Cell Typespecific Expression of ß-Carotene 9',10'-Monooxygenase in Human Tissues
Departments of Obstetrics-Gynecology and Biochemistry (AL,SA), and Department of Ophthalmology (Y-GH), University of Texas Southwestern Medical Center, Dallas, Texas
Correspondence to: Stefan Andersson, University of Texas Southwestern Medical Center, Obstetrics-Gynecology, F2.106 5323 Harry Hines Blvd., Dallas, TX 75390-9032. E-mail: stefan.andersson{at}utsouthwestern.edu
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Summary |
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Key Words: ß-carotene 9', 10'-monooxygenase immunohistochemistry human eye heart pancreas
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Introduction |
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Recently, Kiefer et al. (2001) cloned and characterized a second carotenoid-cleaving enzyme termed ß-carotene 9',10'-monooxygenase (BCO2) because it catalyzes asymmetric cleavage of carotenoids, thus yielding one molecule of ß-apo-10'-carotenal and one molecule of ß-ionone when ß-carotene is used as substrate. Interestingly, the BCO2 enzyme appears to accept a wider variety of substrates as compared with BCO1, including the acyclic carotenoid lycopene, which implies that this enzyme may have physiological roles other than providing precursors for vitamin A synthesis.
BCO1 and BCO2 belong to a superfamily of nonheme ironcontaining oxygenases, many of whose functions are unknown. The physiological role of BCO1 is now well established; however, the function of BCO2 is still unclear. To gain some insight into the physiological role of human BCO2, we investigated the tissue-specific expression by RNA blotting and the cell typespecific expression by immunohistochemical analysis. The expression pattern of BCO2 was compared with that of BCO1 in an effort to learn more about the potential interplay between the two interesting enzymes. The results indicate that the BCO2 enzyme may play a role in biological processes other than vitamin A synthesis.
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Materials and Methods |
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Immunoblotting
The human BCO enzymes were expressed in Sf-9 cells maintained in Insect-Xpress (Cambrex; Walkersville, MD), using baculovirus containing the respective BCO cDNA to infect the cells. SDS-PAGE was performed according to (Laemmli 1970), and the proteins were transferred onto Immobilon-P PVDF membrane (Millipore; Bedford, MA) using a previously published method (Towbin et al. 1979
). For detection of BCO1 and BCO2 proteins, respective monoclonal antibodies were incubated in tissue culture medium, followed by a secondary horseradish peroxidaseconjugated goat anti-mouse IgG polyclonal antibody as described previously (Lindqvist and Andersson, 2002
). Antibody binding was detected by chemiluminescence using an ECL kit following the manufacturer's protocol (Amersham Pharmacia Biotech; Piscataway, NJ).
Immunohistochemistry
Normal human tissues were obtained during routine surgical biopsies or autopsies (ProPath Laboratory Inc.; Dallas, TX), fixed in neutral buffered formalin, and processed routinely into paraffin blocks. Representative histological sections of each specimen stained with hematoxylin and eosin were examined by experienced diagnostic pathologists to confirm the preservation of the histological structure and to validate the normalcy. For immunostaining, the blocks were cut at 4 µm, and sections were mounted on charged adhesive slides and dried in a 1000 W microwave oven (set on "high") for 3 min, followed by 10 min in a 56C oven. The immunostaining procedure has been described elsewhere (Lindqvist and Andersson 2004). Briefly, slides were deparaffinized and rehydrated, the endogenous peroxidase activity quenched, and the epitopes retrieved by a pressure cooking method. The slides were then incubated with monoclonal antibody (MAb) BCO2-1-9, MAb BCO2-2-25, or MAb BCO1-1-11. For negative control, an irrelevant mouse IgG1/
MAb (catalog no. M9269; Sigma, St Louis, MO) was used in place of primary antibody. After rinsing and incubation with a poly-horseradish peroxidaseconjugated anti-mouse IgG (PowerVision reagent; ImmunoVision Technologies Co, Daly City, CA), the slides were immersed in diaminobenzidine (Research Genetics; Huntsville, AL) enhanced with 0.5% copper sulfate, counterstained in hematoxylin, dehydrated in graded alcohol, cleared in xylene, and cover slipped.
