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. Author manuscript; available in PMC: 2022 Sep 3.
Published in final edited form as: Methods Enzymol. 2020 Apr 9;637:561–590. doi: 10.1016/bs.mie.2020.03.013

Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity

Reza Zolfaghari a,*, Floyd J Mattie a, Cheng-Hsin Wei a, David R Chisholm b, Andrew Whiting b, A Catharine Ross a,c
PMCID: PMC9440639  NIHMSID: NIHMS1832035  PMID: 32359660

Abstract

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.

1. Introduction

Retinoids refer to a class of chemicals, which are natural and synthetic derivatives of vitamin A. As a natural retinoid, all-trans retinoic acid (at-RA), the major active metabolite of vitamin A, functions as an endogenous ligand for three family members of the nuclear receptors, the retinoic acid receptors RARα, β, and γ, which dimerize with retinoid X receptors (RXRα, β, γ) and bind to specific DNA sites, known as retinoic acid response elements (RARE), typically located in the promoters of genes that are transcriptionally regulated by at-RA (Carrier & Rochette-Egly, 2015; Mendoza-Parra, Bourguet, de Lera, & Gronemeyer, 2015; Urban, Ye, & Davidson, 2015). These nuclear receptors are the key regulatory molecules, which are usually targeted by synthetic retinoids for combating the diseases such as cancers, immune disorders, and skin abnormalities (Altucci, Leibowitz, Ogilvie, de Lera, & Gronemeyer, 2007; Chien, 2018; de Thé & Fenaux, 2015; Spinella, Freemantle, & Dmitrovsky, 2015).

Of the various procedures for vitamin A analysis, almost all use chemical determination, principally using HPLC for separation through either reverse or normal phase columns, coupled to UV or electrochemical detection and/or mass spectrometry for quantification (Kane & Napoli, 2010). However, a few genetic tools using synthetically designed promoters containing RARE/RXRE elements have also been designed to analyze retinoid activity (Jurutka & Wagner, 2019; Lee, Tam, McCaffery, & Shum, 2019). Among numerous mammalian genes regulated by vitamin A, CYP26A1 has been shown to be the most highly inducible one that is regulated by at-RA as well as other retinoids (Isoherranen & Zhong, 2019; Ross & Zolfaghari, 2011). In this chapter, we describe how the native promoter of this human gene, modified for use in a reporter construct, is potentially useful in human cultured cells to determine the retinoid activities of novel synthetic retinoids. This tool may also be used as an alternative to estimate vitamin A activity of tissues including, in our examples, rat serum, liver and lung.

2. The promoter of CYP26A1 gene is highly responsive to retinoids in cultured cells

CYP26A1, a member of the CYP26 subfamily of cytochromeP450 enzymes, specifically catalyzes the oxidative inactivation of all-trans retinoic acid (at-RA) by converting it to more oxidized products. The CYP26A1 gene is highly responsive to the presence of at-RA as shown by the elevated expression of CYP26A1 mRNA during embryonic development as well as in retinoid-exposed adult tissues and cultured cells (Pennimpede et al., 2010; Ross & Zolfaghari, 2011). This induction is contributed mainly by the presence of four functional RARE elements contained in the full-length (FL) promoter, including one RARE in the region proximal to the transcription start site, and a cluster of 3 elements located in the distal region of the promoter further away from the proximate region of the transcription start site (Loudig, Maclean, Dore, Luu, & Petkovich, 2005; Zhang, Zolfaghari, & Ross, 2010) (see Fig. 1). By elimination of the intermediate region between these elements to bring the distal region closer to the proximate region, we created a construct made in a pGL3-basic-luc vector (a promoter-less vector with luciferase as the reporter gene), named as E4 (Fig. 1) (Zhang et al., 2010). This construct which was shown to have a higher response to at-RA as compared to the FL promoter when transiently cotransfected with RARα/RXRα into HepG2 cells, a human hepatoma cell line (Zolfaghari et al., 2019; Zolfaghari & Ross, 2014). We have tested this construct transiently in HepG2 cells for: (1) kinetics and dose response to at-RA; (2) response to retinol and RAR and RXR specific ligands; and (3) determination of the retinoid activity of novel synthetic retinoids.

Fig. 1.

Fig. 1

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Schematic representation of the full-length (FL) and deleted forms of human CYP26A1 gene promoter in pGL3-basic vector containing luciferase (E4) and mCherry (E4.2) as the reported genes. R1, R2, R3 and R4 represent retinoic acid response elements (RARE). TSS is the transcription start site.

2.1. Equipment

All equipment and reagents are specified by manufacturer/vendor as in the authors’ laboratory, but other comparable items may suffice.

  1. Nano-Drop instrument (Thermo)

  2. Step One Plus Real-Time Thermo-Cycler System (Applied Biosystem)

  3. Luminometer TD-20/20 (Promega)

  4. CO2-air incubator

2.2. Material

  1. Ethanol

  2. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich)

  3. All-trans-retinol (1 and 10mM in ethanol) (Sigma-Aldrich)

  4. All-trans-retinoic acid (at-RA) (1 and 10mM in ethanol) (Sigma-Aldrich)

  5. 9-Cis-retinoic acid (1mM in ethanol) (Sigma-Aldrich)

  6. LG-268, an RXR specific ligand (1mM in ethanol) (Sigma-Aldrich), Am580, an RARα specific ligand (1mM in ethanol) kindly provided to us by Dr. K. Shudo and Y. Kagechika (Tokyo Medical and Dental University, Tokyo, Japan)

