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Experimental Biology and Medicine 228:590-595 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Does Heme Oxygenase-1 Have a Role in Caco-2 Cell Cycle Progression?

Aliye Uc*,1 and Bradley E. Britigan{dagger}

* Department of Pediatrics and
{dagger} Internal Medicine, Veterans Administration Medical Center and University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242

Abstract

Intestinal epithelium undergoes a rapid self-renewal process characterized by the proliferation of the crypt cells, their differentiation into mature enterocytes as they migrate up to the villi, followed by their shedding as they become senescent villus enterocytes. The exact mechanism that regulates the intestinal epithelium renewal process is not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role. Heme oxygenase-1 (HO-1) is involved in endothelial cell cycle progression, but its role in the intestinal epithelial cell turnover has not been explored. With its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process. In this study, we examined the role of HO-1 in the proliferation and differentiation of Caco-2 cells, a well-established in vitro model for human enterocytes. After confluence, Caco-2 cells undergo spontaneous differentiation and mimic the crypt to villus maturation observed in vivo. In preconfluent and confluent Caco-2 cells, HO-1 protein expression was determined with the immunoblot. HO-1 activity was determined by the ability of the enzyme to generate bilirubin from hemin. The effect of a HO-1 enzyme activity inhibitor, tin protoporphyrin (SnPP), on Caco-2 cell proliferation and differentiation was examined. In preconfluent cells, cell number was determined periodically as a marker of proliferation. Cell viability was measured with MTT assay. Cell differentiation was assessed by the expression of a brush border enzyme, alkaline phophatase (ALP). HO-1 was expressed in subconfluent Caco-2 cells and remained detectable until 2 days postconfluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the ALP enzyme activity of confluent cells. HO-1 may be involved in intestinal cell cycle progression.

Key Words: cell cycle • cell proliferation • apoptosis • differentiation

Intestinal mucosa undergoes a process of self-renewal characterized by the initial proliferation of crypt cells, the later cessation of proliferation and the differentiation into mature absorptive enterocytes with upward migration in the crypt–villus axis (1, 2). The differentiation process is defined by marked changes in the cell ultrastructure and expression of brush border enzymes, alkaline phosphatase and sucrase-isomaltase (3). The differentiated intestinal cells then undergo a programmed cell death and exfoliate into the gut lumen. This entire cycle takes 3–5 days. Signaling pathways regulating this process remain largely unknown (4).

The Caco-2 cell line provides an excellent model to study intestinal epithelial cell proliferation and differentiation (3, 5, 6). Derived from a human colon carcinoma, these cells spontaneously differentiate after they reach confluency and acquire structural and biochemical properties of small intestinal enterocytes (6–10). This is analogous to the process by which immature enterocytes morphologically and functionally differentiate in their crypt to villus migration. Factors, such as hormones, growth factors, cytokines, and cell-matrix interactions have been proposed as modulators of Caco-2 cell differentiation. However, the exact mechanism has not been identified (11–16).

The exact process that regulates the renewal of the intestinal epithelium is also not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role (11). With its differential expression along the crypt villus axis (17), HO-1 may be involved in the intestinal epithelial cell turnover. Although the role of HO-1 in cell proliferation has been identified in vascular endothelial cells (18, 19), to our knowledge, its involvement in the intestinal epithelial cell turnover has not been explored. We hypothesized that, with its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process.

Materials and Methods

Tissue Culture.
Caco-2 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and used between passages 30 and 65. Stock cultures were grown to confluency at 37°C in 5% CO2 using Dulbecco’s Minimal Essential Medium containing 4.5 g/l glucose, 10% fetal calf serum, penicillin (50 U/ml), streptomycin (50 µg/ml), and HEPES (15 mM). Fresh medium was added every 2 days. Cells were released from stock plates by brief EDTA–trypsin treatment and plated at 50,000–100,00 cells/ml on plastic wells. At this concentration, cells become confluent in 4–6 days and form domes in 8-9 days.

Assay of Alkaline Phosphatase (ALP) Activity.
The activity of ALP was determined as described previously (20). Briefly, cells were lysed with 10 mM Tris buffer (pH 7.4) and protein concentration was measured. The cell homogenates containing 100–200 µg/ml protein were incubated with 8.44 mM p-nitrophenol phosphate mixed in glycine buffer (0.2 M glycine, 0.5 mM MgCl2, and 1.6 µM ZnCl2, pH 9.8) at 37°C for 30 min. The absorbance was measured at 405 nm to determine the amount of p-nitrophenol released by the enzyme.

Cell Count.
Cells were released by brief EDTA–trypsin application during the first 7 days of rapid growth. Cells were counted under the microscope with a hematocytometer.

