and modest down regulation of cytokeratin 1 were also detected. Late appearance of filaggrin was another abnormal phenotype of the ras-expressing epidermis. Overexpression of bcl-2 had no effect on epidermal differentiation. Together, these findings suggest that constitutive expression of oncogenic Ha-ras and bcl-2 are important determinants of epidermal proliferation, viability and differentiation. In summary, our results demonstrated that the disruption of epidermal homeostasis by overexpressed ras and bcl-2 predisposes to hyperplastic growth of the epidermis and to papilloma development and that these proteins with distinct mechanisms for oncogenesis are functionally synergistic for malignant transformation of chemically induced skin carcinogenesis. ^

Disruption of the CD4–major histocompatibility complex class II interaction blocks the development of CD4+ T cells in vivo

The experiments presented in this report were designed to specifically examine the role of CD4–major histocompatibility complex (MHC) class II interactions during T cell development in vivo. We have generated transgenic mice expressing class II molecules that cannot interact with CD4 but that are otherwise competent to present peptides to the T cell receptor. MHC class II expression was reconstituted in Aβ gene knock-out mice by injection of a transgenic construct encoding either the wild-type I-Aβb protein or a construct encoding a mutation designed to specifically disrupt binding to the CD4 molecule. We demonstrate that the mutation, EA137 and VA142 in the β2 domain of I-Ab, is sufficient to disrupt CD4–MHC class II interactions in vivo. Furthermore, we show that this interaction is critical for the efficient selection of a complete repertoire of mature CD4+ T helper cells as evidenced by drastically reduced numbers of conventional CD4+ T cells in animals expressing the EA137/VA142 mutant I-Ab and by the failure to positively select the transgenic AND T cell receptor on the mutated I-Ab. These results underscore the importance of the CD4–class II interaction in the development of mature peripheral CD4+ T cells.

Unique molecular surface features of in vivo tolerized T cells

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vβ8.1 T cell antigen receptor (TCR) transgenic mice with a Vβ8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1a) induces tolerance to Mls-1a by clonal anergy. CD4 lymph node T cells from Mls-1a inoculated transgenic mice enriched for the 6C10− phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10+ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10− T cells remain 3G11hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+ T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8+ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+ T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new myc collaborating oncogene

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2αA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2αA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2αA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin

Infection with Helicobacter pylori is associated with different human gastric diseases. Biochemical studies, in vitro adherence assays, and in vivo animal models revealed that epithelial attachment of H. pylori can be mediated by the blood-group antigen-binding adhesin (BabA) targeting human Lewisb surface epitopes. Studies with transgenic mice expressing the Lewisb epitope have shown that such attachment can alter disease outcome. In the current study, the presence of the babA2 gene encoding the adhesin was investigated in clinical isolates from a German population by using PCR and reverse transcription–PCR. A positive genotype was correlated to allelic variations in the genes encoding VacA and CagA and also to the prevalence of duodenal ulcer, distal gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma, and antral gastritis. The presence of babA2 was significantly associated with duodenal ulcer (P = 0.0002) and adenocarcinoma (P = 0.033). In contrast, type 1 strains (vacAs1- and cagA-positive) were associated with only duodenal ulcer (P = 0.004) but not adenocarcinoma (P = 0.235). Genotype presence of babA2, vacAs1, and cagA (“triple-positive” strains) showed a highly significant correlation to the prevalence of ulcer (P = 0.000002) and adenocarcinoma (P = 0.014) and discriminated significantly better between disease outcome than did the current type 1 classification. These results indicate that the babA2 gene is of high clinical relevance and would be a useful marker to identify patients who are at higher risk for specific H. pylori-related diseases.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Targeted overexpression of protein kinase C β2 isoform in myocardium causes cardiomyopathy

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the β isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCβ isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCβ2 isoform in the myocardium. These mice overexpressed the PKCβ2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCβ-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCβ2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes

Synthesis of mouse metallothionein (MT)-I and MT-II is transcriptionally induced by the synthetic glucocorticoid, dexamethasone (DEX) or both in vivo as well as in numerous cell lines. However, the location(s) of a glucocorticoid response element (GRE) has not been described. The observation that a marked MT-I gene, as well as heterologous genes, when placed in the context of 17 kb of flanking sequence from the MT locus, are inducible by DEX and lipopolysaccharide in transgenic mice renewed the search for the GRE. Analysis of a series of deletion constructs from this 17-kb region in cultured cells identified a single 455-bp region that conferred DEX induction on a reporter gene. This 455-bp region contains two GREs that bind to the glucocorticoid receptor as assessed by gel mobility shift. Deletion of this fragment from the 17-kb flanking region eliminates the DEX responsiveness of reporter genes. The two GREs, which are located ≈1 kb upstream of the MT-II gene and ≈7 kb upstream of the MT-I gene, are necessary for induction of both genes and can function independently of elements within the proximal promoter region of either gene.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing dl-aminophosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, β-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of β-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Aminopeptidase A inhibitors as potential central antihypertensive agents

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT1 receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.