Changes in redox network expression during Caco-2 cell differentiation into enterocytes

in Redox Experimental Medicine
Authors:
Wei Zhu Department of Nutrition, University of California, Davis, California, USA

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Eleonora Cremonini Department of Nutrition, University of California, Davis, California, USA

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Patricia I Oteiza Department of Nutrition, University of California, Davis, California, USA
Department of Environmental Toxicology, University of California, Davis, California, USA

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https://orcid.org/0000-0001-7462-1641

Correspondence should be addressed to P I Oteiza: poteiza@ucdavis.edu
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Graphical abstract

Abstract

Objective

This work characterized fluctuations in cell components involved in the regulation of cell redox homeostasis during Caco-2 cell differentiation into enterocytes.

Methods

Caco-2 cells were differentiated for 10 days. Gene expression of NADPH oxidases; enzymes that metabolize superoxide anion and hydrogen peroxide, proteins involved in the production and/or regeneration of glutathione, thioredoxin, and in NADPH production, and NRF2-dependent genes were measured by qPCR at 0, 1, 4, 7, and 10 days post-confluence.

Results

NADPH oxidase 1 mRNA levels decreased with Caco-2 cell differentiation, in agreement with its role in regulating cell proliferation. NADPH oxidase 4, DUOX2, superoxide dismutase 1, and catalase mRNA levels increased with differentiation. NRF2 mRNA levels increased with differentiation up to day 4 post-confluence, reaching a plateau until day 10. A similar pattern was observed for the NRF2-regulated genes: NAD(P)H quinone dehydrogenase 1, glutathione reductase 1, and thioredoxin reductase 1. On the contrary, glutamate-cysteine ligase catalytic subunit mRNA levels decreased after reaching a maximum 4 days post-confluence. This and the finding of a correlation between glutathione reductase 1 and thioredoxin reductase 1 mRNA levels suggest that recycling of glutathione and thioredoxin is more relevant than their synthesis during Caco-2 cell differentiation.

Conclusion

Results support the relevance of redox homeostasis for cell fate decisions and in preparing enterocytes to interact with their environment.

Significance statement

Current findings resemble changes in redox components previously characterized in vivo. This stresses the concept that Caco-2 cells are an appropriate model to be used to evaluate redox-regulated mechanisms in human enterocytes.

Abstract

Graphical abstract

Abstract

Objective

This work characterized fluctuations in cell components involved in the regulation of cell redox homeostasis during Caco-2 cell differentiation into enterocytes.

Methods

Caco-2 cells were differentiated for 10 days. Gene expression of NADPH oxidases; enzymes that metabolize superoxide anion and hydrogen peroxide, proteins involved in the production and/or regeneration of glutathione, thioredoxin, and in NADPH production, and NRF2-dependent genes were measured by qPCR at 0, 1, 4, 7, and 10 days post-confluence.

Results

NADPH oxidase 1 mRNA levels decreased with Caco-2 cell differentiation, in agreement with its role in regulating cell proliferation. NADPH oxidase 4, DUOX2, superoxide dismutase 1, and catalase mRNA levels increased with differentiation. NRF2 mRNA levels increased with differentiation up to day 4 post-confluence, reaching a plateau until day 10. A similar pattern was observed for the NRF2-regulated genes: NAD(P)H quinone dehydrogenase 1, glutathione reductase 1, and thioredoxin reductase 1. On the contrary, glutamate-cysteine ligase catalytic subunit mRNA levels decreased after reaching a maximum 4 days post-confluence. This and the finding of a correlation between glutathione reductase 1 and thioredoxin reductase 1 mRNA levels suggest that recycling of glutathione and thioredoxin is more relevant than their synthesis during Caco-2 cell differentiation.

Conclusion

Results support the relevance of redox homeostasis for cell fate decisions and in preparing enterocytes to interact with their environment.

Significance statement

Current findings resemble changes in redox components previously characterized in vivo. This stresses the concept that Caco-2 cells are an appropriate model to be used to evaluate redox-regulated mechanisms in human enterocytes.

Introduction

Differentiated Caco-2 cells are an accepted model of human enterocytes, and they have been essential in the understanding of the intestinal absorption and metabolism of nutrients, drugs, and toxicants in humans (Angelis & Turco 2011, Erlejman et al. 2011). Caco-2 cells are also used to study other functions of enterocytes, e.g. barrier permeability, which involve redox-regulated mechanisms (Cremonini et al. 2018, Iglesias et al. 2020, Wang et al. 2020, Zhu et al. 2024), and the interactions of enterocytes with the luminal microbiota (Liu et al. 2023). Like all other cell types, enterocytes produce reactive oxygen species as a result of normal cell metabolism and need to be equipped with the different proteins involved in oxidant removal and damage repair. During migration from the bottom to the top of the villi, differentiating enterocytes are increasingly exposed to external luminal prooxidant and proinflammatory aggressions from, among others, toxins present in food or originating from the commensal microbiota, food components, pathogens, and toxicants. Additionally, regulated production of select oxidants, e.g., hydrogen peroxide (H2O2), is central to the regulation of cell signaling. Thus, a changing cell redox balance during the proliferation-to-differentiation transition can modulate signaling pathways that regulate cell fate and function. In this regard, Jones & Sies (2015) defined a redox code that influences cell decisions during development and in its adaptation to environmental changes.

