induction of heme oxygenase in intestinal epithelial cells: studies in caco-2 cell cultures

Molecular and Cellular Biochemistry 129: 93-98,1993. 0 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Induction of heme oxygenase in intestinal epithelial cells: studies in Caco-2 cell cultures

Julia W. Cable, Edward E. Cable and Herbert L. Bonkovsky Departments o f Medicine, Biochemistry and Molecular Biology and the Center for Study of Disorders of Iron and Porphyrin Metabolism, University of Massachusetts Medical School, Worcester, MA 01655, USA

Received 30 June 1993; accepted 28 September 1993

Abstract

Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heine catabolism, is present in abundance in Caco-2 cells, and that levels of its m RNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production. (Mol Cell Biochem 129: 93-98, 1993)

Key words: biliverdin reductase, Caco-2 cells, heme, heme oxygenase, intestinal cells

Abbreviations: BVR - biliverdin reductase; D M E M - Dulbecco's modified Eagles medium; DMSO - dimethyl sulfoxide; H O - heme oxygenase; 1 x SSC - a solution of 0.015 M sodium citrate/0.15 sodium chloride

Introduction

In many tissues, heme oxygenase (HO) (EC.1.14.88.3) is the rate-controlling enzyme for the breakdown of heine to biliverdin and subsequently bilirubin [1-4]. The products of the H O reaction are iron, carbon monoxide, and biliverdin. In liver, spleen, and kidney, activities of biti- verdin reductase are higher than those of HO, such that the rate of bilirubin formation is chiefly a function of the oxygenase [5-7]. H O can be induced by its substrate heme, as well as by other chemical agents or physical changes (e.g., heat) [8-16]. H O activities can be induced by at least two different mechanisms. One is dependent

Address for offprints: H.L. Bonkovsky, Division of Digestive Disease 01655, USA

on heme, or nascent heme synthesis, and the other, produced by certain metal ions (e.g., Cd, Co, Fe), is inde- pendent of heine synthesis [8].

Intestinal epithelial cells are known to absorb metal ions, including those known to induce HO. They also are known to absorb the iron of heme; indeed net GI absorption of heme iron generally exceeds that of nonheme or inorganic iron absorption [17-21], indicating that heine may be a preferred iron source for iron reple- tion, or, conversely, a source of dietary iron to be avoid- ed in those with hemochromatosis. It is unknown wheth-

and Nutrition, University of Massachusetts Medical Center, Worcester, MA

94

er heme take up by enterocytes is absorbed intact or bro- ken down by HO, such that its iron enters intracellular non-heme iron pools.

Caco-2 cells, cloned from a human colon cancer, spon- taneously undergo differentiation in culture to form po- larized monolayers with structural and functional fea- tures that resemble small intestinal epithelial cells [22, 23]. Such cultures are being used to study intestinal absorption and metabolism of ions and other chemicals [24]. To aid studies on iron and heme uptake and metabolism by enterocytes, we tested whether cultures of Ca- co-2 cells could take up and metabolize heine, iron, or cobalt, and whether HO and biliverdin reductase could be detected in such cultures.

Materials

Caco-2 cells were a generous gift from Drs. Susan and Robert Baker, Department of Pediatrics, University of Massachusetts Medical Center, Worcester, MA. They may also be obtained from the American Type Culture Collection (Rockvi]lle, MD). Dulbecco's modified Ea- gle's medium (DMEM) was obtained from Fisher (Springfield, IL). Penicillin/streptomycin, L-glutamine, and gentamicin were from Flow ICN (Costa Mesa, CA). Bovine calf serum was from Hyclone Laboratories (Lo- gan, UT). William's E medium was from Gibco BRL (Grand Island, NY). Dimethyl sulfoxide (DMSO), non- essential amino acids, trypsin, cobalt chloride, cadmium chloride, iron chloride, sodium nitrolotriaceate, NADPH, deferoxaraine, bovine serum albumin (BSA), dexamethasone, soybean trypsin inhibitor, triiodothyronine, and insulin were from Sigma (St. Louis, MO). Heine was from Porphyrin Products (Logan, UT). RNAzol | was from Biotex (Houston, TX). All other chemicals were of the highest purity commercially avail- able.

