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LETTERS TO THE EDITOR
Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire
REPLY: The main purpose of generating a mouse strain with 11
-hydroxysteroid dehydrogenase-2 (11-HSD2) promoter-driven Cre expression was to facilitate in vivo studies aimed at assessing the role of aldosterone-regulated genes in electrolyte balance and cardiovascular function. Aldosterone responsiveness is restricted to cells that simultaneously express both the mineralocorticoid receptor (MR) and 11-HSD2 in physiologically relevant amounts. Consequently, in an ideal deleter strain for such studies, Cre activity should be restricted both spatially and temporally to a pattern that corresponds to the overlap between the expression domains of these two genes. Since regulatory elements that recapitulate such a pattern have not yet been identified (and may not even exist), a "second best" approach is to constrain recombinase activity to a pattern that conforms to the expression profile of either the MR or the 11-HSD2 gene. An unavoidable consequence of this compromise is that recombination may also occur in cells that are not aldosterone responsive since the two genes have overlapping but distinct expression domains. Consequently, with a Cre construct driven by MR regulatory elements, recombination is also expected to take place in tissues that are normally not regulated by aldosterone, such as the hippocampus (and possibly at other sites, given the relatively widespread expression of the MR) (1, 9). Conversely, if Cre expression is placed under the control of the 11-HSD2 gene, besides aldosterone target cells, one should anticipate somatic mutagenesis to occur wherever and whenever the 11-HSD2 gene is active. Thus one of the goals of our study was to map such sites of recombination, so that the utility and limitations of this new model can be considered. To facilitate the interpretation of these findings, we emphasized in our Discussion that "Since in our studies iCre driven by 11-HSD2 is already active during embryonic life, these findings might reflect 11-HSD2 expression during development" and that "X-gal staining can represent 11-HSD2 expression at any time during development, since the 11-HSD2-driven iCre activity results in permanent excision of the stop codon from the -galactosidase gene present in the ROSA26 reporter mouse" (8).
Besides providing aldosterone specificity to mineralocorticoid target cells, 11-HSD2 also seems to be employed to limit glucocorticoid activity at other sites, such as certain regions of the developing brain (2, 5, 7, 10) and some reproductive organs (3, 13). Thus it should come as no surprise that these sites and/or their derivatives also became LacZ positive in our studies, since Cre-mediated recombination by design is irreversible. The expression of 11-HSD2 at several of these loci, including the cerebellum and thalamus, is well documented in the literature (2, 5, 7, 10) while activity at other sites like the cartilage was unexpected.
The issue of 11-HSD2 expression in the brain was not the main focus of our study. This topic is linked to a substantial but somewhat confusing literature. Our selection of references was guided by their relevance to our findings and by space limitations and not by an attempt "to disregard prior data that were not consistent with (our) interpretation" as Geerling and Loewy (6a) assert.
Initial as well as some more recent studies failed to detect 11-HSD2 mRNA in the CNS (4, 11, 12) while others reported relatively widespread distribution especially during the pre- and perinatal period (2, 5, 7, 10). The lack of consistency is likely related to one of the few observations on which there seems to be a consensus; i.e., that compared with classic MR target sites, 11-HSD2 expression in the brain is quite low, which makes an evaluation of its distribution in the central nervous system challenging. This is particularly true for immunohistochemistry, especially when the antibody used recognizes multiple proteins on Western blots and if specific staining is demarcated from spurious binding by a less than a twofold difference in antibody concentration (6). Furthermore, even a clear-cut demonstration of 11-HSD2 protein does not guarantee that its presence is physiologically relevant, and conversely, levels that fall below the detection limit may still mediate a biological function. In this context, it is interesting to note that targeted disruption of the 11-HSD2 gene leads to abnormal cerebellar morphology (7) and that 11-HSD2 activity in the paraventicular nucleus also seems to be functionally important (14), even though no 11-HSD2 protein was detected at these sites by immunohistochemistry (6).
FOOTNOTES
Address for reprint requests and other correspondence: A. Náray-Fijes-Tóth, Dept. of Physiology, Dartmouth Medical School, Lebanon, NH 03756-0001 (e-mail: aniko.fejes-toth{at}dartmouth.edu)
REFERENCES
-hydroxysteroid dehydrogenase-2-expressing cells. Am J Physiol Renal Physiol 292: F486–F494, 2007.
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