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. 2014 May 23;289(21):14868-80.
doi: 10.1074/jbc.M114.552257. Epub 2014 Apr 14.

The Retinaldehyde Reductase Activity of DHRS3 Is Reciprocally Activated by Retinol Dehydrogenase 10 to Control Retinoid Homeostasis

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Free PMC article

The Retinaldehyde Reductase Activity of DHRS3 Is Reciprocally Activated by Retinol Dehydrogenase 10 to Control Retinoid Homeostasis

Mark K Adams et al. J Biol Chem. .
Free PMC article

Abstract

The retinoic acid-inducible dehydrogenase reductase 3 (DHRS3) is thought to function as a retinaldehyde reductase that controls the levels of all-trans-retinaldehyde, the immediate precursor for bioactive all-trans-retinoic acid. However, the weak catalytic activity of DHRS3 and the lack of changes in retinaldehyde conversion to retinol and retinoic acid in the cells overexpressing DHRS3 undermine its role as a physiologically important all-trans-retinaldehyde reductase. This study demonstrates that DHRS3 requires the presence of retinol dehydrogenase 10 (RDH10) to display its full catalytic activity. The RDH10-activated DHRS3 acts as a robust high affinity all-trans-retinaldehyde-specific reductase that effectively converts retinaldehyde back to retinol, decreasing the rate of retinoic acid biosynthesis. In turn, the retinol dehydrogenase activity of RDH10 is reciprocally activated by DHRS3. At E13.5, DHRS3-null embryos have ∼4-fold lower levels of retinol and retinyl esters, but only slightly elevated levels of retinoic acid. The membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are decreased by ∼4- and ∼2-fold, respectively, in Dhrs3(-/-) embryos, and Dhrs3(-/-) mouse embryonic fibroblasts exhibit reduced metabolism of both retinaldehyde and retinol. Neither RDH10 nor DHRS3 has to be itself catalytically active to activate each other. The transcripts encoding DHRS3 and RDH10 are co-localized at least in some tissues during development. The mutually activating interaction between the two related proteins may represent a highly sensitive and conserved mechanism for precise control over the rate of retinoic acid biosynthesis.

Keywords: Dehydrogenase; Metabolism; Reductase; Retinoid; Vitamin A.