Methods

The human colon adenocarcinoma cell line Caco-2 was obtained at the 71 ~t passage and was studied through the 84 th passage. Cells were routinely grown in DMEM with 20% bovine calf serum, 1% non-essential amino acids, 1.74mM sodium bicarbonate, 2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 rag/ ml gentamicin. The cells were grown and treated in 6 cm or 10 cm tissue culture plates in a 37 ~ C incubator with

5% CO z [24]. The medium was changed every 2-3 days. After about 10 days of growth and the cells reached con- fluence, monolayers were gently rinsed with, and exposed to serum-free William's E medium containing dexamethasone (300pg/ml), triiodothyronine (1gg/ ml), and insulin (1 gg/ml) [16]. Chemicals were prepared and added at the indicated times and concentrations after which cells were harvested into 100 mM potassium phosphate pH7.50, 20% glycerol (v/v), containing I mM EDTA and sonicated for 3 sec at a setting of 1.5 using a Branson sonicator equipped with a microtip. Heine was administered as a BSA complex (ca. 1.66 moles heine/1 mole albumin). The complex was prepared by adding 100 gl of 10 mM heme, dissolved in dimethyl sulfoxide (DMSO), to 3.9 ml of a solution of BSA, ll.lmg/ml, dissolved in 40mM Tris.HCL (pH 7.4). Heme oxygenase was assayed as described previously [8, 16], except that a 14,000 x g I rain spin su- pernatant was used [25] as the source of the enzyme. 250 units (pmole bilirubin formed~ -1) of exogenous biliverdin reductase (BVR) was added to all HO assays except for those in Fig. 4, in which the exogenous BVR activity was as indicated. One unit of heme oxygenase or biliverdin reductase activity is defined as one pmole bilirubin formed per minute.

Total RNA was isolated by using RNAzol | according to the manufacturer's instructions. 20 Bg of RNA in a final volume of 40 gl were treated with 24 pl of 20 x SSC and 16 gl of 37% formaldehyde (by volume) and incu- bated at 65 ~ C for 15 min. The samples were diluted to 600 gl with 10 x 0.015 M sodium citrate, 0.15 M sodium chloride (SSC) and 10 lxg of RNA were loaded per dot. The blots wre crosslinked by exposure to 2.4 gjoules of UV radiation using a UV-Stratalinker (Stratagene, La Jolla, CA) and prehybridized at 42 ~ C in 15 ml of a buffer containing 50% formamide, 5 x SSC, 100 gg/ml salmon sperm DNA, 5 x Denhardt's solution and 1% SDS. After 2 hr the solution was removed and replaced with a small- er volume (5-7 mls) of the same buffer and a boiled radioactive probe for human heme oxygenase-1 [26] was added to 30 x 106 counts per blot. After hybridization for 18 hr at 42 ~ C, the blots were washed with 0.1 x SSC at room temperature for 30 rain with 3 buffer changes and counted on a beta-counter (Beta-gen, Millvale, CA). To- tal mRNA loaded onto dot-blots was normalized by probing duplicate blots with a poly-T probe, as previously described [27, 28]. Protein concentrations were assayed by the bicinchoninic method [29] using bovine serum albumin as a standard.

All results are shown as mean + SEM, n = 3. Statistical

E

x o

cl') E E - r ' . ~

o m

._~ X::}

< o E l:)..

150

100

- A. Heme

50

0 I I I I 0 40 80 120

[Heme] (I.tM)

200- B. Cadmium

150

100

50 -

. . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . 1 . . . . . . . . . . . . 1 . . . . . . . . . . . . . 1 . . . . . . . . . 1 . . . . . . . . . . .

0 5 10 15 20 25 30 [Cadmium Chloride] (t.tM)

Fig. i. Effect of heme (A) and cadmium chloride (B) on activity of heme oxygenase in Caco-2 cells. Cells were cultured, harvested, and heme oxygenase activity measured as described in Materials and Me- thods. Heme was added as an albumin complex at the indicated concentrations 18 hr prior to harvest. Cadmium chloride was added at the indicated concentrations 18 hr prior to harvest. Data are mean _+ SEM, n = 3; when no error bars are shown the SEM lies within the size of the symbol.

analyses were done using Student ' s t-tests, Dunne t t ' s test, or analysis of variance, with Scheff6 's correc t ion for

multiple comparisons, as appropriate . With all tests, a 95% conf idence level was the lower limit for signifi-

cance.