  7. Synthetic retinoids including EC23, DC360, and DC324 (Fig. 2) were synthesized as reported (Chisholm et al., 2019) and prepared in at 1mM solution in DMSO

  8. Human hepatoma HepG2 cells (ATCC)

  9. Human embryonic kidney HEK293T cells (ATCC)

  10. Eagle’s minimum essential medium (EMEM) (ATCC)

  11. Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO)

  12. Heat inactivated fetal bovine serum (FBS) (GIBCO)

  13. Antibiotics (100 × penicillin-streptomycin) (ATCC)

  14. Phosphate-buffered saline (PBS) (Corning)

  15. Trypsin 0.25%-EDTA 2.2mM (Corning)

  16. pGL3-basic-luc (Promega)

  17. pRLTK vector (Promega)

  18. pcDNA3.1(+) (Invitrogen)

  19. Restriction enzymes including StuI, SmaI, and BstBI (NE Biolab)

  20. T4-DNA ligase (Promega)

  21. Trizol reagent (Invitrogen)

  22. Diethyl pyrocarbonate (DEPC), (Sigma)

  23. MMLV-Reverse transcriptase (Promega)

  24. OligodT nucleotide (Promega)

  25. dNTP (Promega)

  26. Real time PCR kit (Applied Biosystem)

  27. Lipofectamine 2000 (Invitrogen)

  28. DRL luciferase assay system including lysis buffer (Promega)

Fig. 2.

Fig. 2

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Structure of synthetic retinoids. EC23 and DC360 are synthetic analogues of at-RA, and DC324 is a non-active analogue that exhibits poor binding affinity for RARs due to the extended structure of the compound.

3. Methods

3.1. Cultured cells

HepG2 cells were grown in EMEM and HEK293T cells, human embryonic kidney cells, were maintained in DMEM, each supplemented with 10% FBS and 0.5% penicillin-streptomycin at 37°C in a 5% CO2-air incubator. The cells were plated and used at 60–70% confluency.

3.2. RNA isolation and analysis

Total RNA was extracted from the cells using Trizol reagent and then dissolved in DEPC-treated autoclaved water for analysis (Zolfaghari et al., 2019). The first stranded cDNA was synthesized from total RNA and then analyzed by real-time PCR (Zolfaghari et al., 2019) for CYP26A1 mRNA using primer pair of sense, 5’-GCTGCCTCTCTAACCTGCAC-3′, and antisense 5′-TGCTTTAGTGCCTGCATGTC-3′ and for 18S ribosomal RNA as an internal control using the primer pair of sense 5′-CGCGGTTCTATTTTGTTGGT-3′ and antisense, 5′-AGTCGGCATCGTTTATGGTC-3′. The PCR program was set to run first at 94°C for 10 min for activation of the polymerase and then 40 cycles of 20s at 94°C, 30s at 60°C, 30s at 72°C (Zolfaghari et al., 2019) using Step One Plus Real-Time PCR System.

3.3. Plasmid clones

The FL and E4 promoters of the CYP26A1 gene cloned in pGL3-basic-luciferase vector are reported previously (Zhang et al., 2010). Open reading frames of human RARα, RARβ, RARγ, and RXRα were synthesized from poly A+ RNA from human liver tissue sample and each was cloned into pcDNA3.1 as described (Zolfaghari et al., 2019). For construction of pcDNA3.1-hRARα-hRXRα expression vector, the pcDNA3.1-RAR was first double digested with StuI or SmaI/BstBI to remove the DNA reading frame for neomycin and replaced with ORF for RXRα as described (Zolfaghari et al., 2019). Clones created in this section have been deposited to the Addgene repository (Table 1).

Table 1.

Relevant plasmid clones deposited with Addgene.org.

Addgene ID Clone name
135397 pcDNA3.1-hRARα
135400 pcDNA3.1-hRXRα
135411 pcDNA3.1-hRARα.hRXRα
135415 pcDNA3.1-hRARβ.hRXRα
135416 pcDNA3.1-hRARγ.hRXRα
135478 pGL3-Basic-hCYP26AlP-E4.2-mCherry
135566 pGL3-Basic-hCYP26A1-FL-luciferase
135592 pGL3-Basic-hCYP26A1P-E4-luciferase
135910 pcDNA3.1-hRXRα
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3.4. Transient transfection and luciferase assay protocol

  1. Grow the cells in full growth medium with 10% FBS in T-75 flask at 37°C in 5% CO2-air incubator.

  2. Trypsinize and transfer the cells into 12- or 24-well plates 1–2 day before transfection when the cells are about 60–70% confluent.

  3. Aspirate out the medium and replace it with the same medium containing 3% FBS with no antibiotics about 1h before transfection.

  4. Prepare DNA samples as follow: For each well of the 24-well plates mix 0.7μg of FL or E4 promoter construct of the human CYP26A1 gene and pRLTK (7:1 ratio by weight), 0.1μg of pcDNA3.1-hRARα. hRXRα in 50μL EMEM or DMEM without FBS and antibiotics and for each well of the 12-well plate double the amount for each in 100μL of EMEM or DMEM without FBS and antibiotics. For cells without expression vector add pcDNA3.1 empty vector DNA.

  5. Mix 2μL of Lipofectamine-2000 in 50μL of EMEM or DMEM without FBS and antibiotics for each well of 24-well plate or 4μL of Lipofectamine-2000 in 100μL of EMEM or DMEM without FBS and antibiotics for each well of 12-well plate and incubate at room temperature for 5min.