To bleach retinal pigment in human eyes, copper sulfate treatment sections were incubated with 3% hydrogen peroxide in 1% (w/v) dibasic sodium phosphate for 18 hr at 25C, followed by a distilled water wash. Slides were then counterstained with hematoxylin and cover slipped.
RNA Blotting
A human digestive system 12-Lane MTN blot (catalog no. 7782-1) and a human 12-lane MTN blot (catalog no. 7780-1) containing 1 µg of poly(A)+ RNA per lane, and a human MTN blot II (catalog no. 7759-1) containing
2 µg of poly(A)+ RNA per lane were purchased from Clontech Laboratories Inc. (Palo Alto, CA). The RNA used in the blots was isolated from tissues removed from previously healthy Caucasian individuals postmortem (sudden death), with the exception of the peripheral blood leukocytes, in which blood was drawn from living donors. The number of donors, gender, and age range for the donors for each lane in the order they appear in Figure 2 is as follows: esophagus, 39, MF, 1772; stomach, 7, MF, 3153; duodenum, 33, MF, 1872; ileocecum, 33, MF, 1872; ileum, 8, MF, 1857; jejunum, 6, MF, 2057; ascending colon, 27, MF, 2259; descending colon, 10, MF, 1860; transverse colon, 10, MF, 1860; cecum, 29, MF, 1863; rectum, 22, MF, 1870; liver, 1, M, 35; spleen, 14, MF, 3066; thymus, 9, F, 1525; prostate, 47, M, 1457; testis, 45, M, 1464; ovary, 4, F, 2048; small intestine, 11, MF, 1560; colon (no mucosa), 16, MF, 1853; peripheral blood lymphocytes, many, MF, 1840; brain, 1, F, 15; heart, 10, MF, 2151; skeletal muscle, 11, MF, 2559; colon (no mucosa), 20, MF, 1776; thymus, 12, MF, 1525; spleen, 6, MF, 3058; kidney, 14, MF, 1859; liver, 4, MF, 4450; small intestine, 3, MF, 2160; placenta, 17, F, 1933; lung, 2, MF, 3040; and peripheral blood lymphocytes, many, MF, 1840. A 315-bp fragment, corresponding to residue 11461460 in the human BCO2 cDNA (GenBank accession no. AJ290393) was obtained by PCR and radiolabeled with [
-32P]dCTP (Amersham Pharmacia Biotech) using a Rediprime II kit (Amersham Pharmacia Biotech). Hybridization and autoradiography was performed as described previously (Lindqvist and Andersson 2002
). The signal was normalized after hybridization of the membrane with a human ß-actin cDNA probe.
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Results |
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To determine the cell typespecific expression of BCO2, we performed immunohistochemistry using the two anti-BCO2 monoclonal antibodies. BCO2 immunostaining was scored as positive only if both BCO2-1-9 and BCO2-2-15, each recognizing different epitopes of the protein, stained the same cell type in a given tissue. The irrelevant IgG1 control did not stain any of the cell types in which BCO2 staining was detected.
We started by examining tissues that have previously been shown to stain positive for BCO1 (Lindqvist and Andersson 2004). In four of the BCO1-positive tissues, small intestine, testis, liver, and adrenal gland, the BCO2-positive staining was very similar in distribution and intensity rating to what was seen for BCO1. In small intestine, BCO2 was found in the mucosal epithelium, exactly as for BCO1, both in immature enterocytes in the crypts and more mature enterocytes at the tip of the villi, in goblet cells, and in Paneth cells in the bottom of the glands (Figures 3A and 3B). There was also weak staining seen in some endothelial cells. We did not find any BCO2 mRNA in small intestine when analyzed with the Northern blot technique. However, BCO2 activity has been demonstrated in pig (Nagao et al. 1996
) and rat intestinal mucosa preparations (Barua and Olson 2000
), and the BCO2 immunostaining detected in the human small intestine in this study is in agreement with the activity studies performed in rat.