Figures

FIGURE 1.
FIGURE 1.
Silencing of human DHRS3 expression. A, Western blot analysis of HepG2 cells stably transfected with scrambled shRNA (Ctrl), DHRS3 shRNA 2 (clones 3 and 4), or DHRS3 shRNA4 (clone 1). Note the reduced levels of DHRS3 protein in two separate clones transfected with shRNA2, but not shRNA4. B, normal phase HPLC analysis of retinol (10 μm, overnight) metabolism to retinaldehyde (RAL) and atRA (RA) in DHRS3-silenced (shRNA2 clone 4, gray bars) versus control HepG2 cells (white bars). The results are representative of three independent experiments: *, p < 0.02. C, qPCR analysis of gene expression in DHRS3-silenced HepG2 cells (gray bars) versus control cells (white bars). ***, p < 0.001, mean ± S.E., n = 3. Two independent clones, 3 and 4, showed similar results.
FIGURE 2.
FIGURE 2.
Co-expression of DHRS3 or Y188A DHRS3 with RDH10 in HEK293 cells. A, effect of DHRS3 on metabolism of retinol. B, effect of DHRS3 on metabolism of retinaldehyde. C, activation of RDH10 by Y188A DHRS3 mutant. D, effect of Y188A* rescue construct. A–D, a, Western blot analyses of cell homogenates (50 μg); *, a nonspecific band recognized by RDH10 rabbit polyclonal antiserum. Y188A* stands for rescue construct encoding the Y188A mutant of DHRS3. Note similar expression levels of DHRS3 and RDH10 transfected individually and in combination. Ab, Ac, Bb, Bc, Cb, Cc, Db, and Dc, normal phase HPLC analysis of retinaldehyde (RAL), retinol (ROL), and retinoic acid (RA). Cells were incubated with 2 μm retinol for 9 h (A and B), 10 μm retinol for 10 h (C and D) or 5 μm retinaldehyde for 3 h (A-C). A, b and c, **, p < 0.001 for RDH10 versus RDH10 + DHRS3 cells. B, b and c, *, p ≤ 0.03 for RDH10 versus RDH10 + DHRS3 cells. C, b, *, p ≤ 0.03 for RDH10 + DHRS3 versus RDH10 or empty vector. C, c, ***, p ≤ 0.0001 for RDH10 + DHRS3 versus RDH10 + Y188A. The Y188A DHRS3 does not increase the production of retinol, indicating that it lacks the retinaldehyde reductase activity, but at the same time it activates the conversion of retinol to RAL and RA catalyzed by RDH10. D, note that WT DHRS3 co-transfected with RDH10 has no effect on retinoid metabolism in these DHRS3-silenced cells because its transcript is degraded by stably transfected shRNA. All experiments were performed in triplicates.
FIGURE 3.
FIGURE 3.
Subcellular localization of DHRS3 and RDH10 proteins. HEK293 cells were co-transfected with constructs encoding DHRS3-FLAG and RDH10-HA (upper panels in A and B) or with RDH11-FLAG and RDH10-HA (lower panels in A and B). Localization of the proteins was detected by immunofluorescence and analyzed by confocal microscopy. Both a single z-slice (A) and the composite z-stack (B) are shown. Scale bar, 10 μm. Note the colocalization of RDH10 with DHRS3 (yellow ring structures), but not with RDH11.
FIGURE 4.
FIGURE 4.
Co-expression of WT or mutant DHRS3 and RDH10 in Sf9 cells. A, DHRS3 + RDH10 microsomes. B, RDH10 + Y188A DHRS3 or Y210RDH10 + DHRS3 microsomes incubated with 2 μm retinol (ROL) and 1 mm NAD+. C, RDH10 + Y188A DHRS3 or Y210RDH10 + DHRS3 microsomes incubated with 2 μm retinaldehyde (RAL) and NADPH. A-C (top panels), Western blot analyses of Sf9 microsomes (3–5 μg). In Sf9 cells DHRS3 protein appears as three bands when probed with either ProteinTech antibodies or our custom-made DHRS3 antibodies. HA-tagged Y210A RDH10-HA runs higher than RDH10. The His6-tagged DHRS3 and FLAG-tagged DHRS3 have similar molecular weight. B and C, bottom panels, reaction rates for microsomes (2 μg each). The results are representative of several measurements using the same preparation of microsomes. Note that both the oxidative and reductive activities are significantly higher for microsomes containing RDH10 + DHRS3. The results demonstrate that DHRS3 itself does not utilize NAD+ as a cofactor but its WT and mutant forms both activate the NAD+-dependent retinol dehydrogenase activity of RDH10, whereas WT and mutant RDH10 both activate the retinaldehyde reductase activity of DHRS3.
FIGURE 5.
FIGURE 5.