  6. Mix DNA solution (Section 4) with diluted Lipofectamine-2000 (Section 5) and incubate at room temperature for 20min before adding to the cells.

  7. Add the solution prepared in the Section 6 to the cells, mix by swirling and incubate overnight at 37°C.

  8. Following transfection aspirate the medium and incubate the cells with the full-growth medium containing 10% FBS and 0–1μM at-RA, or retinol in ethanol as the vehicle or the synthetic retinoids in DMSO as the vehicle. The final concentration of either vehicle in the growth media was 0.001%.

  9. After incubation at 37°C for up to 24h (incubation time was chosen for convenience) wash the cells with PBS and then lyse to assay for Firefly- and Renilla-luciferase activities using the DRL luciferase assay system from Promega in Luminometer following the protocol from manufacturer.

  10. Promoter activity is defined as the ratio of Firefly- to Renilla-luciferase activity. Each reported activity is an average of 3 wells with standard error of the mean.

4. Results

4.1. RXRα enhances the effectiveness of RARα on the promoter activity of CYP26A1 gene

We have shown here that RXRα alone had no significant effect on the promoter activity of E4 promoter in HepG2 cells in response to at-RA; however, it increased the effectiveness of RARα by about twofold in response to at-RA (Fig. 3A). Based on this result, an expression vector containing the open reading frames for both RARα and RXRα (pcDNA3.1-RARα.RXRα) was constructed (Zolfaghari et al., 2019) and used for cotransfection with the promoter of CYP26A1 gene into HepG2 cells for a better response to at-RA treatment.

Fig. 3.

Fig. 3

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CYP26A1 gene promoter is highly responsive to at-RA in HepG2 cells. (A) HepG2 cells cotransfected with E4 construct together with an expression vector containing both RARα and RXRα were more responsive to at-RA (1nM) than with the expression vector containing only RARα (Zolfaghari et al., 2019). (B) Kinetic response to at-RA (1μM) of HepG2 cells cotransfected with the E4 promoter construct of hCYP26A1 gene without or with the RARα.RXRα expression vector. A similar pattern of kinetic with maximum response at 8h was observed when a lower concentration of at-RA, 100nM, was used. (C) Full-length (FL) or E4 promoter constructs of hCYP26A1 gene responded dose dependently to at-RA in HepG2 cells cotransfected with RARα.RXRα expression vector. (D) At all doses of at-RA, the E4 promoter responses were higher in the presence than the absence of FBS. (E) The response of the E4 promoter to a range of concentrations of at-RA was in parallel with the expression levels of endogenous CYP26A1 mRNA in HepG2 cells.

4.2. CYP26A1 gene E4 promoter response to at-RA is rapid and linear

In a kinetic experiment, the E4 promoter construct responded to at-RA within 2h (Fig. 3B), reached to maximum at 8h, and then decreased probably due to the at-RA induction of the expression of endogenous CYP26A1 which inactivates the at-RA untaken by the cells. Similar kinetic behavior was observed when lower at-RA concentration (100nM) was used (data not shown). The E4 promoter response was much higher than that of the full-length over different concentrations of the at-RA ranging from 10−10 to 103 M (Fig. 3C). In the logarithmic dose-response study, which encompassed up to 1μM of at-RA concentration, the E4 promoter response was nearly linear in the range of 0.1–10nM (Insert of Fig. 3C).

In a separate experiment we also did at-RA dose responses in the absence and presence of FBS. At zero concentration of at-RA the average promoter response was 3.44 ± 0.02 in the presence of FBS as compared to 1.889 ± 0.013 in the absence of FBS. At all doses of at-RA, the promoter responses were significantly higher in the presence than the absence of FBS (Fig. 3D).

The promoter response to at-RA might be higher if an effective inhibitor for CYP26A1 were used during incubation of the cells. However, we did not make any attempt to use any inhibitor because there is not any specific inhibitor for CYP26A1 available in the market and, in addition, we did not want to complicate the system by using a non-specific inhibitor.

The E4 promoter responses to different concentrations of at-RA were shown to be in parallel with the expression levels of endogenous CYP26A1 mRNA in HepG2 cells (Fig. 3E). Among three RAR subtypes, RARα and RARβ, compared to RARγ, were shown to have greater effect on the E4 promoter in response to at-RA in HepG2 cells as well as in HEK 293T cells (Zolfaghari et al., 2019).

4.3. CYP26A1 promoter responds to retinol (ROH) and to various retinoid analogues

The E4 promoter of CYP26A1 gene did not respond significantly to retinol treatment at or below a concentration of 10nM (Fig. 4A), however, at 100nM retinol the relative luciferase activity increased significantly to a level similar to that obtained by 1nM at-RA. It is not known whether retinol acts as a potential ligand for retinoic acid receptors, or if it needs to be oxidized first for formation of at-RA, although it has been reported that all-trans-retinol may act as a functional ligand for RARs (Repa, Hanson, & Clagett-Dame, 1993).

Fig. 4.

