Transgenic Models

The GH axis has been a popular target for transgenic manipulations. A number of transgenic lines of mice have been generated expressing GH or GHRH under the control of heterologous promoter sequences (124-127). The resultant phenotype in the majority of lines is increased growth, again usually associated with increased SRIF and/or decreased GHRH, as would be expected (120). However, transgenesis is not the only way to model in rodents, the type of GH hypersecretion normally seen in humans in acromegaly;

it can be conveniently induced experimentally in rodents using implants of a GH-secreting tumor cell line (128). The effects of such cell implants are comparable to excess exogenous administration of GH with raised SRIF and decreased GHRH mRNA levels, and these changes can be reversed after removal of the GH-secreting tumors (128).

Transgenesis has also been used to induce dwarfism, either by widespread expression of a functional GH antagonist (129) or by targeting, directly or indirectly, the somatotrophs. Dwarf mice have been produced by selective or total ablation of GH-expressing cells (130), whereas transgenic dwarf rats have been produced by expressing an antisense GH transgene in the pituitary to suppress endogenous GH synthesis (131). Paradoxically, dwarfism can also be induced by targeted expression of GH itself. This was first noticed in a line of mice that fortuitously expressed hGH in the CNS (71). In contrast to the giantism generated by peripherally overexpressed hGH transgenes, mice with such central hGH expression exhibited a dwarf phenotype caused by local short-circuit feedback on hypothalamic GHRH and SRIF (71). This has prompted the production of another mouse model in which hGH was targeted more specifically to the CNS and peripheral nervous system using the tyrosine hydroxylase promoter (72). As expected, the resultant mouse also had a dwarf phenotype and exhibited similar hypothalamic GHRH suppression (72).

The authors have also exploited this GH-negative feedback mechanism by targeting hGH to the CNS under the control of the GHRH promoter (73). This was achieved in transgenic rats, and the resulting transgenic growth retarded (Tgr) rat also displays the expected raised SRIF and low GHRH expression (Fig. 8). Rats have some advantages over the mouse because their larger body size made it possible to carry out blood sampling to observe the effects of transgenes on pulsatile GH secretion and to study the growth responses to patterns of hormone administration (132). Although the transgenic mice expressed hGH in many areas of the CNS (71) and in peripheral tissues (72), the Tgr rat has highly restricted expression of the hGH transgene to GHRH neurons that are sensitive to GH feedback so that transgene activation is itself subject to physiological feedback control by GH. The effect of this GHRH-hGH transgene in Tgr rats is also sexually dimorphic with dwarfism much more marked in males than in females. The reason for this is unknown, but may relate to the sex differences already shown for GH feedback in the rat (78,133).

The authors have recently succeeded in adapting the GC cell implant method for maintaining high GH exposure to these dwarf rat models, in order to study the hypotha-lamic changes in GHRH and SRIF expression in the same animals as their GH status changes rapidly from dwarfism to acromegaly (134). It is interesting to compare this model of secondary dwarfism (Tgr rats) with that of primary pituitary dwarfism (dw/dw rats) since they both have low GH output, but exhibit diametrically opposite hypotha-lamic GHRH activity (Fig. 8). GC cell implants can usually only be made in normal female rats of the Wistar-Furth strain, whereas both dwarves were raised on an AS background that rejects GC cell implants. The authors overcame this problem in two different ways. GC cells survived in dw/dw rats when they were given cyclosporin to prevent rejection. An even simpler solution was possible in the Tgr rats, since the Tgr transgene is dominant. By breeding Tgrs with Wistar/Furth animals, the transgene conferred dwarfism on the F1 progeny whereas these retained sufficient Wistar/Furth background to accommodate GC cell implants. In both cases, rapid growth followed implantation of GC cells. Figure 9 compares the effects of chronic GH exposure from

Dw Normal Tgr

SRIH mRNA

GHRH mRNA

Fig. 8. GHRH and SRIF expression in Dw and Tgr rats. Comparison of the hypothalamic distribution and expression of SRIF (Top) and GHRH (Bottom) in PeN and ARC respectively, of dw/dw dwarf, normal, and transgenic growth-retarded (Tgr) rats (100x magnification). From ref. 134.

these implants on GHRH expression in the two dwarf models studied by in situ hybridization of the ARC. As expected, GHRH expression is high in dw/dw rats and was markedly suppressed by GC cell implants. There was a smaller, but significant suppressing effect in the normal animals implanted with GC cells and no effect in the Tgr animals, whose GHRH expression is already maximally suppressed by the local transgene hGH. Thus the effects of central and peripheral GH feedback can be dissociated in these models. Targeting of hGH to other neurons has already been achieved with different neurone-specific promoters (I. C. A. F. Robinson, unpublished observations), and may in the future prove to be a useful means of targeting direct effects of GH in other CNS sites that express GHRs without altering peripheral GH or IGF-1 status, and thereby reveal other potential physiological targets for GH action within the brain.

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