The Vitamin A derivative, all-trans
retinoic acid (atRA), is a potent signaling molecule necessary for patterning, morphogenesis, and organogenesis during embryonic development (Clagett-Dame and DeLuca, 2002
; Duester, 2008
; Ross et al., 2000
; Zile, 1998
). The level and tissue distribution of atRA must be tightly regulated during embryogenesis as excessive or insufficient atRA signaling can cause a variety of congenital abnormalities and fetal death (Clagett-Dame and DeLuca, 2002
; Duester, 2008
; Ross et al., 2000
; Zile, 1998
). During embryogenesis the precise regulation of atRA levels and tissue distribution are accomplished through a balance of synthesis and degradation. Because atRA signaling controls transcription of numerous essential developmental genes, it is important to gain a clear understanding of the enzymes, binding proteins and cellular conditions that impact the metabolism of this essential compound.
Synthesis of atRA from its precursor Vitamin A (all-trans
-retinol; atROL) occurs in a two-step process. First, atROL is oxidized to generate all-trans
-retinal (atRAL), a reaction facilitated by an enzyme with retinol dehydrogenase (RDH) activity. Once atRAL is generated, it is subsequently oxidized to form retinoic acid, a reaction facilitated by an enzyme with retinaldehyde dehydrogenase (RALDH) activity. RALDH activity is known to be mediated by the three members of the RALDH family, RALDH1, RALDH2, and RALDH3 (Niederreither et al., 2002a
; Niederreither et al., 1997
). Each of the RALDH enzymes is expressed in a distinct spatiotemporal pattern during embryogenesis and is required for synthesis of atRA in distinct tissues. RALDH2 is the most widely expressed of the RALDHs, and is most crucial for embryonic atRA synthesis, as Raldh2−/−
embryos die around E10.5 and lack axial rotation, neural tube closure, heart looping and cardiac atrial chamber formation (Niederreither et al., 1999
mice exhibit normal survival to adulthood, are fertile, and exhibit no obvious defects (Fan et al., 2003
mice exhibit mild ocular defects and lethality at birth due to a blockage of the nasal passages, which prevents efficient respiration (Dupe et al., 2003
Whereas the enzymes responsible for the RALDH oxidation of atRAL to atRA have been definitively known for some time, the identity of the enzymes essential for the oxidation of Vitamin A (atROL) to atRAL during embryogenesis have remained elusive. Biochemically, members of two large enzyme families are capable of catalyzing RDH activity in vitro
: the microsomal short-chain dehydrogenase/reductases (SDR) and the cytosolic medium-chain alcohol dehydrogenases (ADH) (Duester, 2008
; Pares et al., 2008
). Because several ADHs are widely or ubiquitously expressed during embryogenesis it was originally proposed that the initial oxidation of Vitamin A during retinoic acid synthesis was carried out ubiquitously by ADH family members (Ang et al., 1996a
; Ang et al., 1996b
; Duester et al., 2003
; Molotkov et al., 2002
). However, our recent finding that a point mutation in Rdh10
) impairs RA synthesis and causes numerous embryonic abnormalities resulting in embryonic lethality between E10.5–E13.0 demonstrates clearly that RDH10 plays a predominant role in the first reaction of the two-step process of atRA synthesis (Sandell et al., 2007
). The phenotype of the Rdh10trex
mutant is consistent with the known phenotypes of retinoid deficiency, although not quite as severe as the phenotype of Raldh2−/−
embryos. This is likely due to the reduced enduring level of atRA produced in the Rdh10trex
mutant embryos, which may result from RDH activity of other enzymes or from residual activity of the destabilized RDH10 protein produced by the point mutation.
RDH10 and ADH family members could each conceivably contribute to embryonic Vitamin A metabolism. RDH10 is expressed in a spatiotemporally-restricted pattern within the embryo, coinciding closely with sites of RALDH gene expression (Cammas et al., 2007
; Romand et al., 2008
; Sandell et al., 2007
). ADH1 (a class I enzyme) and ADH7 (a class IV enzyme with robust RDH activity in vitro
) are expressed in distinct tissue-specific patterns in mouse embryos while ADH5 (a class III ADH enzyme with low RDH activity) is found broadly expressed throughout the embryo (Duester et al., 1995
; Molotkov et al., 2002
). Given that RDH10 and ADH family members are each present within embryonic tissues and are each capable of RDH activity in vitro
, we sought to understand why ADHs have little or no physiologically-relevant RDH activity in embryos and what distinguishes RDH10, making it the dominant RDH in atRA metabolism during embryogenesis.