Kinetic analysis of DHRS3 + RDH10 microsomes. DHRS3 + RDH10 microsomes were incubated with 1 mm NADPH and varied concentrations of retinaldehyde (RAL) (A); with retinaldehyde (5 μm) and varied concentrations of NADPH (B) or NADH (D); or with 1 mm NADP+ and varied concentrations of retinol (ROL) (C). Open circles represent data points obtained with DHRS3 + RDH10 microsomes. Closed circles in A represent RDH10-only microsomes; open squares, DHRS3-only microsomes; closed squares, RDH10-only microsomes plus DHRS3-only microsomes. The amount of microsomal protein in each reaction was equalized by adding microsomes from uninfected Sf9 cells. Note that simple mixing of microsomes containing each protein separately does not lead to activation of DHRS3. Closed circles in D represent data points for RDH10-only microsomes. The kinetic data were fitted to one-enzyme (dashed lines) or two-enzyme (solid lines) models. The difference in the Vmax values between RDH10 microsomes and DHRS3 + RDH10 microsomes is consistent with the activation of RDH10 by DHRS3.
FIGURE 6.
FIGURE 6.
Expression pattern of Dhrs3 relative to other retinoid genes in E13.5 mouse hindlimb buds. Top panel, autopods were incubated in digoxigenin-labeled antisense Dhrs3 riboprobe and developed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Dhrs3 mRNA localizes to interdigital tissue. Middle and bottom panels, autopods were incubated in fluorescein-labeled antisense Rdh10, Raldh2, or Cyp26b1 riboprobes and developed using Vector Red substrate. Autopods were further probed with digoxigenin-labeled antisense Dhrs3 riboprobe and developed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Purple hue indicates colocalization of transcripts. Note the overlapping expression domains of Dhrs3 with those of Rdh10 and Raldh2.
FIGURE 7.
FIGURE 7.
Generation of Dhrs3−/− mice. A, a diagram showing the wild-type (WT) and knock-out (KO) Dhrs3 alleles. Exons (black boxes) are labeled E1 through E6. Targeting homology is represented by gray boxes. In the KO allele, a 6001-bp fragment, which contains coding exons E2–E5, is substituted with the 8540-bp NorCOMM targeting cassette. Arrows indicate the location and orientation of genotyping primers (WT-F, WT-R, and KO-R), and long range PCR primers (N01359-s1, GH717, KO-F, and 3′flankR). B, long range PCR amplification of the 5′ homology arm (expected size 2671 bp) and 3′ homology arm (expected size 7892 bp). C, PCR analysis of WT (primers WT-F and WT-R, product 816 bp) and KO (primers WT-F and KO-R, product 682 bp) mouse tail genomic DNA. D, RT-PCR analysis of exons 2–5 (product 587 bp) using primers mDHRS3ex2F and mDHRS3ex5R. E, Western blot analysis of E14.5 WT and KO MEFs (15 μg of 10,000 × g fraction) treated with 200 nm atRA or vehicle for 12 h using DHRS3 ProteinTech antibody or β-actin antibody (lower). F, external views of E18.5 mouse embryos. Arrow indicates clefting of the secondary palate.
FIGURE 8.
FIGURE 8.
Analysis of retinoids and atRA-responsive genes in wild type and mutant mice. Retinol (A) and retinyl esters (B) were normalized per wet weight of E13.5 embryos. ***, p < 0.0003, mean ± S.D., n = 5. C, qPCR analysis of RNA isolated from WT (white bars) or KO (gray bars) E14.5 embryos. *, p < 0.05; **, p < 0.01, mean ± S.E., n = 3. Stra, stimulated by the retinoic acid gene; Rdh, retinol dehydrogenase; Raldh, retinaldehyde dehydrogenase; Cyp, cytochrome P450; Rar, retinoic acid receptor.
FIGURE 9.
FIGURE 9.
Analysis of retinaldehyde reductase and retinol dehydrogenase activities in Dhrs3−/−versus WT mice. MEFs isolated from E14.5 embryos were incubated with 5 μm retinaldehyde (A) or 10 μm retinol (C) for 12 h. Extracted retinoids were separated by normal phase HPLC and normalized per amount of cellular protein. **, p < 0.01, mean ± S.D., n = 3. Note that the conversion of retinol (ROL) to retinaldehyde (RAL) and atRA (RA) was reduced in Dhrs3−/− mice, supporting the in vitro data on activation of RDH10 by DHRS3. Membrane fractions (30 μg) isolated from wild-type (WT) or Dhrs3−/− (KO) E14.5 embryos were incubated with 1 μm retinaldehyde (B) or 2 μm retinol (D). **, p < 0.01, mean ± S.D., n = 3 embryos. Note that the membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are both decreased in E14.5 Dhrs3−/− embryos.
FIGURE 10.
FIGURE 10.
A model of gradual adjustment of the atRA rate of biosynthesis through mutually activating interaction between RDH10 and DHRS3. Note that the rate of ROL conversion to RAL is increased (thicker arrow) in the presence of DHRS3 compared with RDH10 alone, because DHRS3 activates RDH10. However, because this interaction also activates DHRS3 itself, the overall flux from RAL to RA is decreased (thinner arrow). The simultaneous activation of both RDH10 and DHRS3 would allow for a more measured adjustment of atRA biosynthesis (e.g. 25 versus 50%) in response to induction of DHRS3 expression. The exact rate would be determined by the relative ratios of RDH10 and DHRS3 proteins.

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