Cell Viability Assay.
The MTT assay was used to determine the cell viability. This assay relies on production of a colored formazan by the action of mitochondrial enzymes on 3-[4,5-demethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) by living cells, and correlates well with other measures of cell number (21). Cells were exposed to the desired concentration of hemin or SnPP, then incubated with MTT (0.5 mg/ml) for 2 hr. The amount of formazan formed was solubilized in propanol and the absorbance was measured at 550 nm.

HO-1 Immunoblot Analysis.
Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using acrylamide resolving gels. After electrophoretic transfer to the nitrocellulose membrane, the blots were blocked by 10 mM Tris, pH 7.2, 150 mM NaCl, and 5% BSA (blocking buffer) for at least 1 hr and subsequently incubated for 1 hr with HO-1 antibody (1:2,000; StressGen Biotechnologies, San Diego, CA) diluted in TTBS buffer (0.14 M NaCl, 2.7 M KCl, 25 mM Tris, pH 7.4). The specific protein was detected by using goat-anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (1:10,000; Upstate Biotechnology, Lake Placid, NY). Blots were washed several times with TTBS buffer. Antibody-labeled bands were visualized by incubating the blots for 1 min with ECL chemiluminescent substrate (Amersham, Arlington Heights, IL), and exposing Kodak XAR film for 1–10 min. The percent increase in protein induction was obtained by densitometric scanning (Alpha-Innotech, San Leandro, CA).

HO Activity Assay.
Caco-2 cells were harvested and centrifuged at 2000g for 10 min. The pellet was resuspended in 100 mM phosphate buffer and 2 mM MgCl2. It was frozen and thawed three times and then sonicated briefly on ice. The cells were centrifuged for 10 min at 4°C at 18,800g and the supernatant was saved. The samples were then treated with 2 mg of rat liver cytosol, 20 µM heme, 2 mM glucose 6-phosphate, 0.2 U glucose 6-phosphate dehydrogenase, and 0.8 mM NADPH in a final volume of 400 µl. Samples were incubated at 37°C for 60 min in the dark. One milliliter of chloroform was then added to the samples and absorbance of bilirubin was measured spectrophotometrically at 464 and 530 nm.

Statistics.
One-way analysis of variance and Tukey’s method are used for comparison of different treatment groups. Results are expressed as mean ± SEM.

Results

HO-1 Expression Decreases After Confluency in Caco-2 Cells.
To determine the baseline HO-1 protein synthesis in Caco-2 cells at various differentiation levels, cells were harvested daily for 14 days and HO-1 protein levels were detected by immunoblot. Caco-2 cells became confluent on day 4. HO-1 was expressed in subconfluent Caco-2 cells and remained detectable at 2 days postconfluency (Fig. 1Go). From postconfluency day 3 forward, no HO-1 could be detected.



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  		Figure 1. HO-1 expression decreases after confluency in Caco-2 cells. Caco-2 cells were harvested at various differentiation levels and immunoblots performed with antibodies against HO-1. The far right lane contains 50 ng of HO-1 protein as a positive control. HO-1 is stably expressed in subconfluent Caco-2 cells and remains detectable for 2 days postconfluency. This timing would correspond to the cells starting their differentiation and taking the features of normal intestinal epithelial cells. These results are the representative of n = 3.

 
This timing parallels the cell pattern for differentiation and acquisition of the features of normal intestinal epithelial cells (22). It is also consistent with the change in HO-1 expression observed in crypt (immature) versus villus epithelial (mature) cells in animal models (17).

Hemin Induces HO-1 in Caco-2 Cells in a Time- and Dose-Dependent Manner.
To determine whether HO-1 protein levels could still be induced in confluent Caco-2 cells, cells were exposed to hemin, the substrate for the enzyme, and a known inducer of HO-1 in other cells (23–25). Hemin (Sigma, St. Louis, MO) was solubilized in 1 N NaOH with pH adjusted to 7.4 with media (Dulbecco’s Minimal Essential Medium + 10% fetal calf serum) and 14.7 M HCl. These concentrations of hemin were non-toxic as demonstrated with MTT assay. The amount of protein formed was measured by HO-1 immunoblot. Enzyme activity was measured as the ability of HO-1 to generate bilirubin from hemin, the end product of heme degradation (26, 27). Hemin induced HO-1 and increased its activity in a concentration-dependent manner (Fig. 2Go). The induction started at 10 µM and was maximal at 500 µM. Hemin did not induce inducible nitric oxidase synthase, catalase, copper zinc superoxide dismutase and manganese superoxide dismatase proteins as assessed by immunoblot analysis.



