| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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A transgenic mouse line expressing ODC cDNA under the control of a MMTV-LTR promotor was generated and used to study the pathological and physiological effects of deregulated ODC expression during the life of transgenic animals. When ODC activities were systematically assayed in tissues of transgenic and control animals, the most significant difference was detected in some of the reproductive organs of male mice. Testis displayed two times higher, seminal vesicle about 20 times higher and fat pads in preputial glands about 400 times higher ODC activity when compared to age-matched non-transgenic animals. In contrast, there were no significant changes in the enzyme activity in the reproductive organs of females. Interestingly, the ODC activity was significantly lower in the prostate and kidney tissues of transgenic males. The lower ODC activity in kidneys was observed also in the female transgenic mice. Changes in ODC activity in other tissues (lung, liver, heart, brain displayed altered activity) were statisticly significant but moderate.
The first reported transgenic mouse lines were produced by using the human ODC gene containing its own promoter (Halmekytö et al. 1991a, Halmekytö et al. 1991b, Halmekytö et al. 1991c). Those transgenic mice overexpressed ODC in all tissues studied, with the exception of small intestine and male kidney. Later ODC overexpression has been targeted successfully to keratinocytes (Megosh et al. 1995) or to heart (Shantz et al. 2001) by using tissue-specific promotors and metallothionein I promotor has been used to control inducible overexpression of ODC (Alhonen et al. 1996). In these cases ODC expression patterns were as expected. Most transgenic mouse lines carrying MMTV LTR fusiongenes express transgenes at variable levels in different tissues, but the levels are particularly high in the mammary glands (Sinn et al. 1987, Müller et al. 1988, Tsukamoto et al. 1988, Bouchard et al. 1989, Matsui et al. 1990, Müller et al. 1990, Suda et al. 1990, Berard et al. 1994). In our study, however, mice carrying the MMTV/ODC cDNA fusiongene showed no elevation in ODC activity in the mammary gland. It is apparent that out transgene was integrated to genomic locus that could be transcribed significantly only in certain tissues where the responsiveness to glucocorticodes or/and post-translation regulation of ODC determined the level of ODC overexpression. It is possible that the transciptional activity of the genomic area around the integration site depends also on the age of mice. The single transgenic pup we analysed displayed increased ODC activity compared to non-trangenic pup in all tissues examined except in pancreas – although this kind of analysis is of course not statistically valid. Our study showed both increases and decreases in enzyme activity in tissues of transgenic mice. This might imply that the integration of transgene has had in some tissues effect on the expression of endogenous ODC genes either directly or indirectly. Decreases in ODC activity were relatively moderate and could be explained by the changes in general metabolism, signaling pathways or “well being” of tissue which have decreased the growth potential of tissue.
In addition to the integration site, there are other factors that could have contributed to the moderate only overexpression of ODC. The high ODC activity found in other ODC transgenic mice (Halmekytö et al. 1991a, Halmekytö et al. 1991b, Halmekytö et al. 1991c) might have been caused by the human construct used, which contains ODC"s own strong promoter (Brabant et al. 1988, Palvimo et al. 1991), or by the lack of putative silencer elements on the 5’ flanking region further upstream (Halmekytö et al. 1991a, Halmekytö et al. 1991c). It has been suggested earlier that the responsive elements in the codin region of ODC cDNA are involved in the regulation of the ODC expression (van Daalen Wetters et al. 1989a). Human elements might not be functional in mice, but our transgene was of murine origin and may have contained active elements.
Transgenic mice with high polyamine biosynthesis activity have regulatory mechanisms preventing accumulation of polyamines (Halmekytö et al. 1993, Heljasvaara et al. 1997). In contrast, in transgenic mice that constitutively and strongly overexpress deregulated ODC in the skin (Megosh et al. 1995, O"Brien et al. 1997), the polyamine homeostasis is disturbed and the mice develop spontaneous skin tumors. These observations are in agreement with the suggestion that the effects of ODC overexpression are dependent on the cell type and on the level of overexpression. We carried out a histological analysis of our mice in order to study whether the life-long deregulated ODC expression effected on physiology and pathology of tissues. We found several abnormalities in transgenic mice ranging from inflammatory processes to spontaneous tumors. These could be caused by deregulated ODC expression since increased occurrence of spontaneous tumors has been reported in other transgenic mice overexpressing ODC (Megosh et al. 1995). Similarly, pancreatis we found in several mice has been detected in rats overexpressing SSAT (Alhonen et al. 2000). Overexpression of SSAT increases putrescine levels as does ODC overexpression. Another alternative is that the integration of transgene has changed expression of some endogenous gene(s) leading to pathological alterations in tissues.
The number of infertile transgenic mice was high. Histological examination suggested that alterations in maturation of gonadal cells might have been responsible for the high rate of infertility. Previously, reduced spermatogenesis in infertile transgenic mice has been reported (Halmekytö et al. 1991a). At the same study, also a dramatic increase in the glandular tissue of the preputial gland was described. In our material, the structure of the preputial gland was highly variable both in the control and transgenic animals and we speculate that the reduction of glands observed in our infertile transgenic male mice may have been caused by excessive secretion and consequent tissue depletion. The increased infertility of transgenic males in our study is very likely due to overexpression of ODC, since we detected significant increase in ODC activity in testis and polyamines are postulated to be of great importance in spermatogenesis (reviewed in Coffino 2000). The role of polyamines is spermatogenesis is further emphasized by the existence of testisspecific antizyme form, antizyme 3. Furthermore, tissue-specific variant of hypusinated eIF-5A, eIF-5A2 (Jenkins et al. 2001), and a recently discovered ODC-like protein (Pitkänen et al. 2001) are expressed strongly only in brain and testis. In contrast to most male sexual organs, the ovaries of the transgenic ODC mice did not overexpress ODC, suggesting that the observed degeneration of ovaries of infertile transgenic mice were not directly related to ODC gene expression.