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BCO2 mRNA was also detected in the liver. Our immunohistochemistry study demonstrated that human BCO2 is expressed in hepatocytes (Figure 3F), with no immunostaining observed in the sinusoid endothelial cells. The immunostaining for BCO1 is identical to that for BCO2.
As seen for BCO1, we found that BCO2 is expressed in the cortex of the adrenal gland, both in the mineralocorticoid-producing glomerulosa and in the glucocorticoid-producing fasciculata (Figure 3H) and in the reticularis (data not shown). The capsule was negative for BCO2 immunostaining.
In some tissues, the staining pattern for BCO2 was the same as for BCO1, but of lower intensity. One of those tissues is human kidney, where weak positive staining with the anti-BCO2 antibodies was seen in the cuboidal/columnar epithelium of the proximal convoluted tubule and distal convoluted tubules (Figure 3J). We did not detect any BCO2 mRNA in human kidney by RNA blotting, but BCO2 mRNA was previously found in mouse kidney using RNA blotting and RT-PCR techniques (Kiefer et al. 2001).
Another location where the same but weaker staining pattern for BCO1 and BCO2 was seen was in the stomach, with immunostaining present only in the epithelium (Figure 3L). The mucus-secreting columnar epithelium that covers the surface and lines the pits and the cells in the glands emptying into the pits were all positive for BCO2, but the positive reaction was less intense than seen with the BCO1 antibodies, especially in the surface lining. No immunostaining in lamina propria, muscularis mucosae, or submucosa was observed.
BCO2 mRNA was detected in human skeletal muscle, and strong immunostaining was observed in the skeletal muscle fibers (Figure 3N). BCO1 staining has also been shown in the skeletal muscle, but weakly, and only in a small subset of muscle fibers.
There are other tissues in which both BCO1 and BCO2 immunostaining was seen, but the distribution or the intensity of the staining varied for the two proteins. For example, both prostate and endometrium showed staining for BCO1 in the epithelium of the glands. Even though the RNA blot was negative for presence of BCO2 mRNA in prostate, immunostaining in the prostate indicated that BCO2 is expressed not only in the epithelium of the glands, but also in the fibromuscular stroma (Figure 4A). Examination of the endometrium showed the same pattern, with BCO2 expression in the epithelium of the glands and in the highly cellular connective tissue (Figure 4C).
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Some tissues that stained positive for BCO1 showed no staining at all for BCO2. The granulosa cells and the theca interna of the follicles of the ovary, epidermis of the skin, and the epithelial lining in colon were all negative for BCO2 staining (not shown). The BCO2-1-9 antibody did stain the epithelial lining in colon strongly; however, we believe this to be an artifact, because the epithelium was negative when the BCO2-2-15 antibody was used. This is the only tissue in which a discrepancy between BCO2-1-9 and BCO2-2-15 staining was observed, and is probably because of a colon-specific protein cross-reacting with the BCO2-1-9 antibody. Alternatively, it is conceivable that a colon epithelial-specific protein may mask the epitope recognized by the BCO2-2-15 antibody. Additionally, spleen, brain, and lung showed no immunostaining for BCO2 and were also negative in the BCO2 RNA blot. These three tissues were not included in the BCO1 immunohistochemistry study.
Strong staining for BCO2 was seen in the muscle fibers of the myocardium (Figure 4H), and the result of the RNA blot also indicated that BCO2 was expressed in this organ (Figure 2). We have, however, not been able to detect the presence of BCO1 in human heart using immunohistochemistry (Figure 4J) or RNA blot (Figure 2).