Results

In initial studies, the possible effect of selected culture

condit ions on activity of H O in Caco-2 cells were as- sessed. Since the cells were initially cul tured with D M E M and 20% bovine serum, various doses of h e m e

were added in this m e d i u m and in Wil iam's E m e d i u m with added insulin, dexamethasone and t r i iodothyronine. N o H O induct ion was observed in cells exposed to heine in D M E M with 20% serum (data not shown). In

95

2.5-

Z

2.0- I - -

-6

~ 1 . 5 - ~ 7 ~ e r

1.0-

0

o~ 0.5-

0.0 ~

, , , , , , ' , , , , ' , , , ,

,;,;,;,;,;,;,;

Control I

50 gM Heme

% g k l % g % g % g % 1 % I ,

% g % l % l % g % l k l k g ,

. , . , . . . . . . . . >i 1 10 IxM Cd

Fig. 2. Effects of heme and cadmium on heme oxygenase mRNA levels in Caco-2 cells. Cells were cultured, harvested, and total RNA isolated and probed as described in Materials and Methods. Heme (50 gM), added as the albumin complex, or cadmium chloride (10 gM) was added to some cultures for 10 hr prior to harvest. RNA was isolated and assayed as described in Materials and Methods. Data are mean + SEM, n = 3. The counts per minute for the blots hybridized to human heme oxygenase were normalized by dividing by the counts per minute from duplicate blots probed with radioactive poly-T. An asterisk (*) denotes a significant increase above control (Dunnett's test).

contrast , H O activity was induced in the cells exposed to

heme in Will iam's E m e d i u m containing dexametha-

sone, t r i iodothyronine and insulin (Fig. 1). The hormon- es that were added to the Will iam's E med ium were no t

essential for induction, but their inclusion increased the

activity of H O by 20% (data not shown).

H e m e oxygenase activity in Caco-2 cells, grown in

Will iam's E medium, was inducible by some metal ions,

as well as by heme (Fig. 1). H O activity was greatest in

Caco-2 cells t rea ted for 18 hr with 50--100 ktM heme or

10 gM cadmium. 25 g M cadmium chloride decreased

H O activity, while even higher concent ra t ions (50-

100 gM) resulted in no detectable activity, apparent ly

due to toxicity to the cell cultures. 200 ~tM cobalt chloride or ferric nitr i lotr iacetate (prepared as in [16]) pro-

duced slight (ca. 1.5 to 2-fold), but significant increases

in H O activity (data not shown). Glu te th imide (50 gM),

a phenobarbi ta l - l ike drug that induces H O in cul tured

hepatocytes [8, 16], had no detectable effect on activity

of the enzyme in Caco-2 cells (data not shown). A t 10 hr, 50 g M heme or 10 gM cadmium produced significant in-

creases in H O m R N A levels (Fig. 2). Induc t ion of heme oxygenase activity by heme oc-

curred rapidly and was maximal at 12 hr. The first signif-

icant increase in activity occurred at 8 hr. This heme-de- penden t induct ion was short-lived, with activities signif- icantly decreased f rom m a x i m u m at 16 hr (Fig. 3).

Act ivi ty of H O after heme t rea tment (50 gM) was

96

'.o

c O55

E 7- �9

o

E t ~

150

100

50

0 4 8 12 16

Hours after Treatment

Fig. 3. Time course of induction of heine oxygenase by heme in Caco-2

cells. Cells were cultured, harvested, and heme oxygenase activity

measured as described in Materials and Methods. Heme (50/aM) was

added as an a lbumin complex and cells harvested at the t imes indica-

ted (solid symbols). Controls treated with a DMSO/a lbumin solution

identical to that used to carry the heme had no change in activity (open

symbols). Data are mean + SEM, n = 3; when no error bars are shown,

the SEM lies within the size of the symbol.

measured with and without added BVR (Fig. 4). When no BVR was added, the measured HO activity was 42 +_ 4 units per mg protein. However, the apparent activity of HO increased to 93 units per mg protein when 500 units of exogenous BVR was added to the assay. The maximum amount of added BVR yielded a 5/1 ratio of BVR/HO activities. Thus, the endogenous BVR activity was rate limiting for bilirubin formation when heme oxygenase was induced.

