The EMBO Journal vol.10 no.8 pp.2119-2124, 1991

Gene dosage of CuZnSOD and Down's syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice D.Minc-Golomb, H.Knoblerl and Y.Groner Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, and 'Department of Internal Medicine, Kaplan Hospital, Rehovot, Israel Communicated by Y.Groner

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD. Key words: Down's syndrome/gene dosage/prostaglandin E2/transgenic CuZnSOD mice

Introduction Down's syndrome (DS), a genetic abnormality associated with the presence of three copies of chromosome 21 (Lejeune et al., 1959), is one of the most important human congenital diseases, occurring in one per 700-1000 live births (Hook, 1981). DS patients suffer from a wide range of symptoms. Most obvious among these are morphological defects such as hypotonia in the newborn, short stature and the epicanthic eye folds which give rise to the eye shape characteristic of the syndrome (Benda, 1969). Patients are mentally retarded and those who survive past their mid-thirties usually develop Alzheimer's disease (Epstein, 1986a,b). Premature aging and an increased incidence of leukemia and other hematological disorders are common in affected individuals, as are cardiac defects, a high susceptibility to infections and several types of endocrinological disorders (Epstein, 1986b). Although DS was described more than a century ago and the relationship between trisomy 21 and the Down's phenotype has been

known for over 30 years, very little is known about the way in which the additional chromosome 21 causes the disease (reviewed in Cooper and Hall, 1988). The current concept is that the presence of extra copies of genes that reside on chromosome 21 results in the synthesis of increased amounts

which creates an imbalance in various biochemical pathways; this in turn causes the physiological defects giving rise to the clinical picture of DS. One of the main research efforts in the molecular genetics of DS is directed towards cloning of genes residing on chromosome 21 and relating the consequences of their enhanced expression to the clinical symptoms of the syndrome through the use of model systems (for review see Groner and Elson,

of gene products

1991).

The human copper zinc superoxide dismutase (hCuZnSOD) is a key enzyme in the metabolism of oxygen free radicals (reviewed in Fridovich, 1986). It is encoded by a gene residing on chromosome 21 at the chromosomal region 2 1q22 known to be involved in DS (Cooper and Hall, 1988) and elevated levels of the enzyme are commonly found in DS patients (Francke, 1981; Sherman et al., 1983). This overexpression of the CuZnSOD gene, due to gene dosage, may disturb the steady-state equilibrium of active oxygen species within the cells resulting in oxidative damage to biologically important molecules (Badwey and Karnovsky, 1980; Cerutti, 1985; Halliwell and Gutteridge, 1985). Indeed, recent reports have demonstrated that CuZnSOD is able to catalyze hydroxyl radical production from hydrogen peroxide (Yim et al., 1990) and that elevated levels of SOD enhance the cytotoxicity of active oxygen species (FinazziAgro et al., 1986; Scott et al., 1987; Iwahashi et al., 1988). Such processes may in part be responsible for certain clinical symptoms associated with the Down's phenotype (Balazs and Brooksbank, 1985; Groner et al., 1985, 1986a,b). The possible involvement of CuZnSOD overproduction in the etiology of the syndrome was investigated by us in transfected cell lines and transgenic mice harboring the human gene (Groner et al., 1985, 1986b; Epstein et al., 1987). The transgenic CuZnSOD mice displayed several abnormalities found in DS patients, e.g. diminution of blood serotonin (Schickler et al., 1989) and abnormalities in neuromuscular junctions (Avraham et al., 1988, 1991; Yarom et al., 1988). The transfected cells overexpressing the hCuZnSOD gene had altered properties with increased lipid peroxidation, higher resistance to the toxic effects of paraquat (Elroy-Stein et al., 1986) and a lesion in the uptake mechanism responsible for the transport of biogenic amines (Elroy-Stein and Groner, 1988). Prostaglandins (PGs), a family of fatty acid derivatives, are important local chemical mediators participating in a variety of biological activities (reviewed in Needlman et al., 1986). PGs are continuously synthesized in membranes from precursors that have been cleaved from membrane phospholipids. Because the metabolism of oxygen free radicals and in particular the process of lipid peroxidation are tightly connected to the biosynthesis of PGs (Lands et al., 1984), we analyzed their formation in the two model systems: transfected PC 12 cells possessing elevated levels of CuZnSOD and transgenic CuZnSOD mice. In addition we examined trisomy 21 cells obtained from DS patients.

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D.Minc-Golomb, H.Knobler and Y.Groner

It was found that the biosynthesis of the prostaglandins E2 (PGE2), D2 (PGD2) and 6-keto-PGFoe was significantly reduced. The results suggest that CuZnSOD gene dosage is associated with alterations of arachidonic acid metabolism in trisomy 21.