In terms of oxidant defenses, cells protect themselves from oxidative stress by a system composed of enzymes that metabolize oxidants, produce and recycle their substrates, e.g. glutathione (GSH), thioredoxin (TRX), NADPH, and repair damage (Forman & Zhang 2021, Halliwell 2024). In terms of cell fate and adaptation, the transition from proliferating to fully differentiated Caco-2 cells mimics that of progenitor cells in the bottom of the intestinal crypt, which migrate and differentiate toward the top of the villi. Thus, changes in components of the redox network can participate in the commitment of Caco-2 cells to differentiate and in the adaptation of the enterocyte to interact with the luminal environment. Different studies have looked at particular aspects of Caco-2 redox balance during differentiation, including the expression of select antioxidant enzymes, and variations in GSH and TRX concentrations (Nkabyo et al. 2002, Speckmann et al. 2011). However, to our knowledge, there is no study describing multiple key components of redox regulation in Caco-2 cells during differentiation into enterocytes. Thus, this study characterized during the transition from proliferating to fully differentiated Caco-2 cells, the variations in the expression of genes encoding proteins involved in the production of oxidant species, i.e. superoxide anion (O2.-) and H2O2, enzymes that metabolize reactive oxygen species, those involved in the production and/or regeneration of antioxidant substances, e.g. GSH, TRX, and in NADPH production, and additional nuclear factor erythroid 2-related factor 2 (NRF2)-dependent genes.

Materials and methods

Materials

Caco-2 cells were purchased from the American Type Culture Collection. Cell culture media, including Minimum Essential Medium (MEM), 100× non-essential amino acids, sodium pyruvate, and fetal bovine serum were, obtained from Thermo Fisher Scientific Inc.. Primary antibodies for sucrase isomaltase (SI) (sc-393424), caudal type homeobox 2 (Cdx2) (sc-393572), and heat shock cognate 70 (HSC-70) (sc-7298) were from Santa Cruz Biotechnology. The secondary antibody, horse anti-mouse IgG (#7076P2), was purchased from Cell Signaling Technology. All reagents and materials for the quantitative PCR (qPCR), PVDF membranes, and Clarity Western ECL Substrate were obtained from Bio-Rad.

Cell culture and incubation

Caco-2 cells between passages 5 and 20 were cultured at 37°C in a 5% (v/v) CO2 atmosphere. The cells were grown and differentiated in MEM supplemented with 10% (v/v) fetal bovine serum, antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin), 1% (v/v) of 100× non-essential amino acids, and 1 mM sodium pyruvate. For the experiments, Caco-2 cells were plated in 60 mm dishes (0.5 × 106 cells/dish) and differentiated for 10 days. At 10 days post-confluence, major makers of enterocyte differentiation had reached a maximum (Buhrke et al. 2011). The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. Day 0 was considered as the day that cells reached confluence. At the different time points, the medium was removed, cells were washed with PBS, and processed for total protein and mRNA extraction as described below.

Western blot

Total proteins were isolated and quantified using the Bradford assay as previously described (Wang et al. 2020). Aliquots containing 30 μg of protein were separated by 7–15% (w/v) PAGE and electroblotted onto PVDF membranes. Dual standards (colored (Bio-Rad Laboratories) and biotinylated (Cell Signaling Technologies)) were run simultaneously. Membranes were blocked for 5 min using EveryBlot (Bio-Rad), then incubated at 4°C overnight with the antibodies for SI and Cdx2 (1:1000 dilution) in EveryBlot. After incubation for 1.5 h at room temperature with the secondary antibody (HRP-conjugated, 1:10,000 dilution), conjugates were visualized by chemiluminescence detection in a ChemiDoc Imaging System (Bio-Rad). TRIzol reagent was purchased from Invitrogen, and all other reagents for qPCR were from Bio-Rad.

RNA isolation and qPCR

RNA was extracted from the cells using TRIzol reagent. cDNA was generated using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). mRNA levels of the assessed proteins were evaluated using the qPCR system (iCycler, Bio-Rad) with the primers described in Table 1. The relative mRNA levels were normalized to β-actin as the housekeeping gene and calculated using the 2−ΔΔCt method (Rao et al. 2013).

Table 1

Primers used for q-PCR analysis.