The precise enzymatic role of RDH10 in embryonic atRA synthesis has not been fully demonstrated, although our previous work showed that RDH10 catalyzes the oxidation of atROL to atRAL in vitro
(Wu et al., 2002
). Since that reaction is the first step of atRA synthesis, and Rdh10trex
mutants have a deficiency in atRA synthesis, we proposed that RDH10 is necessary to produce sufficient levels of atRAL to serve as substrate for RALDH enzymes that catalyze the second step of atRA synthesis. However, it was surprising that the widely expressed ADHs, which have RDH activity, did not compensate for the loss of RDH10. The inability of ADHs to compensate for the loss of RDH10 could indicate that ADHs do not normally function in the capacity of an RDH in vivo
despite in vitro
evidence to the contrary. An alternative explanation could be that RDH10 is essential for both the first and second oxidative steps of atRA synthesis. Although SDRs and ALDHs utilize completely distinct modes of catalysis and the alcohol oxidation is chemically reversible, whereas aldehyde oxidation is not, we decided to systematically explore all possible developmental reasons behind the embryonic requirement for RDH10. Thus, in order to fully explore the precise enzymatic activity of RDH10 it was necessary to determine the capacity of RDH10 to catalyze the oxidation of atRAL.
The importance of resolving the precise role of RDH10 in embryonic atRA synthesis is underscored by studies in RARE-lacZ reporter mice that have indicated that there are sites of RALDH-independent atRA synthesis in the developing embryo, which include areas in the forebrain, hindbrain, spinal cord, neural tube and heart (Mic et al., 2002
; Niederreither et al., 2002b
). Such areas of “RALDH-independent” activity could be accounted for by unidentified RALDH(s). Alternatively, several studies have identified non-RALDH enzymes with the capability of synthesizing atRA in vitro
, including members of the Cytochrome P450 (CYP) family (CYP1A1, CYP1A2, CYP3A6, and CYP1B1) (Chambers et al., 2007
; Ding et al., 2001
; Roberts et al., 1992
; Tomita et al., 1996
; Tomita et al., 1993
), and these enzymes may account for RALDH-independent atRA synthesis. CYP1B1 is particularly interesting, because it possesses the unique enzymatic capacity to catalyze the stepwise oxidation of atROL to atRA in vitro
, and thus combines the function of a RDH and a RALDH into one enzyme (Chambers et al., 2007
). However, it remains to be determined if CYP1B1 can directly synthesize atRA from atROL in vivo
. The CYP enzymes generally have much lower activity than the RALDHs, but they may contribute to sites of RALDH-independent atRA synthesis during embryogenesis. Most interestingly, RDH10 is expressed in at least two areas which have RALDH-independent atRA synthesis, including the floor plate of the neural tube and the hindbrain (Romand et al., 2008
; Sandell et al., 2007
), raising the possibility that RDH10 may contribute to RALDH-independent atRA synthesis, either on its own or in concert with CYPs.
Another aspect of atRA metabolism that is not well understood is the sub-cellular location of various reactions and intermediates. The precursor molecule atROL is derived initially from the maternal diet, distributed to the embryo through the circulation and transported into embryonic cells via the cell surface receptor STRA6 (Bouillet et al., 1997
; Kawaguchi et al., 2007
). The second reaction in atRA synthesis, the conversion of atRAL to atRA by the RALDH family of enzymes takes place in the cytosol. Thus, it is reasonable to expect that the initial RDH reaction, the conversion of atROL to atRAL, would also occur within the cytosol. However, the finding that RDH10, which is membrane-bound, is the dominant embryonic RDH, and the fact that ADHs, which are cytosolic, do not apparently contribute substantially to the process, led us to hypothesize that the first enzymatic step of atRA synthesis occurs not in the cytosol but, instead, takes place primarily in a membrane compartment. We therefore sought to define the sub-cellular location of the first step of atRA synthesis and determine the relative contribution of ADHs and RDH10 to this important process.
In the present study we investigated the role of RDH10 in the stepwise synthesis of atRA during embryogenesis. We demonstrated that RDH10 does not catalyze conversion of atRAL to atRA in vitro and rescued Rdh10trex mutants by supplementation of the maternal diet with atRAL. Taken together these data demonstrate that RDH10 is essential only for the first step of embryonic atRA synthesis. We measured total embryonic RDH activity and showed that membrane-bound RDH activity is abolished in Rdh10trex mutants, demonstrating that RDH10 is the primary enzyme carrying out RDH activity in the membrane compartment within embryos.
We show that RDH10 is unable to oxidize atROL bound to the cytosolic cellular retinol-binding protein (RBP1) in vitro and demonstrate this inhibitory protein is expressed during the stages of development when RDH10 is active. We also demonstrate that ADH7, which exhibits high RDH activity on atROL in aqueous solution, is unable to oxidize atROL that has been incorporated into liposomes. Taken together, these data suggest that RDH10 oxidizes a RBP1-free pool of atROL present in cellular membranes in vivo, and that the ADH enzymes are unable to easily access membrane pools of atROL. Thus, we speculate that ADHs do not contribute to embryonic synthesis of atRA because they may function only in the aqueous cytosolic compartment where inhibitory RBP1 is present. Importantly, the data indicate that that the first step in the two-step embryonic atRA synthesis is carried out predominantly by RDH10 in a membrane-bound environment protected from inhibitory RBP1.