Results Reduced synthesis of PGE2 in trisomy 21 fibroblasts and in PC12-CuZnSOD cells Cell cultures of trisomy 21 and diploid fibroblasts (see Materials and methods) were assayed for CuZnSOD activity and production of PGE2. The trisomy 21 cells contained approximately twice as much CuZnSOD activity as normal fibroblasts (Table I) in agreement with previous reports (Francke, 1981; Sherman et al., 1983; Epstein, 1986a). When the ability of the cells to secrete PGE2 was analyzed by radioimmunoassay it was found that trisomy 21 fibroblasts displayed a 50% reduction in released PGE2 (Table I). To examine whether the impaired biosynthesis of PGE2 was related to the elevated level of CuZnSOD activity in the trisomy 21 fibroblasts, rat PC 12-SOD cells transfected with hCuZnSOD gene were analyzed for PGE2 release. Construction of PC12 cell clones harboring hCuZnSOD gene was previously described (Elroy-Stein and Groner, 1988). The 'P' pool analyzed here represents 14 independently isolated clones that overexpress approximately twice the normal level of CuZnSOD (Figure 1 and Table I). The magnitude of this increase in CuZnSOD activity is similar to the values found in the trisomy 21 fibroblasts (Table I). Pool 'N', the control cells, contained four transfected clones which exhibited resistance to G418 but did not express the hCuZnSOD gene. The PC12-CuZnSOD cells closely resembled the parental cells in their morphology, growth rate and response to nerve growth factor. Analysis by TLC of arachidonic acid (AA) metabolites released from these cells had indicated, as previously reported by others (DeGeorge and Walenga, 1989), that PGE is the main PG produced by PC12 cells (Figure 2). In the transfected PC12-CuZnSOD cells the amount of PGE2 was lower compared with the control pool (Figure 2). Quantitation by densitometry showed that the amount of PGE2 extracted from PC12-CuZnSOD cells was 30% that of control while the level of other AA derivatives in the two cell types did

not differ significantly. Similar to the trisomy 21 fibroblasts, lower production of PGE2 by PC12-CuZnSOD cells was also manifested in a diminished secretion as determined by radioimmunoassay (RIA) (Table I). The cleavage of AA from cellular phospholipids, a step considered to be rate limiting in PG biosynthesis (Conklin et al., 1988), is enhanced by calcium ionophores and cholinergic agonists (DeGeorge et al., 1986). When the PC12 cells were treated with the calcium ionophore A23 187 or with the cholinergic agonist carbachol a significant increase in the level of PGE2 was detected (Figure 3) and carbachol even augmented the difference between PC 12-CuZnSOD and PC 12-control. Nevertheless, even in the presence of these inducers the formation of PGE2 by PC 12-CuZnSOD amounted to only 50% of that of the non-induced PC 12-control, indicating that it is not the process of generating free AA which was impaired in PC12-CuZnSOD cells (Figure 3). The human fibroblasts and PC12 cells were also assayed for secretion of several other PGs, e.g. 6-keto-PGF1a and thromboxan B2 (TXB2), but the amount was found to be too low to allow a reliable detection of differences (not shown). Paraquat is a herbicide known to act in vitro as superoxide generator perturbing the steady state equilibrium of oxygen radicals within the cell (Autor, 1977; Elroy-Stein et al., 1986). Pretreatment of the human fibroblast cultures with 0. 1 mM paraquat resulted in a marked reduction ( > 40%) of PGE2 release from normal fibroblasts while the release from the trisomy 21 cells remained low with no significant change. This result further pointed to the involvement of oxygen metabolism in the biosynthesis of PGE2.

Diminished synthesis of prostaglandins in cells and tissues of transgenic CuZnSOD mice

Transgenic mice with elevated CuZnSOD activity. Transgenic harboring the hCuZnSOD gene were produced as

mice

previously described by microinjecting fertilized eggs with a linear 14.5 kb fragment of human genomic DNA containing the entire CuZnSOD gene, including its regulatory sequences (Epstein et al., 1987; Avraham et al., 1988). The transgenic mice did not have any obvious physical or behavioral abnormalities and there was no significant difference between the body weight of the transgenic CuZnSOD mice and non-transgenic littermates. Three

Table I. CuZnSOD activity and PGE2 release from normal, DS fibroblasts and transfected PC 12 cells Culture Normal cells Culture CuZnSOD activity PGE2 released (U/mg protein) (pgl,g protein)

Fibroblasts

no.1 no.2 no.3 no.4

0.785 0.648 0.494 0.687

+ ± + ±

0.074 0.043 0.004 0.019

0.50 0.53 0.54 0.61

+ ± ± ±

0.01 0.06 0.08 0.06

Fibroblasts

Fibroblasts

no.5 no.6 no.7 no.8 no.9

DS fibroblasts CuZnSOD activity (U/mg protein)

PGE2 released (pg/14g protein)

1.530 0.925 1.300 1.300 1.230

0.24 0.14 0.31 0.32 0.35

i ± ± ± ±

0.198 0.037 0.048 0.029 0.119

i 0.03 ± 0.03 s 0.04 0.01 ± 0.06

mean ± SEM

0.654 ± 0.052

0.55 ± 0.02

Fibroblasts mean SEM

1.257 ± 0.087*

0.27 ± 0.03*

PC12-Con

5.15 ± 0.49

0.55 ± 0.15

PC12-SOD

8.49

0.23

+

0.72

4

0.06

From each culture 105 fibroblasts were seeded per well (12-well plates) and assays were performed after 36 h. The cell cultures used for experiments were maintained in culture for

Gene dosage of CuZnSOD and Down's syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investig...
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