Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase Adh3

Results

Retinol Rescue of Adh3^−/− Growth Deficiency.

To determine whether the growth deficiency of Adh3^−/− mice could be rescued by retinol treatment, we compared Adh3^−/− and WT mice generated on standard mouse chow with mice generated on a retinol-supplemented diet containing 10-fold higher vitamin A than standard mouse chow. Whereas standard mouse chow allowed growth of Adh3^−/− mice to achieve only 72% the weight of WT mice (24.7 ± 0.8 g vs. 34.3 ± 1.2 g at 14 weeks of age), the retinol-supplemented diet increased growth of Adh3^−/− mice nearly to normal compared with WT (30.7 ± 2.0 g vs. 33.9 ± 1.2 g at 14 weeks of age; no statistically significant difference) (Fig. 1). The growth of WT mice was not significantly changed by retinol supplementation, indicating that the effect was specific for mice lacking Adh3. Thus, retinol treatment can significantly rescue the growth deficiency phenotype of Adh3^−/− mice. These studies establish that ADH3 is essential for optimal usage of standard dietary levels of retinol to promote normal growth, and that other enzymes cannot replace this function unless provided supraphysiological amounts of retinol.

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Figure 1

Retinol rescue of Adh3^−/− growth deficiency. Mice were propagated for one generation on either Purina 5015 Standard Mouse Chow containing the standard amount of vitamin A (30 units/g) or Purina 5755 Basal Diet supplemented with additional retinyl acetate to bring the total vitamin A concentration to 300 units/g. Reported are the weights of male offspring at 14 weeks of age. All values are mean ± SEM (n = 14); *, P < 0.001 (Student's t test, Adh3^−/− vs. wild-type, both on standard mouse chow).

Metabolism of Retinol to RA in Vivo by ADH3.

Because ADH3 has not been reported to have retinol oxidation activity, we tested all strains of mice for metabolism of a dose of retinol to RA, the signaling molecule needed for growth. Mice were treated orally with a 50 mg/kg dose of all-trans-retinol and 2 h later all-trans-RA was quantitated in serum (36). Whereas WT mice generated high levels of serum all-trans-RA (1.37 μg/ml) in response to this dose of retinol, we observed that Adh1^−/− mice had a 23-fold reduction (0.06 μg/ml), Adh3^−/− mice had a 3.6-fold reduction (0.38 μg/ml), and Adh4^−/− mice had a small reduction (1.26 μg/ml) that was not statistically significant (Fig. 2A). These findings indicate that ADH3 contributes significantly to RA generation in vivo, and under these conditions makes a larger contribution than ADH4 perhaps because of its ubiquitous expression compared with the much more limited expression pattern of ADH4. ADH4 is not expressed in liver or intestine and instead is limited to epithelia of the stomach, esophagus, skin, adrenal, and reproductive tract (31, 39–41). The observed major role of ADH1 in metabolism of a dose of retinol to RA is consistent with its expression at very high levels in liver, intestine, and several other organs (31). To ensure that all strains had been successfully treated, we also examined retinol. Serum all-trans-retinol levels in treated mice ranged from 3.2 to 4.2 μg/ml among the five strains showing no significant difference (Fig. 2B). Because untreated mice generally have serum all-trans-retinol levels between 0.3 and 0.4 μg/ml (see below; Fig. 3D), the treatment resulted in an approximate 10-fold increase for each strain.

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Figure 2

Effect of Adh genotype on metabolism of a dose of retinol to RA. (A) For WT mice and each Adh null mutant strain indicated, all-trans-RA levels were quantitated by HPLC in serum 2 h after a 50 mg/kg oral dose of all-trans-retinol; all values are from adult female mice (n = 4). (B) Serum all-trans-retinol levels quantitated by HPLC are shown for the same mice examined above. All values are mean ± SEM. *, P < 0, 03; **, P < 0.05; (Student's t test, null mutant vs. WT).

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Figure 3

Adh3^−/− mice undergo postnatal lethality during gestational VAD. (A) After exposure to VAD, the number of congenitally VAD F₂ generation offspring born for WT mice and each Adh null mutant strain is shown, plus the numbers surviving until postnatal day 40. (B) The previous data are plotted as the percent survival for each mouse strain. Less than 20% survival was observed for Adh3 and Adh4 mutants by postnatal day 6. (C) The weight gain for the WT and null mutant mice above is shown (*, all mice had died by the day indicated). (D) Depletion of serum retinol in WT and Adh null mutant mice during gestational VAD is shown. Serum all-trans-retinol levels were quantitated by HPLC. Control values are shown for 14-week-old vitamin A-sufficient (VAS) female mice (n = 3) maintained on standard mouse chow. Values indicated as VAD-parents refer to the original female parents used to begin the VAD studies (n = 3); these mice were placed on the VAD diet at 6 weeks of age, mated to produce one litter; then at 14weeks of age, serum retinol was measured. Values indicated as VAD-F₁ refer to the F₁ generation of congenitally VAD female offspring (n = 8) of the original VAD female parents; these females were mated at 6 weeks of age to produce the F₂ generation mice described in A–C; then at 14 weeks of age, serum retinol was measured.