Gene Forward Reverse
SI 5′- TATTTTGGCAGCCTTATCCAAGT -3′ 5′- CTTGCCATCTTATTTCATAGCCTGT -3′
CDX2 5′- GACGTGAGCATGTACCCTAGC -3′ 5′- GCGTAGCCATTCCAGTCCT -3′
NOX1 5′- GTACAAATTCCAGTGTGCAGACCAC -3′ 5′- CAGACTGGAATATCGGTGACAGCA -3’
NOX4 5′- CTCAGCGGAATCAATCAGCTGTG -3’ 5′- AGAGGAAGACGACAATCAGCCTTAG -3’
DUOX2 5′- CCGGCAATCATCTATGGAGGT -3’ 5′- TTGGATGATGTCAGCCAGCC -3′
GCLC 5′- GGAGACCAGAGTATGGGAGTT -3′ 5′- CCGGCGTTTTCGCATGTTG -3′
GCLM 5′- CATTTACAGCCTTACTGGGAGG -3′ 5′- ATGCAGTCAAATCTGGTGGCA -3′
GPX1 5′- CAGTCGGTGTATGCCTTCTCG -3′ 5′- GAGGGACGCCACATTCTCG -3′
SOD1 5′- GGTGGGCCAAAGGATGAAGAG -3′ 5′- CCACAAGCCAAACGACTTCC -3′
CAT 5′- TGGAGCTGGTAACCCAGTAGG -3′ 5′- CCTTTGCCTTGGAGTATTTGGTA -3′
TXNRD1 5′- ATATGGCAAGAAGGTGATGGTCC-3′ 5′- GGGCTTGTCCTAACAAAGCTG -3′
G6PDH 5′- ACCGCATCGACCACTACCT -3′ 5′- TGGGGCCGAAGATCCTGTT -3′
GSR1 5′- CACGAGTGATCCCAAGCCC -3′ 5′- CAATGTAACCTGCACCAACAATG -3′
ACTB 5′-TCATGAAGTGTGACGTGGACATCCGC -3′ 5′-CCTAGAAGCATTTGCGGTGCACGATG -3′

Statistical analysis

Data were analyzed by one-way ANOVA using GraphPad Prism 10.0 (GraphPad Software), after being tested for normal Gaussian curve (bell-shape) distribution by the Shapiro–Wilk test. Fisher's least significance difference test was used to examine differences between group means. Results are shown as means ± s.e.m. of three to six independent experiments. A P value < 0.05 was considered statistically significant.

Results and discussion

Caco-2 cell differentiation

Caco-2 cells begin to differentiate once they reach confluence. Full Caco-2 cell differentiation is achieved between days 10 and 17 post-confluence (Buhrke et al. 2011). Thus, mRNA levels of the brush border hydrolases alkaline phosphatase and SI, recognized biomarkers of enterocyte differentiation (Smith 1991), reach a maximum already at day 10 (Buhrke et al. 2011). We initially assessed Caco-2 cell differentiation by measuring the mRNA and protein levels of biomarkers of enterocyte maturation, i.e. SI and CDX2, from the time the cells reached confluence (day 0) until day 10 post-confluence. SI mRNA levels increased significantly 7 days post-confluence and kept increasing on day 10 (58-fold, P < 0.0001) (Fig. 1A). On the other hand, SI protein levels increased significantly 4 days post-confluence, reaching maximum levels after 7 days (14.4-fold, P < 0.0001), and remaining constant up to day 10 (Fig. 1C). CDX2 is a transcription factor involved in the inhibition of progenitor cell proliferation and in the promotion of differentiation to mature enterocytes (Mutoh et al. 2005). While CDX2 mRNA levels significantly increased from days 4 to 10 post-confluence (3.8- to 6.0-fold, P < 0.04), Cdx2 protein levels were significantly higher 7 days (2.2-fold, P = 0.03) post-confluence, plateauing at day 10 (Fig. 1B and D). SI protein variation pattern supports that, in the used model, Caco-2 cell differentiation is mostly accomplished 10 days post-confluence.

Figure 1
Figure 1

Biomarkers of Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) Sucrase-isomaltase (SI) and (B) caudal type homeobox 2 (CDX2) mRNA levels were measured by qPCR. mRNA levels were normalized to β-actin as the housekeeping gene. (C) SI and (D) CDX2 protein levels were evaluated by Western blot. Bands were quantified and values were referred to HSC-70 levels (loading control). Results are shown as mean ± s.e. of three to four independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0009

The redox balance of enterocytes

The production of reactive oxygen occurs as a part of normal cell metabolism but can also be triggered by cells’ exposure to environmental factors. During the transition from proliferation into mature enterocytes, redox-regulated mechanisms can participate in cell fate decisions, in the regulation of cell interactions with the environment, e.g. crosstalk with luminal microbiota to sustain immunological homeostasis, and in supporting relevant intestinal epithelium functions, e.g. intestinal barrier physiology. Physiological redox homeostasis is attained by regulating the production of oxidant species, managing their removal, and through the repair of oxidized molecules (Forman & Zhang 2021). The current study is focused on characterizing the fluctuation of important cell components that sustain cell redox homeostasis during Caco-2 cell differentiation. The results will be discussed with a focus on cell fate decisions and mature enterocyte physiology, and within the frame of previous intestinal epithelium in vivo and in vitro evidence.