Discussion

Our results show that Caco-2 cells contain the enzymes required for catabolism of heme to bilirubin, namely, HO and biliverdin reductase. Indeed, basal levels of activity of HO are equal those of the reductase. Further- more, HO activities are induced by heme or by metal ions, such as cadmium (Fig. 1), but not by glutethimide, a phenobarbital-like drug that induces the enzyme in liver cultures (data not shown, [8,16]). After induction of HO, biliverdin reductase activity becomes rate-limiting for bilirubin formation (Fig. 4), in contrast to liver, for example, in which the reductase is present in large excess [5-7].

The increase in activities of HO produced by heine or cadmium treatments were accompanied by, and probably due to, increased amounts of mRNA for HO (Fig.

100

~ i 80 ~-.~ O ~ 60

"6~, 40 ._Z, ,=

"6"~ 20 < O E

0

# r

I . p 4

10 20 30 40 50

BVR in Assay (pl)

Fig. 4. Effects of exogenous biliverdin reductase (BVR) on apparent

activity of heme oxygenase in extracts of Caco-2 cells. Cells were cultu-

red, harvested, and heine oxygenase activity measured as described in

Materials and Methods. Heine (50 gM) was added as an albumin com-

plex 18 hr prior to harvest. 40 gl equals 500 units of biliverdin reducta-

se activity. Data are mean _+ SEM; n = 3. w represents a significant in-

crease over 0 l-tI BVR, and * represents a significant increase over tl3 gl

BVR, Scheffe's test for multiple comparisons. The difference between

20 and 40/_tl BVR was not statistically significant.

2). Thus, as for other types of cells studied by us [16, 28] and others [12, 13], induction of HO in Caco-2 cells by heme or metals probably occurs chiefly as the result of an increase in gene transcription rate. The lack of strict correspondence between the increases in HO mRNA and activity is not surprising, since the mRNA amounts have been measured at only one time point (10 hr). Re- cent results from our laboratory [28] established that berne-dependent induction of HO mRNA in liver cells was transient, with a peak at 5-7 hr, and short-lived. Fur- thermore, the half-life of the message (3 hr) was much shorter than that of the enzyme protein (15 hr).

The ready inducibility of HO makes it unlikely that heme could pass through the Caco-2 monolayer intact, escaping breakdown by the oxygenase. Therefore, heine must apparently be administered parenterally in order to gain access to the circulation or internal organs with the iron-protoporphyrin IX macrocycle intact. Further- more, administration of large doses of heme enterally, in an effort to exceed the capacity of enterocyte HO, is not likely to be feasible, since induction of HO will occur. Thus, despite the difficulties that may attend the administration of heme parenterally [30, 31], the parenteral route is the only one that currently is known to be effica- cious [32-34]. Some of the potentially adverse side-effects of parenteral heme may be obviated by its administration complexed to albumin [34] or arginine [33].

It is possible that heme could pass through enterocytes intact if the HO of these cells were strongly inhib- ited. Some non-iron metalloporphyrins, especially tin or zinc-diiododeuteroporphyrin, -mesoporphyrin, or -protoporphyrin, are potent inhibitors of HO [35-39], including HO of the intestinal mucosa [40-43]. However, recent preliminary results indicated that tin protoporphyrin blocked transport of heme iron across Caco-2 cells, suggesting that catabolism of heme by HO may be required for iron from heme to be transported through the cell [44].

The mechanism of intestinal iron absorption and delivery of iron to the basolateral side of the intestinal mucosa is still uncharacterized. The ability of heme to induce heme oxygenase, and the recent demonstration of directional transport of radioactive iron in Caco-2 cells [44], shows that such cells can be used as a model to in- vestigate heme and iron metabolism of the intestinal epithelium. These cells could also be used to test possible enteral heine delivery systems, as an alternative to pa- rentheral heme administration, for example, in patients with acute porphyria [32-34].

In summary, Caco-2 cells contain HO and biliverdin reductase activities, and, as in other tissues, the mRNA levels of HO are increased by heme or by transition metals. Further studies to establish the molecular mechanisms responsible for this induction are in progress in our laboratory.

Acknowledgements

We thank R. and S. Baker for their generous gift of the Caco-2 cells, S. Shibahara for the generous gift of the plasmid (pHHO1) containing human heme oxygenase cDNA, K. Laruso for assistance with tissue culture, and R. Lambrecht for helpful discussion. Supported by grants from NIH (DK 38825) and from the American Porphyria Foundation.

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induction of heme oxygenase in intestinal epithelial cells: studies in caco-2 cell cultures

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