Discussion

The findings above establish that ADH3 performs an essential role in the retinoid-signaling pathway, i.e., generation of retinal to be used as a substrate by any of the RALDHs to produce RA. ADH3 fits this role quite nicely because it, unlike other ADHs or SDRs, is expressed ubiquitously. In mouse embryos from embryonic day 7.5 (E7.5) to E9.0, the expression pattern of the RA-generating enzyme RALDH2 is very similar to the RA localization pattern, i.e., in trunk mesoderm up to the junction with the hindbrain and in the optic vesicle (17–20). Raldh2^−/− mice totally lack RA in the trunk and have only a small amount of RA remaining in the eye region, thus demonstrating that RALDH2 is indeed the major RA-generating enzyme at this stage of development (17, 18). Because ADH3 mRNA has been detected at high levels in all cells at E7.5–E9.5 by whole-mount in situ hybridization (24), it could produce retinal for all tissues expressing RALDH2 or any other RA-generating enzyme. ADH3 mRNA remains ubiquitous in late-gestation mouse embryos (25), and by adulthood ADH3 protein is easily detected by Western blotting in all mouse tissues analyzed (31). In cells that do not express RALDH2 or another RA-generating enzyme, the retinal produced by ADH3 would not accumulate because it would simply be in equilibrium with retinol due to the reversible nature of this reaction.

It is clear that other retinol-oxidizing enzymes must exist or Adh3^−/− mice would have suffered completely penetrant mid-gestation lethality as Raldh2^−/− mice do (17, 18). ADH4 mRNA has been detected in mouse embryos at stages E7.5–E9.5, but it is not expressed in the optic vesicle, and expression in trunk mesoderm is centered at a more posterior location than that seen for RALDH2 mRNA (24). As for the other candidate enzymes, either very limited or no overlap occurred with RALDH2 mRNA from E7.5–E9.5 as summarized. ADH1 mRNA is first observed at E9.5 limited to the mesonephric ducts (24); RDH5 mRNA at E8.5 is localized in neuroepithelia (15); reverse transcription–PCR studies have shown that ADH1 plus CRAD1 mRNAs are first detected at E9.5, and that RoDH plus CRAD2 mRNAs are undetectable from E6.5 to E9.5, and further that ADH4 plus RDH5 mRNAs are detected from E6.5 to E9.5 with RALDH2 mRNA detected from E7.5 to E9.5 (26). Thus, whereas additional retinol-oxidizing enzymes exist, a combination of them may be needed to approach the efficiency of ADH3 for providing retinal throughout the body for RA synthesis. In fact, the efficiency of ADH3 is not quite achieved by all remaining enzymes, because we have shown here that Adh3^−/− mice suffer reduced survival and growth deficiency, requiring supraphysiological levels of retinol to gain back some of these losses.

Efficiency here is not measured in terms of how much activity the enzyme has, for ADH3 retinol activity is lower than that of ADH1 or ADH4, but rather in terms of whether it is expressed properly (as ADH3 is) to provide the small amount of retinal needed to fuel local production of RA wherever and whenever needed. Because RA is usually present in tissues at only nanomolar quantities (26, 36), and because these quantities are sufficient to regulate RA receptors (2), the level of ADH3-catalyzed retinol oxidation observed here could be sufficient to generate this amount of RA. The activity of 105 pmol⋅min⁻¹⋅mg⁻¹ we observe for ADH3 is comparable with the 115 pmol⋅min⁻¹⋅mg⁻¹ reported for partially purified RoDH, which has been hypothesized to play a significant role in RA synthesis (11). Other SDRs (CRAD1, CRAD2, and RDH5) overexpressed in cell lines have been reported to have all-trans-retinol activities between 20 and 200 pmol⋅min⁻¹⋅mg⁻¹ of cell extract (13, 14, 16). Although it is difficult to compare the activities of ADHs directly with SDRs because of differences in purity, we conclude that ADH3 has retinol oxidation activity that is much less than the oxidation activity of other ADHs, but in the same order of magnitude as the activity seen for microsomal SDRs. However, the physiological impact of SDRs on RA synthesis during development may be less than the impact of ADH3, because they lack the ubiquitous expression pattern that ADH3 possesses (however, see Note Added in Proof).