NADPH oxidases expression during Caco-2 cell differentiation

Excess activation of NADPH oxidases (NOXs), as that occurring in chronic inflammatory conditions, e.g. inflammatory bowel diseases (IBD), can lead to oxidative stress. On the other hand, they are major players in the regulation of multiple physiological processes (Bedard & Krause 2007). Thus, NOXs produce pulses of O2 .−/H2O2 that prolong cell signaling and are involved in, among others, the regulation of cellular metabolism, cell decisions to proliferate and differentiate, and host immune defenses. During enterocyte differentiation, fluctuations of oxidant levels suggest the involvement of NOXs and the regulation of the redox tone in this process. In this regard, O2 .−production is high in proliferating colorectal cancer cells, including Caco-2 cells, which decreases upon cell differentiation into enterocytes (Perner et al. 2003). As discussed below, this and other evidence suggest a critical role of the cell's redox balance in cells' decisions to proliferate and/or differentiate, and to adapt to their environment (Jones & Sies 2015, Jones 2024).

The most abundant NOXs in enterocytes are NOX1, DUOX2, and NOX4, with NOX1 and DUOX2 located at the cell membrane and NOX4 intracellularly. We observed that NOX1 mRNA levels did not vary until day 4 post-confluence, decreasing by 32% (P = 0.012) and 53% (P = 0.0002) after 7 and 10 days, respectively (Fig. 2A). This is in agreement with the previously observed decreased gradient of NOX1 expression from the bottom of the crypt, where stem cells reside, toward the top of the crypt during the process of enterocyte differentiation (Coant et al. 2010). On the other hand, Caco-2 cells stimulated to differentiate with 1α,25-dihydroxyvitamin D(3) or interferon gamma show increasing NOX1 expression with differentiation (Geiszt et al. 2003). While this difference can be due to the differentiating stimuli used, our findings are more in agreement with the proposed pro-proliferative role of NOX1. In fact, NOX1 is highly expressed in colorectal cancer (Laurent et al. 2008), and Caco-2 cells are epithelial cells extracted from a human colorectal adenocarcinoma. Additionally, NOX1 was shown to regulate progenitor cell proliferation and commitment of differentiation in the bottom of the intestinal crypt via Wnt/-catenin and Notch1 signals (Coant et al. 2010). NOX1 is also involved in the regulation of intestinal barrier permeability, through the activation of the NF-κB and/or ERK1/2 signaling cascades (Ma et al. 2004, Xiao et al. 2005, Ye & Ma 2008, Ihara et al. 2015). However, proinflammatory conditions and high-fat diets cause chronic NOX1 activation and increased expression, leading to pathological loss of barrier function (Cremonini et al. 2018, Wang et al. 2020, Zhu et al. 2024). Overall, the profile of NOX1 expression during differentiation follows the pattern observed in vivo.

Figure 2
Figure 2

Expression of NADPH oxidases during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) NOX1, (B) NOX4, and (C) DUOX2 mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0009

NOX4 has been described as being located in different cellular organelles, particularly the endoplasmic reticulum. NOX4-regulated generation of O2 .− and H2O2 is necessary for the activation of the Nrf2 pathway and for an adequate antioxidant response (Greatorex et al. 2023). NOX4 mRNA levels increased significantly after 7 and 10 days post-confluence (4.0- (P = 0.014) and 8.3- (P < 0.0001) fold, respectively) (Fig. 2B). In addition, a positive correlation between mRNA levels of NOX4 and NRF2 was observed (r = 0.63, P = 0.02), suggesting a role for NOX4 in NRF2 activation during differentiation.

DUOX2 is located at the apical cell membrane of enterocytes, facing the gut lumen. DUOX2 mRNA levels significantly increased (P = 0.002) after reaching confluence, with levels being 5-, 6-, and 11.8-fold higher at days 4, 7, and 10, respectively, compared to day 0 (Fig. 2C). Furthermore, as observed for NOX4, DUOX2 mRNA levels correlated with those of NRF2 (r = 0.80, P = 0.001), suggesting a potential involvement of DUOX2 in NRF2 activation during enterocyte differentiation. Evidence from metazoans indicates that the H2O2 generated by DUOX is critical in the protection against infections (Chávez et al. 2009). In humans, deleterious variants of the DUOX2 gene are associated with altered microbiota-immune system homeostasis and increased IBD risk (Grasberger et al. 2021). Thus, the importance of DUOX in regulating the interactions between epithelial cells and the luminal microbiota (Grasberger et al. 2015) can also explain its increased expression during the process of Caco-2 cell differentiation into enterocytes. Accordingly, in mouse ileum and colon, DUOX2 expression is high at the tip of the villus (Sommer & Bäckhed 2015).