Fig. 4

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CYP26A1 promoter responds to retinol and to different retinoid analogues in HepG2 cells. (A) HepG2 cells cotransfected with E4 promoter construct together with hRARα.hRXRα expression vector were treated with either at-RA or retinol at different concentrations for 24h and then lysed for luciferase assay. The promoter responded to retinol but at higher concentrations than that of at-RA. (B) E4 promoter did not respond to RXR specific ligand alone unless it was used in combination with RAR specific ligands. (C) HepG2 cells grown in 24-well plates were cotransfected with either FL or (D) E4 promoter of human CYP26A1 promoter without or with retinoid receptors and then treated with vehicle or retinoid compounds including at-RA at 0.1μM in the medium for 24h after which the cells were collected for luciferase assay. (E) HepG2 cells cotransfected with E4 promoter together with hRARα.hRXRα were incubated with either at-RA or EC23 at different concentrations for 24h after which the cells were collected for luciferase activity assay. (F) HepG2 cells grown in 12-well plates and treated with either vehicle or retinoid compounds including at-RA at 3 different concentrations in triplicate for 4h and then collected for RNA analysis by RT-PCR. In the bar graphs, the relative expression of CYP26A1/18S ribosomal RNA level was set as 1.0 in HepG2 cells treated with vehicle only.

The promoter responded comparably to Am580, an RARα specific ligand (Fig. 4B) as well as 9-cis-RA (Zolfaghari et al., 2019), which binds to both RAR and RXR receptors. However, the promoter did not respond to LG-268, an RXR specific ligand, unless it was used in combination with at-RA or RAR specific ligands (Fig. 4B). Nuclear receptors such as RARs, TRs, and VDR are considered non-permissive receptors, which are enhanced by their own agonists but are not affected by RXR ligands (Gilardi & Desvergne, 2014). This is in contrast to the permissive action of RXR heterodimers, which can be affected by either RXR ligands and/or the ligands of the nuclear receptors such as PPARs, LXR, PXR, FXR and CAR (Gilardi & Desvergne, 2014).

The CYP26A1 FL (Fig. 4C) and E4 (Fig. 4D) promoters were both used to determine the retinoid activities of three novel synthetic retinoids, namely, EC23, DC360 and DC324. EC23 was shown to have higher activity than the other two retinoids, DC360 and DC324 (Fig. 4C and D). EC23 is also more effective than at-RA at low concentrations based on the data from promoter activity (Fig. 4E) as well as based on CYP26A1 gene expression in at-RA-treated HepG2 cells (Fig. 4F). Similar to the results obtained using at-RA (Fig. 3E), the promoter responses to the synthetic retinoids were shown to vary in parallel with the expression levels of endogenous CYP26A1 mRNA in HepG2 cells (Fig. 4C–F).

5. mCHERRY RFP may be used as a reporter for the promoter of the CYP26A1 gene

The E4 promoter response to at-RA levels as measured by luciferase activity assay in cells is relatively specific and sensitive, and, in addition, it is reproducible in repeated experiments for measurement of at-RA levels. However, for luciferase activity assay, the cells have to be lysed and processed using an expensive assay kit. To overcome this problem, we first substituted the luciferase open reading frame in our E4 promoter construct with mCherry cDNA, as the reporter gene (Fig. 1), which upon expression produces a red fluorescent protein that can be observed by fluorescence microscopy in live cells. We tested this construct, which we refer to as E4.2 (Fig. 1), to determine how it responds to at-RA in a transient transfection mode in HepG2 cells as well as in HEK293T cells.

5.1. Instruments

  1. Olympus IX70 inverted fluorescence microscope equipped with an Olympus DP72 camera and CellSens standard 1.14 image acquisition software, or a similar microscope

  2. NIH ImageJ 1.49 software for analysis

5.2. Material

  1. pCMV-YFP

6. Methods

6.1. Construction of E4.2 vector

A DNA fragment of the mCherry ORF was first amplified by PCR from a commercial plasmid vector using aaccatggTGAGCAAGGGCGAGG as sense and aatctagaTTACTTGTACAGCTCGTCCATG as antisense primer pairs, and then the fragment was cloned into pGEM-T Easy vector by TA-cloning as described (Zolfaghari et al., 2019). An isolated clone was subjected to DNA sequencing for confirmation and then double digested with NcoI/XbaI to obtain the mCherry fragment for subcloning into pGL3-Basic plasmid vector. For construction of pGL3-Basic-hCYP26A1P-E4-mCherry (E4.2 vector) clone containing the human CYP26A1 promoter, the pGL3-Basic-hCYP26A1P-E4-luciferase was first double digested with NcoI/XbaI to remove luciferase DNA fragment and then replaced with the mCherry ORF as described (Zolfaghari et al., 2019). The pGL3-Basic-hCYP26A1P-E4.2-mCherry clone created in this section has been deposited to the Addgene repository (Table 1).

6.2. Transient transfection and RFP analysis

  1. Grow monolayer cells with the full growth medium containing 10% FBS in 12-well plates 1–2day before transfection.

  2. When the cells are about 60–70% confluent aspirate out the medium and replace it with the same medium containing 3% fetal bovine serum with no antibiotics about 1h before transfection.

  3. Prepare DNA samples as follow: For each well of the 12-well plates mix 1.2μg of E4.2 and 0.2μg pYFP, 0.2μg of pcDNA3.1-hRARα.hRXRα in 100μL EMEM or DMEM without FBS and antibiotics. For the cells without pcDNA3.1-hRARα.hRXRα add pcDNA3.1 empty vector DNA.

  4. Mix 4μL of Lipofectamine-2000 in 100μL of EMEM or DMEM without FBS with no antibiotics for each well and incubate at room temperature for 5min.

  5. Mix DNA solution (Section 3) with diluted Lipofectamine-2000 (Section 4) and incubate at room temperature for 20min before adding to the cells.