To our knowledge, no immunohistochemistry studies have been performed on human eye to determine the cell typespecific expression of BCO1 or BCO2. Presence of BCO1 mRNA in eye has been investigated in two studies, however. BCO1 mRNA presence in retinal pigment epithelium was first demonstrated using RNA blots and RT-PCR (Yan et al. 2001); later, Bhatti and colleagues found BCO1 mRNA in retinal pigment epithelium-choroid using RT-PCR (Bhatti et al. 2003
). In an effort to compare the distribution of BCO1 and BCO2 in the eye, we included antibodies against both enzymes in this study. The results indicated that BCO1 and BCO2 were present in the very same cell types, because strong staining was seen for both proteins exclusively in retinal pigment epithelium (Figures 5A and 5C) and ciliary body pigment epithelia (Figures 5D and 5F).
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Discussion |
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For the larger part of the past century, it was debated if ß-carotene is cleaved symmetrically or asymmetrically in the body (Olson 1983; Ganguly and Sastry 1985
). With the recent cloning of BCO1 and BCO2, it has become clear that both enzymes are expressed in tissues of many vertebrate species. The question remains, though, what role the respective enzymes play in the utilization of carotenoids for vitamin A synthesis. BCO1 has previously been shown to be present, not only in intestinal mucosa where the majority of carotenoid cleavage take place, but also in structures sensitive to vitamin A deficiency, mainly epithelium and classical steroidogenic cells (Lindqvist and Andersson 2004
). It was proposed that BCO1 in extraintestinal tissues constitutes a backup system for local vitamin A synthesis during times of reduced intake of preformed vitamin A. In contrast to what is observed for BCO1, there is no obvious correlation between cell types being sensitive to vitamin A deficiency and expressing BCO2. As reported herein, both skeletal muscle and heart muscle appears to contain BCO2, and, to our knowledge, neither of these tissues is affected during vitamin A deficiency, except during development. The same applies to connective tissue in endometrium and prostate and to the endocrine part of the pancreas. Furthermore, because BCO1 is not expressed in these structures and other enzymatic systems designated to degrade apocarotenals have not yet been proven to exist, the production of vitamin A by way of BCO2 cleavage is questionable in these tissues.
Retinoids, such as retinal and retinoic acid, that are formed when ß-carotene is cleaved by BCO1, are crucial for physiological functions such as vision, development, and cell differentiation. It is conceivable that the primary products formed when the BCO2 enzyme cleaves ß-carotene have a function other than providing precursors for vitamin A synthesis. It is interesting to note that it has been reported that ß-ionone, the smaller product of BCO2 catalyzed ß-carotene cleavage, possesses unique biological activities. ß-Ionone is known to: (1) induce CYP2B1 in rat liver (Jeong et al. 1995) and CYP1A and CYP2B in mice (Aoki et al. 2000
); (2) upregulate the expression of CYP2A3 in rat lungs (Robottom-Ferreira et al. 2003
); (3) inhibit cell proliferation, induce apoptosis, and upregulate metalloproteinase expression in human gastric adenocarcinoma cells (Liu et al. 2004a
,b
); and (4) inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in human breast cancer cells in culture (Duncan et al. 2004
). With regard to the larger product, ß-apo-10'-carotenal, one report described that a carotenoid oxidation mix containing this molecule causes rapid and potent inhibition of Na+/K+-ATPase in vitro (Siems et al. 2000
). Thus the products of BCO2 catalyzed ß-carotene cleavage may play important roles in a number of physiological processes.