Overall, the main enterocyte NOXs are key in the regulation of important cellular processes, including the control of cell decisions to proliferate and/or differentiate, sustaining intestinal barrier function, the regulation of antioxidant responses (NRF2), and the interactions of enterocytes with the luminal microbiota regulating immune responses. The observed profiles of NOXs during Caco-2 cell differentiation are in agreement with the previously described functions of NOXs in stem cells and enterocytes (Coant et al. 2010, Grasberger et al. 2015, Sommer & Bäckhed 2015).

The NRF2 pathway during Caco-2 cell differentiation

The NRF2 pathway is central to the detoxification of electrophiles and oxidant species. The interaction of KEAP1 with NRF2 keeps the transcription factor in the cytoplasm and targets it for degradation by the proteasome (Itoh et al. 2004, Baird & Yamamoto 2020). KEAP1 has multiple sensing mechanisms to different stimuli, which allows NRF2 activation to modulate multiple cellular responses (Baird & Yamamoto 2020). Among the NRF2-regulated genes that help cells manage oxidant-mediated stress, they include those encoding proteins involved in GSH production and regeneration, TRX and TRX regeneration, NADPH production, and others that prevent/mitigate oxidative stress or metabolize toxic substances with the potential to cause redox dysregulation. We observed that NRF2 and NAD(P)H quinone oxidoreductase 1 (NQO1) mRNA levels were significantly higher (2.7- and 3.9-fold, respectively) (P = 0.0004 and 0.01, respectively) after 4 days post-confluence compared to day 0, remaining constant up to day 10 (Fig. 3A and B). A tight (r = 0.90, P < 0.0001) positive correlation was observed between NRF2 and NQO1 mRNA levels. We observed positive and significant (P < 0.05) correlations between the mRNA levels of NRF2 and those of other NRF2-regulated genes included in sections below, i.e. glutamate cysteine ligase catalytic subunit (GCLC), TRX reductase 1 (TXNRD1), GSH peroxidase 1 (GPX1), and GSH reductase 1 (GSR1). On the other hand, when compared to day 0, heme oxygenase 1 (HMOX1) mRNA levels were significantly higher only at day 1 post-confluence (45%, P = 0.035), subsequently decreasing and reaching basal values at days 4 and 7 post-confluence (Fig. 3C). HMOX1 degrades heme into biliverdin, carbon monoxide (CO), and Fe2+. The lack of correlation between NRF2 and HMOX1 levels is explained by the involvement of other transcription factors, e.g. PPARγ and AP-1, regulating HMOX1 gene expression (Gong et al. 2002, Krönke et al. 2007). HMOX1 is involved in the regulation of iron metabolism, protection against oxidative stress, immunomodulatory functions, and regulation of proliferation and differentiation processes of proliferations and differentiation (Campbell et al. 2021). In support of a role for HMOX1 in cell differentiation, and in agreement with our current findings, HMOX1 gene expression decreases during the differentiation of human adipocytes (Moreno-Navarrete et al. 2017).

Figure 3
Figure 3

Expression of NRF2 and NRF2-regulated genes during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) NRF2, (B) NQO1, and (C) HMOX1 mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0009

In summary, while changes in the NFE2L2 gene (encoding for NRF2) expression during the proliferation-to-differentiation transition of Caco-2 cells can be largely related to an increase in antioxidant protection, HMOX1 seems mostly involved in the cell decision to differentiate.

O2 .− and H2O2/lipid peroxides metabolizing enzymes during Caco-2 cell differentiation

Enzymes with the capacity to metabolize oxidants from endogenous or exogenous origin constitute the first line of the cell’s defense against oxidative stress (Forman & Zhang 2021). These enzymes include superoxide dismutase (SOD), which metabolizes superoxide anion O2 .−, catalase, which metabolizes H2O2, glutathione peroxidase (GPX), which can metabolize H2O2 and lipid peroxides, and the peroxiredoxin (PRDX)/TRX system, which catalyzes thiol-based reactions (Forman & Zhang 2021).

SOD1 is located in the cell cytosol and catalyzes the conversion of O2 .− into H2O2. SOD1 mRNA levels were significantly higher (two-fold, P = 0.047) 4 days post-confluence, remaining at this level up to day 10 (Fig. 4A). The lack of correlation between SOD1 and NRF2 mRNA levels is probably due to the fact that NF-κB also regulates SOD1 gene expression (Rojo et al. 2004). Catalase, located in peroxisomes, metabolizes H2O2. We observed that catalase mRNA levels were 3.4- to 5.8-fold higher between days 4 and 10 post-confluence, respectively. While also involved in H2O2 metabolism, the pattern of fluctuation of GPX1 mRNA levels during Caco-2 cell differentiation was different from that of catalase (Fig. 4B and C). GPX is one of eight members of the GPX family. It is a selenium-dependent enzyme that can reduce both H2O2 and lipid peroxides. GPX1 was found to be uniformly distributed throughout the rat intestine, from the crypt to the top of the villi (Esworthy et al. 1998). We observed that GPX1 mRNA levels were only significantly higher at days 1 and 4 post-confluence (1.7- and 2.0-fold, respectively) compared to day 0 (Fig. 4C). A similar pattern was previously observed for GPX4 protein, activity, and mRNA levels in differentiating Caco-2 cells at days 6 and 11 post-confluence (Speckmann et al. 2011). Similarly to the observation for GPX4, while GPX2 did not show variations in expression or sensitivity to added selenium, GPX1 mRNA levels increased 6 days post-confluence, with expression being the expression higher upon selenium supplementation (Speckmann et al. 2011).