  6. Add the solution prepared in the Section 5 to the cells, mix by swirling and incubate overnight at 37°C.

  7. Following transfection aspirate the medium and incubate the cells with the full-growth medium containing 10% fetal bovine serum and 0–1μM at-RA.

  8. Observe and evaluate the cells under fluorescence microscopy for expression of RFP in response to at-RA treatment and compare to the expression of YFP as an internal control for transfection efficiency.

  9. At least three regions of the monolayer cells in each well were evaluated and recorded. The RFP data were analyzed and quantified using NIH ImageJ 1.49d.

7. Results

7.1. Expression results of RFP in response to at-RA are comparable to those of luciferase activities in HepG2 cells

Expression of RFP was almost none (undetectable) in HepG2 cells treated with vehicle, however, treatment of these cells with as low as 0.1nM at-RA resulted in quantifiable expression of RPF in the cells as visualized by microscopy (Fig. 5). A gradual increase in the expression of RFP was observed in the cells treated with up to 10nM at-RA (Fig. 5), similar to that of luciferase used as the reporter gene (Fig. 3C). We also observed that both RARα and RARβ had stronger effects on RFP expression than RARγ at all the at-RA concentrations examined in HepG2 cells and in HEK293T cells, similar to those results obtained by luciferase assay (Zolfaghari et al., 2019).

Fig. 5.

Fig. 5

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E4.2 promoter of the CYP26A1 gene responds to at-RA dose-dependently for expression of RFP in HepG2 cells. Monolayer cells grown in 12-well plates were cotransfected with E4.2 promoter construct together with hRARα.hRXRα expression vector and pCMV-YFP as the internal control for transfection efficiency and then incubated with at-RA at different concentrations at 37°C for 6h. The live cells in three sections of each well were observed by microscopy for relative expression of RFP and YFP. Scale bar represents 200μm.

8. Permanent transfection of E4.2-CYP26A1 vector in HEK293T cells results in production of a suitable cell line for determination of retinoid activity

Based on the quantitative responses of the CYP26A1 promoter to at-RA in transient transfection mode, we decided to produce a permanent cell line possessing the CYP26A1 promoter with RFP, as a reporter gene (E4.2 Vector), which can be directly measured in live cells treated with retinoids without repeated transfection. For this, HEK293T cells were transfected with E4.2 vector and screened for selection of an appropriate cell line, which had to have low background for the expression of RFP in the absence of at-RA, while still responding to at-RA significantly upon treatment with receptor ligand. We selected HEK293T cells over HepG2 cells for several reasons: (1) these cells are easier to clone; (2) they grow rapidly and are easily trypsinized into individual cells for flow cytometry; (3) they have an apparently higher copy number of the reporter gene fused in the right places in the genome, and therefore a higher signal in response to retinoids; and (4) they tolerate high level of retinoids including at-RA. A monoclonal cell line, namely HEK293T 1A1 cells, was isolated and used to estimate the retinoid activities of novel synthetic retinoids as well as of extracts prepared from rat tissue samples, using both retinol and at-RA as the standard references for the assay.

8.1. Instruments

  1. Beckman Coulter MoFlo Astrios EQ Cell Sorter, or an equivalent sorter

  2. Accuri C6 flow cytometer (Accuri, BD Biosciences, San Jose, CA), or equivalent analytical equipment

8.2. Material

  1. Geneticin, G418 (Thermo)

9. Methods

9.1. Permanent transfection of HEK293T cells with E4.2 vector

Monolayers of HEK293T cells grown in 12-well plates were cotransfected with E4.2 vector and expression vectors for hRARα and hRXRα (see Section 6.2). After 2days, the cells were transferred and grown in a new 12-well plate with full growth medium containing 300μg/mL G418 as the selection agent. Following 2weeks, the live cells were transferred into a new plate and incubated with 1μM at-RA for 24h after which the cells expressing RFP were first cloned by limiting dilution, then sorted into individual cells using a Beckman Coulter MoFlo Astrios EQ Cell Sorter, with discrimination based on at-RA-induced RFP expression. The individual cells were grown in 96-well plates for 2weeks and then transferred into 12-well plates with full growth medium containing G418 (300μg/mL as above). The resulting monoclonal lines were assessed for expression of RFP after treatment with at-RA under a fluorescence microscope and a cell line, named HEK293T 1A1, was selected for further processing and analysis (Zolfaghari et al., 2019).

9.2. Treatment of HEK293T 1A1 cells with at-RA for RNA extraction

  1. Grow HEK293T 1A1 cells with full growth medium containing 10% FBS and 300μg/mL G418 in12-well plates for 1–2 days

  2. When the cells are 60–70% confluent aspirate the medium and replace with the fresh full growth medium with at-RA at different concentrations and incubate at 37°C for 24h.

  3. After observing the cells under fluorescence microscope for expression of RFP aspirate out the medium and wash the cells with PBS.

  4. Extract total RNA from the cells with Trizol reagent and analyze for expression of mCherry mRNA by RT-PCR as described on Section 3.2 using primer pairs for mCherry (see Section 6.1) and for18S ribosomal RNA as the control (see Section 3.2).

  5. Analyze the data using Step One Plus Real Time PCR System (see Section 3.2).

9.3. Treatment of HEK293T 1A1 cells with at-RA treated conditioned media from HepG2 cells for RFP expression analysis by flow Cytometry

  1. Treat HepG2 cells with at-RA at different concentrations in a 12-well plate for 24h and collect the medium and store frozen at −80°C.