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  		Figure 2. Hemin dose response in Caco-2 cells. Confluent Caco-2 cells were treated with 10–500 µM hemin for 24 hr and HO-1 induction (immunoblot) or HO-1 activity (bilirubin formation, nmol/mg protein/day) was determined. In the immunoblot, the far right lane contains 50 ng of HO-1 protein as a positive control and the % induction was determined with densitometry. Caco-2 cells exhibited a dose-dependent increase in HO-1 levels in response to hemin exposure. These results are the representative of n = 11.

 
The time response was measured after treating the cells with 100 µM hemin at various time points. HO-1 induction was not apparent until 2 hr of hemin exposure and it was maximal at 12 hr (Fig. 3Go). These results demonstrated that confluent Caco-2 cells do respond to hemin in a time- and dose-dependent way by increasing their HO-1 enzyme levels and activity.



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  		Figure 3. Hemin time course in Caco-2 cells. Confluent Caco-2 cells were treated with 100 µM hemin for various times and HO-1 induction (immunoblot) or HO-1 activity (bilirubin formation, mg/mg protein/day) was determined. In the immunoblot, the far right lane contains 50 ng of HO-1 protein as a positive control and % induction was determined by densitometry. HO-1 induction by hemin in confluent Caco-2 cells peaked at 12 hr. These results are the representative of n = 11.

 
Effect of SnPP on Caco-2 Cell Proliferation.
If HO-1 is stably expressed in immature Caco-2 cells and the expression is downregulated with confluency, HO-1 could play a role in Caco-2 cell proliferation and/or differentiation. To determine the effects of HO-1 on Caco-2 cell proliferation, cells were plated in the presence of SnPP, a competitive HO-1 enzyme inhibitor (28, 29). Cell numbers were determined the first 5 days, during the rapid growth of cells. There were fewer cells if the media contained SnPP 50 µM (Fig. 4Go). Cells grown in media containing SnPP reached confluency later compared with control cells (6.7 ± 0.2 days in control cells vs 7.6 ± 0.3 days in cells treated with SnPP 50 µM, P < 0.01).



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  		Figure 4. Effect of SnPP Caco-2 cell proliferation. Caco-2 cells were grown in media with or without SnPP 50 µM and cell number was determined every other day until the cells reached confluency. There were fewer cells if media contained SnPP ({circ}) vs control cells (•), P < 0.01. These results are the representative of n = 2.

 
The low cell number and delay in cell confluency caused by SnPP appears to be related to cell death, as assessed with the MTT assay (Fig. 5Go). Cells incubated with SnPP were less viable compared with control cells.



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  		Figure 5. Effect of SnPP on Caco-2 cell viability. Caco-2 cells were grown in media with or without SnPP 50 µM and cell viability was determined daily until the cells reached confluency. Cells incubated with SnPP ({circ}) were less viable compared with control cells (•); P < 0.01 for days 2, 3, 4, P < 0.05 for day 7. These results are the representative of n = 4.

 
Effect of SnPP on Caco-2 Cell Differentiation.
After they become confluent, Caco-2 cell start differentiating into mature cells and assume many characteristics of human enterocytes. A brush border enzyme, ALP is commonly used as a marker of Caco-2 cell differentiation (30). ALP activity appears in Caco-2 cells at 6 days after plating and rapidly increases with maximum levels reached on Day 11 or 12 (31). Because the inhibition of HO-1 activity delays the proliferation of Caco-2 cells, we studied the effects of the enzyme inhibition on Caco-2 cell differentiation. Cells were grown in the presence or absence of SnPP and harvested on Day 2 when cells are expected to have no ALP activity and Day 14 when cells reach the maximum amount of enzyme activity. Compared with controls, cells treated with SnPP had less ALP activity on Day 14 (Fig. 6Go).



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  		Figure 6. Effect of SnPP on Caco-2 cell differentiation. Cells were grown in the presence or absence of SnPP 50 µM and harvested on Day 2 and Day 14 for ALP activity. Compared with controls, cells treated with SnPP had less ALP activity on Day 14 (P < 0.01). These results are the representative of n = 4.

 
Discussion

We showed that HO-1 is expressed in immature Caco-2 cells and the expression is downregulated as they become confluent. These results are in concordance with an in vivo study that showed a higher HO-1 enzyme activity in the immature crypt cells of the rat small intestine (17). In that study, HO-1 activity decreased as the cells differentiated and moved to the tip of the villus. Differential expression of gene products along the crypt-villus axis may affect intestinal epithelial cell cycle progression. For example, p42 MAPK is present in the crypt cells and its expression is downregulated as the cells differentiate and migrate to become absorptive villus cells (11). The inhibition of p42 MAPK inhibits cell proliferation and induces cell differentiation in Caco-2 cells, suggesting the role of p42 MAPK in intestinal epithelial cell cycle progression (11).