Figure 4
Figure 4

Expression of O2 .− and H2O2/lipid peroxides metabolizing proteins during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) Superoxide dismutase 1 (SOD1), (B) catalase, and (C) glutathione peroxidase 1 (GPX1) mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0009

In summary, SOD1 and catalase mRNA levels increase throughout Caco-2 cell differentiation, suggesting that the cell needs to prepare for pro-oxidant and pro-inflammatory insults that will face the mature enterocyte. In terms of GPX1 pattern of expression, enzymes required for the recycling of GSH, the substrate of GPX1, may be more relevant in enterocyte antioxidant protection than GPXs per se.

Production and regeneration of GSH and TRX

GSH is the most abundant low molecular weight thiol in cells. While GSH is involved in the metabolism of oxidant species, it is also critically important for sustaining the cell thiol redox balance, which can ultimately modulate cell signaling. In this regard, the switch between cell proliferation and differentiation is partly regulated by the redox potential of GSH/glutathione disulfide (GSH/GSSG), with a reduced potential promoting proliferation and an oxidized potential favoring differentiation (Hutter et al. 1997, Nkabyo et al. 2002). In the developing intestine, active proliferation is accompanied by a significant increase in GSH, thus a more reduced GSH/GSSG redox potential (Reid et al. 2017). In agreement with this, the concentration of GSH in Caco-2 cells decreases during the differentiation while that of TRX is not affected (Nkabyo et al. 2002). Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis. It is composed of two subunits, the catalytic (GCLC) and modulatory (GCLM) subunits, whose expression is regulated by NRF2 and NF-κB (Kensler et al. 2007, Peng et al. 2010). During Caco-2 cell differentiation, we observed that GCLC mRNA levels reached a maximum increase 4 days post-confluence (3.9-fold, P = 0.0001), significantly decreasing at days 7 and 10 compared to post-confluence day 4 (Fig. 5A). GCLM mRNA levels were approximately two-fold higher at days 7 and 10 post-confluence than at day 0 (Fig. 5B) and did not correlate significantly with GCLC mRNA levels. GSH is not only regulated by synthesis but also by recycling. Glutathione reductase (GSR) reduces GSSG to GSH, using NADPH as the electron donor. GSR1 mRNA levels were already significantly (39%, P = 0.042) elevated after 1 day post-confluence, reaching a 2.1-fold increase at day 4, which was sustained up to 10 days post-confluence (P < 0.0001) (Fig. 5C). This rapid and persistent increase in GSR1 mRNA levels suggests that, as previously described for oxidative stress conditions (Harvey et al. 2009), GSH recycling may become more important than synthesis during Caco-2 cell differentiation.

Figure 5
Figure 5

Expression of genes involved in the production and regeneration of GSH and TRX during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Methods. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A, B) Cysteine ligase catalytic (GCLC, A) and modulatory (GCLM, B) subunits, (C) GSH reductase 1 (GSR1), (D) TRX reductase 1 (TXNRD1), and (E) glucose-6-phosphate dehydrogenase (G6PDH) mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test). (F) Linear regression analysis between the mRNA levels of TXNRD1 and GSR1. The solid line represents the regression line and the dotted lines the 95% CI.

Citation: Redox Experimental Medicine 2024, 1; 10.1530/REM-24-0009

GSH functions to regulate the cellular thiol redox status and is complemented by TRX. The TRX system is composed of TRX and TRX reductases (TXNRD). TRX is a small protein (12 kDa) with an active site containing two cysteines separated by two amino acids. It reduces disulfide bonds in many different substrates. While TRX is important as a defense against oxidative stress, given the presence of thiol groups in several signaling proteins, TRX also acts to regulate important cellular processes, including cell survival and growth (Liu & Min 2002, Lee et al. 2013). When TRX is oxidized, TXNRD, which are FAD-containing pyridine nucleotide disulfide oxidoreductases, catalyze TRX reduction using the reduction equivalents provided by NADPH. We currently observed that the mRNA levels of the cytosolic TXNRD1 significantly increased (2.1-fold, P < 0.0001) at day 4 post-confluence, and remained at similar levels up to day 10 post-confluence (Fig. 5D). NADPH is required for the reduction of GSSG and oxidized TRX. We next measured the mRNA levels of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway, critical in the production of NADPH and of precursors of DNA and RNA synthesis. We observed that G6PDH mRNA levels were significantly higher (35%, P = 0.024) 4 days post-confluence, decreasing to day 0 values at days 7 and 10 post-confluence (Fig. 5E). This pattern suggests that changes in G6PDH mRNA levels correspond to metabolic/cell function demands required for differentiation rather than for antioxidant protection.

