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Keywords:

  • HaCaT;
  • keratinocytes;
  • regulation of gene expression;
  • retinoids

Abstract

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

Please cite this paper as: Retinal and retinol are potential regulators of gene expression in the keratinocyte cell line HaCaT. Experimental Dermatology 2010.

Abstract:  Vitamin A is a pivotal regulator of differentiation and growth of developing and adult skin. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes through retinoic acid nuclear receptors. Here, we present evidence that other vitamin A derivates – retinol and retinal – are also capable of functioning as regulators of gene expression in the keratinocyte cell line HaCaT. We have shown that all-trans retinol (ATRol) and all-trans retinal (ATRal) are capable of modulating gene expression in keratinocytes, which is not because of vitamin A metabolism in the cells, and retinol and retinal modulate gene expression through nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Based on the data, we propose that ATRol and all-trans retinal, in addition to all-trans retinoic acid, can function as important regulators of gene expression manifesting their effect through the nuclear receptors RARs and RXRs.


Abbreviations:
ATRA

all-trans retinoic acid

ATRal

all-trans retinal

ATRol

all-trans retinol

ChIP

chromatin immunoprecipitation

DR

direct repeats

RARE

retinoic acid response element

RARs

retinoic acid receptors

RXRs

retinoid X receptors

Background

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

At present, hundreds of genes are known to be regulated by all-trans retinoic acid (ATRA) (1). Two families of retinoid nuclear receptors have been identified: retinoic acid receptors (RAR) and retinoid X receptors (RXR), which are members of the steroid hormone receptor superfamily of ligand-activated transcriptional factors. The retinoid nuclear receptors interact with a specific element of different promoters, retinoic acid response element (RARE) that matches or closely relates to the AGGTCA sequence (2). Direct repeats (DR) of RARE with a spacer of two-five nucleotides (DR2-DR5) are known to be involved in the binding with homodimers of RXR, which is the combinatorial partner in the nuclear receptor family (3). Physiological actions of retinoids are manifested in RAR/RXR-mediated transactivation or transrepression (4). Also, retinoids are important for therapy of some malignancies (5,6). Recently, it has been shown that not only ATRA is an active form of vitamin A, but also all-trans retinal (ATRal) can play an important role in gene regulation (7). There is also evidence that all-trans retinol (ATRol) can indeed function as a ligand for the retinoic receptors, binding to RARα, β and γ (8). These data suggest that ATRol also may be a potential regulator of gene expression.

Questions addressed

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

This study aimed to determine whether ATRol and ATRal can function as regulators of gene transcription in the keratinocyte cell line HaCaT.

Results

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

We first performed a gene expression analysis of HaCaT cells after cultivation with ATRA, ATRal and ATRol using Illumina BeadChips technology. We found that ATRal and ATRol like ATRA are also capable to modulate gene expression (Fig. S1 and Table S1). We selected six genes, which are involved in keratinocyte physiology: KRT1, KRT5, DEFB, CYP26A, CYP26B, OAS. Real-time PCR confirmed that KRT1, KRT2 and DEFB are down-regulated, and CYP26A, CYP26B and OAS are up-regulated by ATRol and ATRal, which correlated with our chip data (Fig. S2a). Next, we analysed the expression of genes that are down-regulated (LGALS1) or up-regulated (CLCA4) only by ATRal and genes that are down-regulated (WNT4) or up-regulated (TNFAIP2) only by ATRol (Table S1). The expression of LGALS1 and CLCA4 could be modulated only by ATRal but neither by ATRol nor by ATRA. Modulation of the expression of WNT4 and TNFAIP2 was found only in case of ATRol treatment (Fig. S2b).

Cultivation of HaCaT cells with ATRol did not lead to the production of either ATRal or ATRA (Fig. 1a), thus the effect of ATRol on gene regulation is attributed to this retinoid itself. To inhibit the ATRal oxidation to ATRol, HaCaT cells were cultivated with citral, a well-known alcohol dehydrogenase inhibitor (9). Subsequent cultivation with retinoids and citral led to the inhibition of ATRol and ATRA production from ATRal (Fig. 1a). Real-time PCR analysis showed that cultivation either with ATRal or with ATRol led to comparable down-regulation of KRT1, KRT5 and DEFB and to up-regulation of CYP26A, CYP26B and OAS in the combined cultivation (Fig. 1b).

image

Figure 1.  Retinoid metabolism in HaCaT has no influence on gene regulation. (a) HaCaT cells were incubated in the dark for 24 h with all-trans retinol (ATRol) (10 μm), ATRal (10 μm) or without retinoids (co), with or without citral (50 μm) as indicated. Then, retinoid fraction was isolated (see Supporting Information), and HPLC analysis was performed (a representative elution profile from three independent experiments is presented). (b) HaCaT cells were cultivated in the dark for 24 h with ATRol (10 μm), ATRal (10 μm) or without retinoids (co), with or without citral (50 μm). Then, RNA was isolated, and relative expression was tested by real-time PCR with specific primers for KRT1, KRT5, DEFB, CYP26A, CYP26B and OAS. Data are present in means ± SEM. Three independent experiments performed in triplicates are depicted. *P < 0.05, significant differences between groups indicated with the lines.