It was hypothesized that ADHs may not be able to contribute significantly to RA synthesis because the vast majority of retinol is physiologically bound to cellular retinol-binding protein type I (CRBPI), and it was reported that ADH4 had no detectable activity with CRBPI-retinol, whereas microsomal RoDH did (42). Unfortunately, this hypothesis is based on ADH4 studies with a spectrophotometric assay having a limit of detection of 2,000 pmol⋅min⁻¹⋅mg⁻¹ (7, 43) and RoDH studies with an HPLC assay with a limit of detection of less than 100 pmol⋅min⁻¹⋅mg⁻¹ (11). Thus, the only conclusion that can be made from these studies is that CRBPI-retinol reduces the activity of ADH4 to some value below 2,000 pmol⋅min⁻¹⋅mg⁻¹, which is significantly lower than its activity with free retinol (<400,000 pmol⋅min⁻¹⋅mg⁻¹), but perhaps still as high or higher than RoDH (115 pmol⋅min⁻¹⋅mg⁻¹). Indeed, CRBPI null mutant mice do not lack RA synthesis, but have excessive RA synthesis and reduced vitamin A storage (44), thus suggesting that CRBPI protects against excessive utilization of retinol by ADHs but does not necessarily reduce activity to zero. Our demonstration that mice need ADH3 and ADH4 to maximize survival and growth during gestational VAD provides evidence that these ADHs can indeed use retinol even when CRBPI-retinol must be the predominant form, and further indicates that SDRs cannot completely fulfill the function of oxidizing retinol to retinal for RA production.

The mammalian ADH gene family was derived from duplications of an ancestral ADH3 gene conserved in lower vertebrate cartilaginous fishes (32) and in chordate invertebrates including Amphioxus (33) and ascidians (45). Because evolutionary data suggest that ADH1 diverged from ADH3 during the advent of bony fishes (32, 46), and that ADH4 diverged from ADH1 during emergence of amphibians (47, 48), ADH1 and ADH4 cannot be responsible for RA synthesis in present-day primitive fishes and their ancestors where retinoid signaling is believed to have originated (1). Instead, our data indicate that ADH3 initially evolved to perform this function, and its relatively low activity with retinol may have actually been advantageous in that it would have spared retinol, particularly important during VAD commonly encountered in the wild, while still making sufficient RA for development. We propose that duplications of an ancestral ADH3 gene have resulted in higher vertebrates obtaining high-activity, tissue-specific forms (ADH1 and ADH4) that function along with low-activity, ubiquitous ADH3 to improve the efficiency of retinoid metabolism for minimization of vitamin A toxicity or deficiency. To support this hypothesis, we demonstrate here that a loss of ADH4 does not result in a significant systemic reduction in metabolism of a toxic dose of retinol, but does result in a significant loss of growth and viability during gestational VAD. Thus, the systemic contribution of ADH4 for oxidation of retinol to RA is relatively low, but evidently localized RA production in ADH4-expressing tissues is very important for growth and survival of higher vertebrates. As for ADH1, the reverse is true, pointing to a role in minimizing retinol toxicity but not VAD in higher vertebrates. ADH3 functions in an intermediate fashion contributing significantly to both systemic retinol clearance and protection against gestational VAD, but not as efficiently as ADH1 or ADH4, respectively. Further support for this hypothesis comes from studies showing that ADH1 and ADH3 are expressed at high levels in liver (as well as ADH3 being ubiquitous) whereas ADH4 is expressed at relatively low levels in peripheral tissues and absent from liver (31, 37, 49). More support comes from studies indicating that Adh1^−/− mice exhibit high sensitivity to acute or chronic retinol toxicity, whereas Adh4^−/− mice exhibit low sensitivity and Adh3^−/− mice exhibit an intermediate level of sensitivity (A.M., X.F., and G.D., unpublished data). Collectively, these findings indicate that low-activity, ubiquitous ADH3 may alone be adequate to catalyze oxidation of retinol to RA under conditions of vitamin A sufficiency, but that it is advantageous to have a high-activity enzyme expressed in peripheral target tissues during VAD (ADH4) and a high-activity enzyme expressed in liver during vitamin A toxicity to increase turnover of excess retinol (ADH1).

We have shown here that ADH4 becomes important during VAD in the mouse suggesting that tissues where ADH4 is expressed (primarily epithelial) may have a greater requirement for RA than other tissues. However, when maintained on a standard diet Adh4^−/− mice do not exhibit a growth deficiency as do Adh3^−/− mice, indicating that ADH3 is the dominant enzyme for systemic retinal generation. A compound null mutant for Adh3 and Adh4 could be used to examine the overlapping functions of ADH3 and ADH4 during development, but because these two genes are closely linked on the same chromosome this compound null mutant cannot be obtained by simple matings of the single mutants and will instead require more complex methodology.

Higher vertebrate ADHs have also evolved to metabolize a variety of toxic alcohols including ethanol (28). Our findings here should spur further interest in determining whether the mechanisms of alcohol toxicity and teratogenicity involve interactions of alcohol with RA-signaling pathways as hypothesized (50).