Overall, these results suggest that during differentiation, the recycling of GSH and TRX to their reduced forms is more relevant than their synthesis. This is supported by: (i) previous findings of decreased GSH and no variations in TRX concentration throughout the differentiation of Caco-2 cells (Nkabyo et al. 2002), and (ii) the tight correlation observed between the mRNA levels of GSR1 and TXNRD1 (r = 0.91, P < 0.0001) (Fig. 5F).

Summary and conclusions

While Caco-2 cells have the advantage of proliferating and differentiating in vitro to mimic human enterocytes, they present the limitation of being derived from colon cancer origin. Current alternatives for in vitro studies of human enterocytes include the use of human organoids (Schutgens & Clevers 2020), which have limitations when studying transport across enterocyte monolayers, and planar cultures of human enterocytes, which require the availability of fresh intestinal tissue (Gomez-Martinez et al. 2022). Given this, differentiated Caco-2 cells are expected to continue being a widely used tool for basic and applied research. Thus, to be able to extrapolate findings in Caco-2 cells to those occurring in human enterocytes, it is essential to know that all the required components of the mechanism or event studied are present in the differentiated cell. The use of Caco-2 cells during the transition from proliferating cells to differentiated monolayers could be also considered a model of what occurs in vivo, from the bottom of the intestinal crypt where progenitor cells reside, through the migration and differentiation of cells into enterocytes toward the top of the crypt. Thus, this study provides relevant information on components of the redox network that characterize the differentiating Caco-2 cell. Several of the current observations support that mature Caco-2 cells are a good model to characterize redox-regulated events occurring in enterocytes.

Additionally, the observed changes in the pattern of expression of redox-related genes suggest that, in differentiating Caco-2 cells: (i) the redox balance plays a major role in the cell’s decisions to transition from proliferation to differentiation, (ii) differentiation prepares the enterocytes for their contact with the luminal environment, including the exposure to luminal pro-oxidant aggressors and their interactions with the microbiota to sustain immune system homeostasis.

Declaration of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. PI Oteiza is a member of the editorial board for Redox Experimental Medicine and was not involved in the peer review of this manuscript.

Funding

This work was supported by NIFA-USDA (grant CA-D-NTR-2819-H) and an unrestricted gift from Brassica Protection Products, LLC. PIO is a correspondent researcher from CONICET, Argentina.

Data availability statement

The data underlying this article cannot be shared publicly due to intellectual property rights and potential misuse. The data will be shared on reasonable request to the corresponding author.

Author contribution statement

WZ: Conceptualization, investigation, formal analysis, and manuscript editing; EC: Conceptualization, formal analysis, and manuscript editing; PIO: Conceptualization, supervision, formal analysis, funding acquisition, and writing the original draft.

Acknowledgements

The graphical abstract was generated with BioRender.com.

References

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  • Figure 1

    Biomarkers of Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) Sucrase-isomaltase (SI) and (B) caudal type homeobox 2 (CDX2) mRNA levels were measured by qPCR. mRNA levels were normalized to β-actin as the housekeeping gene. (C) SI and (D) CDX2 protein levels were evaluated by Western blot. Bands were quantified and values were referred to HSC-70 levels (loading control). Results are shown as mean ± s.e. of three to four independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

  • Figure 2

    Expression of NADPH oxidases during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) NOX1, (B) NOX4, and (C) DUOX2 mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

  • Figure 3

    Expression of NRF2 and NRF2-regulated genes during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) NRF2, (B) NQO1, and (C) HMOX1 mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

  • Figure 4

    Expression of O2 .− and H2O2/lipid peroxides metabolizing proteins during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Materials and methods section. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A) Superoxide dismutase 1 (SOD1), (B) catalase, and (C) glutathione peroxidase 1 (GPX1) mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test).

  • Figure 5

    Expression of genes involved in the production and regeneration of GSH and TRX during Caco-2 cell differentiation into mature enterocytes. Caco-2 cells were cultured as described in Methods. Differentiation day 0 was considered the day that cells reached confluence. The cell culture medium was replaced every 2 days, and cells were collected on days 0, 1, 4, 7, and 10 post-confluence. (A, B) Cysteine ligase catalytic (GCLC, A) and modulatory (GCLM, B) subunits, (C) GSH reductase 1 (GSR1), (D) TRX reductase 1 (TXNRD1), and (E) glucose-6-phosphate dehydrogenase (G6PDH) mRNA levels were measured by qPCR, and values were normalized to β-actin as the housekeeping gene. Results are shown as mean ± s.e. of three to six independent experiments. *Significantly different compared to day 0 (P < 0.05, one-way ANOVA test). (F) Linear regression analysis between the mRNA levels of TXNRD1 and GSR1. The solid line represents the regression line and the dotted lines the 95% CI.