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We proposed that the regulatory effects of ATRol and ATRal could be mediated through the retinoic acid nuclear receptor. We found expression of RARα, β and γ and RXRα and β, but not RXRγ (Fig. S3a). To regulate gene expression, ATRol and ATRal should be able to enter into the nucleus. After cultivation with ATRal for different time periods (1, 3 and 6 h), we detected both the retinoid itself and its derivates (ATRol and ATRA) in the cytoplasm (Fig. S3b and Table S2). In the nuclear fraction, only ATRol and ATRal were detected but not ATRA. Cultivation of the cells with ATRol led to the accumulation of the retinoid in both cytosolic and nuclear fractions. Next, we performed a competitive ELISA analysis using HaCaT nuclear protein extracts as a source of RARs and RXRs and an antibody against ATRA to quantify the amount of ATRA bound to the nuclear extract in the presence of ATRol or ATRal. We found that both ATRol and ATRal possess an equal potential to compete with ATRA for binding with nuclear proteins. Then, we performed a ChIP analysis with antibodies against RARα, β and γ and RXRα, β and γ using HaCaT cells before and after cultivation with the retinoids. Subsequent real-time PCR analysis was conducted using two primer sets selected for the promoter region of KRT1 containing RARE (Fig. S4). Antibodies against RARs precipitated DNA containing RARE in HaCaT cultivated with all the retinoids (except for RARγ with KRT13 primers). Antibodies against RXRs precipitated the DNA, only in the case of cultivation with ATRol and ATRal (except for RXRγ), but not by cultivation with ATRA (Fig. S4). Thus, ATRol and ATRal can really interact with RARs and RXRs. Finally, we incubated HaCaT cells with retinoids with or without 100 nm of Ro-41-5253 a selective RARα antagonist. This inhibitor abolished the effect of retinoids (Fig. S5a).

Finally, we wanted to understand whether these retinoids can bind to different DRs of RARE. For this purpose, we performed a capture-ELISA using double-strand sequences for DR1-5 (Table S3) and the nuclear extract from HaCaT. ATRol and ATRal promoted binding of RARs and RXRs to all DRs. ATRA did not show any binding in the case of RXRs, except for RXRα for DR1, RXRγ for DR2, RXRβ and RXRγ for DR3 and DR4, but the values measured were not significant in these cases (Fig. S5b and Table S3). Hence, ATRol and ATRal might be ligands not only for RXRs and RARs but also for other nuclear receptors, i.e. the peroxisome proliferator-activated receptor (DR1), thyroid hormone receptor (mainly DR4) and the vitamin D receptor (mainly DR3).

Conclusions

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

In this work, we have shown that (i) ATRol and ATRal are capable of modulating gene expression in the cell line; (ii) this modulation is not because of vitamin A metabolism in the cells; (iii) ATRol and ATRal may interact with nuclear receptors.

References

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Background
  4. Questions addressed
  5. Experimental design
  6. Results
  7. Conclusions
  8. References
  9. Supporting Information

Data S1. Materials and Methods.

Figure S1. Retinoids up- and down regulate gene expression in HaCaT.

Figure S2. Real-time PCR verification of up- and down-regulation of gene expression in keratinocytes by retinoids.

Figure S3. ATRol and ATRal can interact with RARs and RXRs.

Figure S4. ChIP analysis with antibodies against RARs and RXRs.

Figure S5. (a) Real-time PCR analysis of gene expression in HaCaT after retinoid treatment with or without a selective RARα antagonist Ro-41-5253. (b) RAR and RXR can interact with different DRs of RARE in the presence of ATRol and ATRal.

Table S1. Genes up- and down-regulated by ATRal, ATRol and ATRA in HaCaT cells.

Table S2. Distribution of retinoids in the cellular fractions of HaCaT.

Table S3. Binding of the RAR and RXR nuclear receptors with DNA DRs in the presence of retinoids.

Table S4. Primers used for conventional (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR).

FilenameFormatSizeDescription
EXD_1127_sm_FigureLegends.doc26KSupporting info item
EXD_1127_sm_fS1.tif218KSupporting info item
EXD_1127_sm_fS2.tif845KSupporting info item
EXD_1127_sm_fS3.tif1950KSupporting info item
EXD_1127_sm_fS4.tif2573KSupporting info item
EXD_1127_sm_fS5.tif3226KSupporting info item
EXD_1127_sm_SupportingMaterialandMethods.doc66KSupporting info item
EXD_1127_sm_tS1-4.doc422KSupporting info item

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