  2. Grow monolayer HEK293T 1A1 cells in full growth medium containing 10% FBS and 300μg/mL G418 in 12-well plates and treat them with either the conditioned media from HepG2 cells (from Section 1) or at-RA as the reference standard at different concentrations for 24h.

  3. Aspirate out the medium and wash the cells with 1mL of PBS, add 150μL of trypsin-EDTA, and incubate at 37°C for 5min.

  4. Add 1mL of the full growth medium and pipet up and down to break the cells into individual ones.

  5. Transfer the cells into each 5-mL tube and wash the wells with 1mL more PBS and transfer to the tubes.

  6. Centrifuge the tubes at 1000 × g for 15min, aspirate out the medium and then resuspend the cells into 150μL PBS. Pipet up and down to disperse the cells.

  7. Analyze the cells with flow cytometer using wild type HEK293T cells treated with at-RA for 24h as the base.

10. Results

10.1. mCherry mRNA was expressed in HEK293T 1A1Cells proportionally in response to retinoic acid levels

HEK293T 1A1 cells were treated with at-RA at different concentrations for 24h and then collected for RNA extraction and analysis. Similar to the RFP signal, the mCherry mRNA level was almost undetectable when the cells were treated with vehicle, however, it was increased proportionally in response to at-RA concentrations (Fig. 6A).

Fig. 6.

Fig. 6

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Red fluorescent protein (RFP) is expressed quantitatively in HEK293T 1A1 cells in response to at-RA. (A) HEK293T 1A1 cells grown in 12-well plates were incubated with at-RA at different concentrations for 24h after which the cells were washed and collected for total RNA isolation. The RNA samples were then subjected to RT-PCR analysis for expression of mCherry RNA in triplicate using 18S ribosomal RNA expression as the internal control. (B) HEK293T 1A1 cells grown in 12-well plates were incubated with either at-RA at different concentrations or 24-h at-RA-treated conditioned media from HepG2 cells. For conditioned media preparation, HepG2 cells were incubated with at-RA at different concentrations in full growth media for 24h. The media were collected and then incubated at 50% with HEK293T 1A1 cells without addition of any at-RA. For reference standard, the HEK293T 1A1 cells were incubated with 50% HepG2 cell 24-h vehicle-treated conditioned media with added at-RA at different concentrations for 24h. The HEK293T 1A1 cells in duplicate wells were then trypsinized and collected for flow cytometry to quantify the cells expressing RFP.

10.2. Expression of RFP in HEK293T 1A1 cells could be measured by flow Cytometry

Monolayer cells grown in 12-well plates were incubated for 24h with either at-RA-treated conditioned media from HepG2 cells or an at-RA reference standard at concentrations from 1 to 500nM and then prepared for flow cytometry. RFP expression levels were linearly correlated in response to at-RA treatment (R2 =0.96, P <0.0001) (Fig. 6B). The conditioned media was as effective as the at-RA reference standard at the lower concentrations, however, they were more effective than the reference standards at the higher concentrations of at-RA. For example, RFP expression induced in the cells by the conditioned medium was about twice that induced by the at-RA reference standard alone at 500nM concentration (Fig. 6B). It should be mentioned that we found no particular by-product of at-RA in the conditioned medium as analyzed by Mass-Spectroscopy (data not shown).

10.3. HEK293T 1A1 cells could be used to measure the retinoid activity of synthetic retinoid analogues

Monolayer HEK293T 1A1 cells in 12-well plates were incubated with either synthetic retinoid analogues or at-RA, each at three different concentrations of 10, 100, and 1000nM for 24h and then analyzed by fluorescence microscopy for expression of RFP. The RFP expressing cells were counted and the relative fluorescence intensity levels were calculated based relative to the expressed RFP in cells treated with vehicle (Fig. 7A). The cells were significantly more responsive in term of RFP expression to EC23 than to at-RA at either 10 or 100nM (Fig. 7B and C). However, at 1000nM, EC23 was less effective than at-RA, probably indicating saturation of EC23 as a potent ligand for retinoic acid receptors. In fact, the cells responded to at-RA by the expression of RFP dose-dependently. Two other retinoid analogues, DC360 and DC324, were significantly less effective than at-RA in inducing expression of RFP in these cells (Fig. 7B and C).

Fig. 7.

Fig. 7

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HEK293T 1A1 cells responds to the synthesized retinoid analogues. (A) and (B) Monolayer HEK293T 1A1 cells in 12-well plates were incubated with either at-RA or retinoid analogues, each at different concentrations for 24h and then observed under a microscope for expression of RFP. (C) The RFP expressing cells were counted using the NIH ImageJ program and the relative fluorescence intensity levels were calculated based on the expression of RFP in vehicle-treated cells. Values represent the mean of 3 different regions of the well ± SD. Scale bar represents 200μm.

In a separate experiment, monolayer HEK293T 1A1 cells in 12-well plates were treated with either EC23 or at-RA at concentrations from 0 to 100nM for 24h, after which the cells were analyzed by fluorescence microscopy for RFP. EC23 was shown to be more effective than at-RA in inducing RFP expression in these cells at concentrations from 0.5 to 100nM (Fig. 8A and B). These results were comparable to those obtained in HepG2 cells transfected with CYP26A1 E4 promoter with luciferase as the reporter gene (Fig. 4E).

Fig. 8.