  • Angelis ID & & Turco L 2011 Caco-2 cells as a model for intestinal absorption. Current Protocols in Toxicology Unit20.6. (https://doi.org/10.1002/0471140856.tx2006s47)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Baird L & & Yamamoto M 2020 The molecular mechanisms regulating the KEAP1-NRF2 pathway. Molecular and Cellular Biology 40. (https://doi.org/10.1128/MCB.00099-20)

  • Bedard K & & Krause K-H 2007 The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87 245313. (https://doi.org/10.1152/physrev.00044.2005)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Buhrke T, Lengler I & & Lampen A 2011 Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2. Development, Growth and Differentiation 53 411426. (https://doi.org/10.1111/j.1440-169X.2011.01258.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Campbell NK, Fitzgerald HK & & Dunne A 2021 Regulation of inflammation by the antioxidant haem oxygenase 1. Nature Reviews. Immunology 21 411425. (https://doi.org/10.1038/s41577-020-00491-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chávez V, Mohri-Shiomi A & & Garsin DA 2009 2009 CE-Duox1/BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infection and Immunity 77 49834989. (https://doi.org/10.1128/IAI.00627-09)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Coant N, Ben Mkaddem S, Pedruzzi E, Guichard C, Tréton X, Ducroc R, Freund JN, Cazals-Hatem D, Bouhnik Y, Woerther PL, et al.2010 NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon. Molecular and Cellular Biology 30 26362650. (https://doi.org/10.1128/MCB.01194-09)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cremonini E, Wang Z, Bettaieb A, Adamo AM, Daveri E, Mills DA, Kalanetra KM, Haj FG, Karakas S & & Oteiza PI 2018 (-)-Epicatechin protects the intestinal barrier from high fat diet-induced permeabilization: implications for steatosis and insulin resistance. Redox Biology 14 588599. (https://doi.org/10.1016/j.redox.2017.11.002)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Erlejman AG, Fraga CG & & Oteiza PI 2011 Caco-2 cells as a model to study the intestinal effects of non-absorbable phytochemicals. In Caco-2 cells and their uses. Ed. Schulz MA. New York: Nova Science Publishers Inc, pp. 116.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Esworthy RS, Swiderek KM, Ho YS & & Chu FF 1998 Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine. Biochimica et Biophysica Acta 1381 213226. (https://doi.org/10.1016/S0304-4165(9800032-4)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Forman HJ & & Zhang H 2021 Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nature Reviews. Drug Discovery 20 689709. (https://doi.org/10.1038/s41573-021-00233-1)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Geiszt M, Lekstrom K, Brenner S, Hewitt SM, Dana R, Malech HL & & Leto TL 2003 NAD(P)H oxidase 1, a product of differentiated colon epithelial cells, can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes. Journal of Immunology 171 299306. (https://doi.org/10.4049/jimmunol.171.1.299)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gomez-Martinez I, Bliton RJ, Breau KA, Czerwinski MJ, Williamson IA, Wen J, Rawls JF & & Magness ST 2022 A planar culture model of human absorptive enterocytes reveals metformin increases fatty acid oxidation and export. Cellular and Molecular Gastroenterology and Hepatology 14 409434. (https://doi.org/10.1016/j.jcmgh.2022.04.009)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gong P, Stewart D, Hu B, Vinson C & & Alam J 2002 Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite. Archives of Biochemistry and Biophysics 405 265274. (https://doi.org/10.1016/s0003-9861(0200404-6)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grasberger H, Gao J, Nagao-Kitamoto H, Kitamoto S, Zhang M, Kamada N, Eaton KA, El-Zaatari M, Shreiner AB, Merchant JL, et al.2015 Increased expression of DUOX2 is an epithelial response to mucosal dysbiosis required for immune homeostasis in mouse intestine. Gastroenterology 149 18491859. (https://doi.org/10.1053/j.gastro.2015.07.062)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grasberger H, Magis AT, Sheng E, Conomos MP, Zhang M, Garzotto LS, Hou G, Bishu S, Nagao-Kitamoto H, El-Zaatari M, et al.2021 DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk. Journal of Clinical Investigation 131. (https://doi.org/10.1172/JCI141676)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Greatorex S, Kaur S, Xirouchaki CE, Goh PK, Wiede F, Genders AJ, Tran M, Jia Y, Raajendiran A, Brown WA, et al.2023 Mitochondria- and NOX4-dependent antioxidant defense mitigates progression to nonalcoholic steatohepatitis in obesity. Journal of Clinical Investigation 134. (https://doi.org/10.1172/JCI162533)

    • PubMed
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