Modeling Human Disease through Targeted Overexpression

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Animal models of human diseases can enhance our understanding of the etiology and progression of a particular disorder and enhance the design of therapeutic strategies. With technical advances in the manipulation of the mouse genome, several transgenic models of human diseases have emerged (see Bedell et al., 1997, for a recent review). These include animal models of cancer (Adams and Cory, 1991; Kappel et al., 1994; Lovejoy et al., 1997), neurodegenerative disorders (Aguzzi et al., 1996; Brown, 1995), atherosclerosis (Bres-low, 1996; Chien, 1996), diabetes (Tisch and McDevitt, 1996), cardiovascular disease (Chien, 1996), Down syndrome (Cabin et al., 1995; Groner, 1995; Mirochnitchenko and Inouye, 1996; Sumar-sono et al., 1996), and others.

Transgenic animal models also help determine the relationship between underlying genetic, epigenetic, and environmental factors and the onset of a disorder. These same factors may also explain the predisposition of some individuals to a particular disease. Thus, transgenic approaches can be particularly informative for complex multifactorial disorders with epigenetic contributions (Erickson, 1996).

Several of the transgenic models described earlier were established by mutation or deletion of a specific gene. Other disease models have resulted from the overexpression of a candidate gene, either in wild type or mutant form. This approach is particularly powerful when targeted expression of a candidate gene is used to rescue a particular phenotype.

When contemplating an overexpression paradigm, much emphasis is placed on selection of a promoter with sufficient strength and cell-type specificity. Although integration site effects seldom compromise promoter specificity, they often interfere with the extent of expression. Furthermore, many candidate promoters are regulated by complex feedback pathways, making their overexpression even more problematic. As described next, most of these limitations can be overcome by careful promoter selection, by consideration of the half-life of the protein encoded by the transgene, and by using the biology of the system to limit feedback effects.

The authors' laboratory has developed transgenic mice that chronically hypersecrete luteinizing hormone (LH) (Risma et al.,

1995). This pituitary hormone belongs to a family of glycoprotein hormones that include thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG). Members of this family are heterodimers containing a common a subunit and a unique (3 subunit. LH and FSH are secreted into the circulation as distinct pulses that ultimately regulate gametogenesis and gonadal steroidogenesis. The secreted pulses of LH and FSH derive from identical pulses of GnRH secreted from the hypothalamus. Although LH and FSH stimulated production of estrogen and androgens from the gonads, these sex steroids also feed back at the level of both the hypothalamus and the pituitary to limit secretion of additional LH.

Several clinical studies have shown a correlation between hypersecretion of LH and functional ovarian hyperandrogenism (FOH), infertility, and miscarriage in women (Barnes et al., 1994; Ehrmann et al, 1995; Franks, 1995; Regan et al., 1990; Shoham et al., 1993), suggesting that chronically elevated LH impairs fertility. Unfortunately, no studies show a direct relationship between hypersecreted LH and reproductive abnormalities. Because LH is secreted in regulated pulses (Gibson et al., 1991) and its serum half-life is short (20-30 min; Niswender et al., 1974), it is difficult to devise protocols for chronic administration of exogenous LH that mimic endogenous pulse patterns of LH. To circumvent this limitation, the authors devised a transgenic approach where elevated hormone levels are maintained chronically, without requiring multiple injections, supraphysiologic dosing, or appreciable dampening of the hypothalamic-pituitary-gonadal axis.

A two-pronged approach was utilized to achieve elevated levels of serum LH. First, to increase delivery of hormone from the pituitary, expression of a transgene encoding an additional LH0 subunit was directed to gonadotropes using a previously characterized bovine a subunit promoter (Hamernik et al., 1992; Kendall et al., 1991). This promoter effectively directs expression of a variety of reporter genes to gonadotropes and renders them responsive to GnRH, estrogen, and androgen (Clay et al., 1993; Keri et al., 1991). Second, the LH(3 subunit encoded by the transgene contained a peptide extension that the authors proposed would slow the elimination of LH from the serum. This peptide is normally found at the carboxyl terminus of the (3 subunit of human chorionic gonadotropin (hCG) and is referred to as the carboxyl terminal peptide (CTP). This peptide is thought to be a major determinant of the serum half-life of hCG (Matzuk et al., 1990) and has been shown to increase the half-life of FSH two- to threefold when fused to its (3 subunit. Accordingly, a transgene was constructed that links the a subunit promoter with the coding region of bovine LH(3 fused in frame to the coding region of CTP (aLH(3-CTP).

Female transgenic mice carrying the aLH(3-CTP transgene chronically hypersecreted LH. Concomitant with elevated LH, serum levels of androgens and estrogens were elevated (Risma et al., 1995). In contrast, FSH and prolactin levels were normal, suggesting that the hypothalamic-pituitary-gonadal axis remains functionally intact. Hypersecretion of LH and androgens occurred early during neonatal development, causing premature vaginal opening and ovarian follicular development (hallmarks of precocious puberty) (Risma et ah, 1997). Although follicles continued to develop, ovulation failed to occur. This led initially to the formation of follicular cysts with pronounced hemorrhagia and ultimately to a large granulosa tumor mass. In addition, some of these mice had enlarged bladders and developed hydronephrosis. In other instances the bladder became herniated. These renal phenotypes probably reflect elevated levels of serum steroids. Hydronephrosis has been reported in rats chronically treated with high levels of estradiol as well as in pregnant women and nonhuman primates (Au et al., 1985; Buhl et al., 1985; Roberts, 1976). Furthermore, bladder hernia has been previously reported in mice that overexpress the wildtype estrogen receptor under the control of the metallothionine promoter (Davis et al., 1994).

Although elevated levels of LH can be attributed to the combined effects of a strong gonadotrope-specific promoter and increased stability of the recombinant subunit, an additional biological component that underlies overexpression was discovered. Normally, high levels of sex steroids reduce the concentration of serum LH. In the case of the aLH(3-CTP mimic, elevated LH in the face of elevated estrogen and androgen suggested that resistance to steroid negative feedback must have occurred. Preliminary data indicate that the aLHp-CTP transgene retains responsiveness to negative feedback from androgen but has developed a selective resistance to estrogen negative feedback (Abbud et al., 1997). This resistance is transgene specific and therefore contributes to the overall elevation in serum LH.

In summary, a transgenic strategy has been successfully developed for demonstrating that chronic hypersecretion of LH causes functional ovarian hyperandrogenism, a leading cause of infertility in women. Although the strategy required targeted overexpression of LH, this outcome relied on designing a composite transgene that capitalized on unique features of a complex physiological pathway. This model of chronic LH hypersecretion joins a growing list of transgenic animal models of human disease. Because most diseases are multifactorial, increasingly novel approaches will be required to provide a full array of disease models. The ability to manipulate the mouse genome and test alterations in gene expression in different genetic background offers further assurance that transgenic mice will remain as invaluable tools in studying complex human diseases and designing efficient strategies for gene therapy.

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100 Pregnancy Tips

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