Fig. 8

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EC23 was shown to have more retinoid activity than at-RA at lower concentrations. (A) Monolayer HEK293T 1A1 cells in 12-well plates were incubated with either EC23 or at-RA, each at different concentrations for 24h and then observed under microscope for RFP expression. (B) The RFP expressing cells were counted using NIH ImageJ program and the relative fluorescence intensity levels were calculated based on the expressed RFP in cells treated with vehicle. The values represent the mean of 3 different regions of the well ± SD. Scale bar represents 200μm.

Compared to at-RA, these synthetic retinoid analogues were shown to exhibit a longer-lasting effect on RFP expression in HEK293T 1A1 cells, measured after treatment with different concentrations for either 1 or 4 days. The RFP signals in the cells treated with EC23, compared to at-RA, were higher in the 4-day treatment than in the 24-h treatment (Fig. 9A, B and E). Likewise, RFP signals were doubled in cells treated with either DC360 or DC324 for 4 days as compared to those in the cells treated for 24h (Fig. 9C–E).

Fig. 9.

Fig. 9

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Synthetic retinoid analogues have apparently more extended retinoid activity than at-RA in expression of RFP in HEK293T 1A1 cells. Monolayer HEK293T 1A1 cells grown in 12-well plates were incubated with either at-RA at 10 and 100nM (A), EC23 at 1 and 10nM concentrations (B), DC360 (C) or DC324 (D), each at 1000nM for either 1 or 4days and RFP expression was observed by fluorescence microscopy, and, (E) measured using NIH ImageJ. The values represent the mean of 3 different regions of the well ± SD. Scale bar represents 200μm.

at-RA is not only the inducer of CYP26A1 but also it is its specific substrate whereas EC23 and other synthetic retinoids used in this study are apparently considered as the inducers of CYP26A1 expression but are not the apparent substrates for CYP26A1.

11. CYP26A1 promoter can be used to estimate the retinoid activity levels in tissue samples

As an alternative to conventional methods we used CYP26A1 promoter to estimate the retinoid activity in tissue samples, using rat tissues as tests. For this, we treated rats orally with either vehicle as the control or at-RA and then euthanized the animals to collect blood, liver, and lung samples. Total retinoid activity was measured in serum and tissue extracts using the E4 plasmid construct in HepG2 cells for luciferase activity determination and HEK293T 1A1 cells for RFP analysis. As the reference standards, the cells were treated with either retinol or at-RA at different concentrations. For comparison to conventional method total retinol levels in tissue samples were measured by UPLC.

11.1. Instruments

  1. CO2 Tank

  2. N2 Tank

  3. Water bath

  4. Surgery tools

  5. Table top centrifuge

  6. Homogenizer

  7. Ultra Performance Liquid Chromatography (UPLC) (Waters), or a comparable instrument system

11.2. Material

  1. Potassium hydroxide (Sigma)

  2. Pyrogallol (Sigma)

  3. Hexane

  4. Trimethylmethoxyphenyl (TMMP)-retinol (Sigma)

  5. Methanol

  6. Acetonitrile

  7. Vegetable oil

12. Methods

12.1. Animal experiment

The protocol for animal use in this study was approved by The Animal Care and Use Committee of The Pennsylvania State University. Adult female rats were orally treated once with either vegetable oil or at-RA at 1mg, 2mg, or 5mg/kg body weight for 6h and then euthanized by CO2 inhalation. Blood was withdrawn and liver and lung tissue samples were snap frozen in liquid nitrogen immediately. Serum samples were prepared by centrifugation and stored frozen at −20°C while liver and lung samples were stored frozen at −80°C until use.

12.2. Retinoid extraction

Individual serum samples (0.1mL) were subjected to ethanol and then hexane extraction for total retinoid partition by UPLC (Zolfaghari & Ross, 2002). Part of the serum samples was used directly to incubate with the cells for retinoid activity determination. Liver and lung samples (about 0.5g) were homogenized in ethanol (1:10, w:v) and then saponified after which the samples were mixed with 12mL of hexane to partition the total free retinoids (Zolfaghari & Ross, 2000). A portion of the ethanol and hexane extract, 1mL from each, was evaporated to dryness under a light flow of N2 gas and immediately dissolved in 1mL of ethanol before dilution for incubation with cells for retinoid determination. Liver (>2000 times diluted) and lung (>80 times diluted) extracts were separately diluted before addition to the cell media. Retinol and at-RA at different concentrations were used as the reference standards. The hexane extract of the tissue samples was also subjected to UPLC for total free retinol determinations (Zolfaghari & Ross, 2000). We used this method because we assume most of the retinoids, if not entirely, in the tissues consists of retinol and retinyl esters.

13. Results

13.1. HEK293T 1A1 cells were used to estimate the retinoid activity levels in rat serum

HEK293T 1A1 cells were directly incubated with rat serum at a 20% dilution in DMEM media without FBS for 24h and then observed under fluorescence microscope for RFP expression; either exogenous retinol or at-RA was included in separate wells as reference standards, added in DMEM media with 20% FBS. As in the previous experiments, there was no significant expression of RFP in 1A1 cells treated with vehicle (Fig. 10A). Whereas HEK293T 1A1 cells responded effectively to at-RA in the expression of RFP at a concentration as low as 1nM, retinol did not have a significant effect on the expression of RFP at a concentration of 100nM (Fig. 10B and C). As compared to the at-RA reference standards, the control serum sample from the vehicle-treated rat was equivalent to ~2.5nM at the 20% serum dilution used (Fig. 10B and C). Then, serum samples from rat treated with 1, 2 and 5mg of at-RA/kg BW were tested. Serum (after 1mg at-RA/kg) increased the RFP expression slightly (Fig. 10B and C), however, serum from rats treated with 2 and 5mg at-RA/kg BW elevated the at-RA concentrations in the serum to about 30 and 180nM, respectively, as determined by RFP expression in HEK293T 1A1 cells (Fig. 10B and C). Total retinol concentrations of serum as determined by UPLC were all in the normal ranges of 0.98 for the control rat and 0.77, 0.74, and 1.74μM for rats treated with at-RA at 1, 2, and 5mg/kg body weight, respectively.