Additionally, BCO2 has been shown to be able to cleave the acyclic carotenoid lycopene (Kiefer et al. 2001). The products of the asymmetric cleavage of lycopene is the C13 molecule pseudoionone and the C27 molecule apo-10'-lycopenal. To our knowledge there are no reports on potential biological effects of any of these molecules. However, carotenoid-rich diets are known to reduce the risk of degenerative diseases, such as cancer and cardiovascular problems. Moreover, epidemiological studies have shown that elevated plasma lycopene levels are associated with a reduced risk of prostate cancer (Gann et al. 1999
) and seems to have a protective role against the development of atherosclerosis (Klipstein-Grobusch et al. 2000
). These effects have been attributed to the antioxidant properties of carotenoids, although the underlying mechanisms remain to be elucidated. A characteristic structural feature of carotenoid molecules is the conjugated polyene chain, which is prone to undergo autoxidation. Autoxidation products of lycopene have been shown to exert biological effects such as growth inhibition of human leukemia cells (Nara et al. 2001
), apoptosis induction in human prostate cancer cells (Kotake-Nara et al. 2002
), and promotion of cell-to-cell communication in rat liver epithelial cells (Aust et al. 2003
). In the case with growth inhibition, the effect was more pronounced with oxidized lycopene than with the intact compound (Nara et al. 2001
). That autoxidation products can be more potent than the carotenoid itself have also been shown for ß-carotene, where in vitro studies of genotoxicity for hepatocytes indicates that ß-carotene has no effect, but an oxidation mixture does (Alija et al. 2004
). It is therefore conceivable that the cleavage products from autoxidized carotenoids catalyzed by BCO2 are, at least in part, responsible for the beneficial effects on health seen with a carotenoid-rich diet. Whether autoxidized carotenoids are substrates for BCO2 (or BCO1) must be evaluated.
The prerequisite for any production of cleavage products is the availability of substrates for BCO2. A substantial amount of the absorbed dietary carotenoids is not cleaved by the BCO enzymes in the intestine (Goodman et al. 1966; Blomstrand and Werner 1967
; Hickenbottom et al. 2002
; Lemke et al. 2003
); hence, humans absorb unchanged ß-carotene and other carotenoids. The carotenoids get incorporated in chylomicrons to enter the circulation. The majority of the lipophilic carotenoids are transported in the core of VLDL and LDL particles (Johnson and Russell 1992
; Ziouzenkova et al. 1996
) and ultimately delivered to peripheral tissues via the LDL receptor. LDL receptors are present at varying amounts, not only in different tissues, but also in different BCO1- and BCO2-expressing cell types (Kovanen et al. 1979
; Spady et al. 1983
), with the adrenal glands and liver having the highest levels of LDL receptors. Therefore, the carotenoid content in various tissues may, at least in part, be a reflection of the degree of local LDL receptor activity. Even though it is known that there are pronounced interindividual differences of carotenoid content in different tissues of normal persons, it has been shown that both heart and skeletal muscle contains carotenoids of undefined identity (Blankenhorn 1957
). Considering that the delivery of carotenoids to tissues are performed by VLDL and LDL particles, the carotenoid profile in tissues depend on the carotenoid distribution in the particles (Gross et al. 2003
). The results from the RNA blot indicates that an interindividual difference in BCO2 expression may exist in certain tissues. Liver was included on two of the blots and, in both cases, 1 µg of poly(A)+ RNA had been loaded, but the RNA was prepared from different individuals. Despite the fact that all blots were hybridized simultaneously in the same solution, a pronounced difference in hybridization signal for the two lanes with liver mRNA was observed, with a much stronger signal detected in Figure 2A than in Figure 2B. A similar obvious difference was not detected in the immunohistochemistry study even though multiple stainings were performed for each tissue. However, because the tissue sources for the specimens were unidentified, each tissue may have originated from the same source. It would be interesting to correlate the BCO2 expression with the carotenoid content for a specific cell type in different individuals.
We found the presence of both BCO1 and BCO2 in the retinal pigment epithelium and in the ciliary body pigment epithelia of the human eye. It has been reported that these cell types contain substantial amounts of carotenoids, with lycopene interestingly being the most abundant (Khachik et al. 2002). Because 11-cis-retinal is synthesized in the retinal pigment epithelium and is an active component of the rhodopsin photoreceptor complex in the rod cells of the retina, these two enzymes may be involved in local vitamin A synthesis and other physiologically relevant processes for normal eye function.
We propose that BCO2 is involved in metabolism of carotenoids, possibly to assist in the production of biologically active metabolites of ß-carotene, lycopene, and other carotenoids. A more thorough investigation of possible substrates for BCO2 and identification of products produced will aid in understanding the physiological role for this enzyme.
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Acknowledgments |
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We thank Dr. Margaret Hinshelwood for critical review of the manuscript.
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Footnotes |
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