HO-1 plays a role in the cell cycle of many eukaryotic cells (18, 19, 32, 33) but, to our knowledge, the role of HO-1 in intestinal cell proliferation has not been examined. The effects of HO-1 on cell cycle progression may be cell specific. For example, the overexpression of the HO-1 gene enhances vascular endothelial cell cycle progression and angiogenesis, possibly through a carbon monoxide-dependent effect (18). Alternatively, HO-1 inhibits the growth of vascular smooth muscle cells (34–36). This dual effect of HO-1 in the vascular system may maintain homeostasis by enhancing the repair of endothelial cells and preventing the proliferation of smooth muscle cells, thus, atheroma formation after injury.

HO-1, with its ability to degrade heme and produce anti-oxidant molecules, bilirubin, biliverdin and carbon monoxide (37, 38), may protect the rapidly proliferating cells from oxidative stress. For example, HO-1 gene expression is upregulated in rapidly growing renal carcinoma cells (39). Intestinal mucosa is constantly exposed to dietary oxidants as well as endogenously generated reactive oxygen species (40) and, at high oxidative stress, cells die with apoptosis (41). Confluent (mature) Caco-2 cells possess antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase and their levels increase slightly with maturation (42). However, subconfluent (nondifferentiated) Caco-2 cells are more sensitive to oxidant-induced injury than mature cells (43). This enhanced susceptibility to oxidative stress in Caco-2 cells is associated with a lower GSH-dependent detoxication capacity (43). The role of oxidative stress in intestinal cell proliferation has recently been proposed (44). In a recent study, Caco-2 cells proliferation was inhibited by agents that decreased the ratio of reduced glutathione to glutathione disulfide (GSH/GSSH; ref. 44). It can be postulated that antioxidant enzymes, such as HO-1, can promote cell proliferation during rapid growth stages by scavenging and/or preventing the formation of oxygen radicals and other oxidant species.

In our study, cell proliferation was inhibited if the cells were grown in media containing SnPP, a HO-1 enzyme activity inhibitor. Cells exposed to SNPP became confluent one day later than control cells. Although SnPP did not totally inhibit Caco-2 cell growth, it significantly delayed the proliferation. This effect was probably secondary to decreased cell viability as a result of HO-1 activity inhibition. We have not yet conducted studies to demonstrate increased oxidative stress in cells with inhibited HO-1 activity; however, the cytoprotective effects of HO-1 are well described. For example, transfecting endothelial cells with the HO oxygenase gene protects against heme-induced toxicity (45). Alternatively, HO-1 deficient endothelial cells are destroyed by severe oxidative stress (46).

Although confluent Caco-2 cells did not express HO-1 protein at basal conditions, the protein and its activity could be induced by the enzyme substrate, hemin. It has been previously shown that Caco-2 cell HO-1 mRNA levels and enzyme activity increase in response to hemin in a dose- and concentration-dependent manner (47). Our results are consistent with this study (47). In addition, we used a broader range of hemin concentrations, established toxicity with higher doses and determined HO-1 protein synthesis.

Caco-2 cells, derived from a human colon carcinoma, spontaneously differentiate into cells with structural and biochemical properties of small intestinal enterocytes (7). They develop polarity, tight junctions, form microvilli and synthesize apical brush border enzymes (ALP, sucrase-isomaltase, etc) as a sign of maturity and differentiation. In our study, Caco-2 cells exposed to SnPP were delayed in their differentiation into more mature cells as defined by the ALP activity. This was probably a reflection of delayed growth caused by SnPP, but this requires additional study.

In summary, we demonstrated that HO-1 was stably expressed in subconfluent Caco-2 cells and downregulated after confluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the alkaline phosphatase enzyme activity of confluent cells. These results suggest that HO-1 may have a role in intestinal cell cycle progression in health and disease. The factors and signaling mechanisms that contribute to downregulation of HO-1 with confluence is a potential area for future study.

Footnotes

1 To whom requests for reprints should be addressed at 2865 JPP Pediatrics, University of Iowa Health Care, 200 Hawkins Drive, Iowa City, IA 52242. E-mail: aliye-uc{at}uiowa.edu Back

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A. Uc, J. B. Stokes, and B. E. Britigan
Heme transport exhibits polarity in Caco-2 cells: evidence for an active and membrane protein-mediated process
Am J Physiol Gastrointest Liver Physiol, December 1, 2004; 287(6): G1150 - G1157.
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