Fig. 10.

Fig. 10

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Total retinoid activity may be estimated in intact rat serum by HEK293T 1A1 cells. Adult female rats were treated orally with either oil as the vehicle control or at-RA at 1, 2, and 5mg/kg body weight for 6h and then sacrificed for blood, liver and lung tissue collection. Serum samples were prepared by centrifugation and stored at −20°C until analysis. Total serum retinol concentrations as determined by UPLC were all in the normal ranges of 0.98 for the control rat and 0.77, 0.74, and 1.74μM for rats treated with at-RA at 1, 2, and 5mg/kg body weight, respectively. (A) and (B) Cells were incubated with intact rat serum samples at 20% in DMEM media without added FBS for 24h, and then observed by fluorescence microscopy for expression of RFP using retinol and at-RA at different concentrations in DMEM media with added 20% FBS as the standards. (C) and (D) RFP expressing cells were counted using NIH ImageJ program and the relative fluorescence intensity levels were calculated based on the expressed RFP in cells treated with vehicle. The retinoid activity levels of the serum samples tested were calculated based on the at-RA standard. The values represent the mean of three different regions of the well ± SD. Scale bar represents 200μm.

13.2. Retinoid activity levels were estimated in tissue extract of rat liver and lung

We also estimated the retinoid activity levels in rat liver and lung samples using luciferase activity assay in HepG2 cells and RFP expression in HEK293T 1A1 cells. For this, total retinoids were extracted from liver and lung samples and then incubated with either HepG2 cells cotransfected with E4 plasmid vector and RARα.RXRα expression vector for luciferase expression or HEK293T 1A1 cells for RFP expression. Based on the retinol reference standards the total retinoid activity levels of liver and lung samples as measured by luciferase expression are comparable to those retinol levels determined by UPLC as the conventional method (Fig. 11). However, the estimated values as determined by RFP in HEK293T 1A1 cells were somehow higher than those determined by UPLC.

Fig. 11.

Fig. 11

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HEK293T cells 1A1 cells were used to estimate total retinol based on the retinoid activity in the rat liver and lung. Liver and lung samples (about 0.5g) from rats treated with either oil or at-RA for 6h (see Sections 12.1 and 12.2 and the legend of Fig. 10) were homogenized in ethanol (1:10 w:v) and saponified before addition of hexane (12mL) for partitioning. The hexane extract samples (2mL) were dried and dissolved in 1mL of ethanol which was then diluted (2000 times for liver extract and 80 times for lung extract) before incubation with HepG2 cells for luciferase assay and HEK293T 1A1 cells for RFP expression using retinol and at-RA at different concentrations as the reference standards. Part of the hexane extract samples was used for determination of total retinol by UPLC.

Although most, if not all, of the vitamin A contents in tissues including liver and lung are in the form of retinol and retinyl esters, we also determined the total retinoid activity levels based on at-RA as the reference standard in the liver and lung samples using luciferase activity in HepG2 cells (Table 2).

Table 2.

Retinoic acid activity levels in the liver and lung of rats.a

Rat no. Treatment of rat with at-RA (mg/Kg body weight) at-RA levels (nmol/g tissue)α
Liver Lung
1 0 189 ± 18 2.62 ± 0.11
2 1 109 ± 07 2.91 ± 0.23
3 2 148 ± 07 2.68 ± 0.30
4 5 197 ± 35 3.62 ± 0.53
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a

The hexane extract from the rat liver and lung samples (see Section 12.2) were dried and dissolved in ethanol before incubation with HepG2 cells for luciferase assay using at-RA at different concentrations as the reference standards (Mean ± SD).

Although both retinol and at-RA may be used as the standard references, we should be cautious that retinol is much less stable than retinoic acid. Whereas at-RA in either oil or ethanol is stable even after 2 years, retinol is not stable even after a few months when it is stored at −20°C. This is probably the reason that TMMP-retinol or retinyl acetate is used as the reference standard in UPLC (Zolfaghari & Ross, 2000). We have not tried either of these compounds in this culture cell system.

14. Summary

A short version of the promoter of the CYP26A1 gene driving the expression of either luciferase (E4) or RFP in a promoter-less vector (E4.2) was used to test its response to at-RA in HepG2 cells and HEK293T cells cotransfected with RARs. The promoter responded nearly linearly to a wide range of the log-10 concentrations of at-RA and was sensitive enough to detect at-RA activity as low as 0.5nM. The promoter responded better to at-RA when the cells were cotransfected with RARα.RXRα as compared to the other RARs. It responded to retinol and was used to estimate the retinoid activities of three synthetic retinoids. A stable cell line of HEK293T cells was established to express RFP under the CYP26A1 E4.2 promoter to respond to at-RA as well as to retinol. This cell line could be observed live for expression of RFP using fluorescence microscopy or trypsinized easily into individual cells for quantification of RFP expression using flow cytometry. This system was used to estimate the retinoid activities of synthetic retinoids as well as biological specimens such as serum and the retinoid extracts obtained from liver and lung.

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