Elicitation of Rice Tissue Culture to Maximize Skin Epigenetic Modulation

Aug 24, 2011 | Contact Author | By: Philip Ludwig, Suellen Bennett and James V. Gruber, PhD, Arch Personal Care Products
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Title: Elicitation of Rice Tissue Culture to Maximize Skin Epigenetic Modulation
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ReGeniStem™ Red Rice (INCI: Ozonized Oryza Sativa (Rice) Callus Culture Extract) was produced by harnessing the power of undifferentiated plant cells through elicitation, as shown in Figure 1. Meristem cells of Himalayan red rice were cultured using the method detailed in Figure 2. These cells are grown in a bioreactor and subjected to an ozone stress, which causes them to produce secondary metabolites. Through an in vitro assay, rejuvenation was shown at the epigenetic level of treated cells. An increased expression of dermatopontin in keratinocytes, besides being a novel finding, implies that ReGeniStem™ Red Rice may be able to firm the skin and improve the skin barrier function.

Secondary Metabolites Induced by Ozone Stress

Plants grown in nutrient media often do not express important secondary plant metabolites because they are not threatened by damaging atmosphere elements, adverse soil conditions or pathogens or insects. However, it was found that certain media components, such as ozone and plant growth regulators, can cause plants grown in culture to express secondary metabolites.1-4 In order for the meristem cultures discussed here to produce secondary metabolites, ozone was chosen as the stressor, as it was the most effective at causing the culture to produce the most secondary metabolites as compared to other elicitors.

High performance liquid chromatography (HPLC) analysis was conducted on the meristem conditioned nutrient media exposed to ozone. Although only one peak has been identified at this time as biotin, multiple peaks were observed in the extract, demonstrating that exposure to ozone causes the production of secondary metabolites by the rice meristem culture. These differences are clearly seen in the chromatograms in Figure 3 and Figure 4.

DNA Methylation as a Type of Epigenetic Modification

Epigenetics is the study of heritable changes in gene expression that is caused by a mechanism other than changes to the DNA sequence. Epigenetic factors and changes have been shown to play an important part in cellular differentiation, development, aging and disease.5-7 DNA methylation in regards to epigenetic changes occurs at the cytosine-5 in CpG dinucleotides. The p represents the phosphate that links the cytosine and guanine together. This helps differentiate CpG from a CG base pair. Methylation of the CpG-rich promoter areas of genes has been identified as an essential mechanism in the regulation of gene transcription.7, 8 For humans, promoters tend to gain methylation with age. This trend can be seen when viewing the variation in methylation in regards to aging and environmental exposure to toxins such as exposure to smoking, arsenic or asbestos.9-14

Epigenetics can be thought of as a “youth switch.” The switch is turned on in young cells and turned off as cells age. DNA methylation could also be considered as a stop sign in gene promoters, as shown in Figure 5. Young healthy cells are thought to maintain their CpG sites in a low methylated state to allow transcription to occur. Less methylation is fewer stop signs to prevent the gene from being expressed. As aging occurs and DNA methylation drift increases the amount of DNA promoter methylation, the gene is expressed less causing lower protein production. Undifferentiated plant cells may help reverse promoter methylation. There may be various plant transcription factors, cellular components and homologous proteins and peptides that may be able to be applied to aged skin and help the skin cells to rejuvenate by decreasing promoter DNA methylation (see Figure 6). By profiling the CpG methylation differences in young and aged skin tissues, characterization of the role of aging related to methylation variation can be observed.

In Vitro Efficacy Studies

Global Decrease of CpG Methylation in Gene Promoter Regions: ReGeniStem™ Red Rice was evaluated using a DNA CpG methylation chip. The evaluation of global CpG methylated sites involved a two-step process wherein three sets of human dermal fibroblast cultures were intrinsically (cell culture passage) and extrinsically (UVB light exposure) aged, as pictured in Figure 7. These cells were divided into three groups: the first group (A) consisted of young cells that were untreated with no UVB (control), the second group (B) were old cells that were extrinsically and intrinsically aged but were not treated (control) and the third group (C) were cells that were extrinsically and intrinsically aged and treated with 2% ReGeniStem Red Rice™.

The resulting data consisted of three sets of approximately 250,000 CpG sites each. Since interest lied in the change of methylation at the promoter level, all CpG sites within all promoters were analyzed, as shown in Graph 1. As expected, there is an increase of global methylation at the promoter level when comparing young cells to old cells, both without ReGeniStem™ Red Rice. The application of 2% w/w ReGeniStem™ Red Rice to old cells had the ability to modulate the epigenetic environment and decrease the global level of promoter methylation, perhaps helping the old cells to function and perform as a younger, healthier cell.

CpG Methylation at Collagen 1A1 (COL1A1) Gene and Pro-collagen 1 Protein Synthesis: When COL1A1 was examined for its promoter methylation levels, the resulting data showed that treatment with 2% ReGeniStem™ Red Rice also reduces CpG methylation at the COL1A1 promoter region on treated aged cells, as depicted in Graph 2.

To correlate DNA demethylation to Collagen 1A synthesis, a protein assay was conducted on human fibroblasts treated with and without 2% w/w ReGeniStem™ Red Rice. The results shown in Graph 3 confirm that treatment with 2% ReGeniStem™ Red Rice increases collagen synthesis in cells when compared to old untreated cells.

Dermatopontin Expression in Keratinocytes: Dermatopontin is a protein found in the extracellular matrix of the dermis. It is known to have multiple and diverse functions in the cells ranging from accelerating collagen fibrillogenesis to fibroblast and neurogenic cell adhesion.15

Another study suggests that dermatopontin is responsible for epidermal adhesion of keratinocytes to the epidermis.16 The microarray assay conducted uncovered that this gene was upregulated in keratinocytes treated with ReGeniStem™ Red Rice, as shown in Graph 4. Dermatopontin protein expression in keratinocytes that were either untreated or treated with 1% or 2% w/w ReGeniStem™ Red Rice were then assayed. Dermatopontin was upregulated in the 2% treated tissue. This suggests that ReGeniStem™ Red Rice may be able to firm the skin and influence the skin barrier function especially in the dermal and epidermal layers where this protein is found. This finding that dermatopontin is expressed in keratinocytes and can be upregulated is also in itself a novel finding since scientific literature has not previously published that dermatopontin is expressed in keratinocytes.

Conclusion

Growing ReGeniStem™ Red Rice in bioreactors is a sustainable process that reduces residual biomass waste, but the benefits of this technology are not limited to environmental impact. Through elicitation of red rice meristematic cell cultures, appreciable quantities of secondary metabolites were produced in the culture. When the culture was tested on fibroblasts in vitro, a reverse in the age related increase of methylation in the promoter region of genes was shown.

This rejuvenation at the epigenetic level may have multiple benefits to the cells and to the skin. Some benefits include anti-aging, the renewal of gene and protein expression levels to that found in younger cells, such as was shown for collagen and the reversal of age related decreases in protein production. In essence, the skin, through an application of ReGeniStem™ Red Rice, is pushed to a younger, healthier state. An increased expression of dermatopontin in keratinocytes, besides being a novel finding, implies that ReGeniStem™ Red Rice may be able to firm the skin and improve the skin barrier function.

References
1. MH Zenk, The impact of plant cell culture on industry, in Frontiers of plant tissue culture, TA Thorpe, ed, Univ. of Calgary Printing Services Intl Assoc Plant Tissue Culture (1978) pp1–13
2. M Wink, Physiology of the accumulation of secondary metabolites with special reference to alkaloids, in Cell culture and somatic cell genetics of plants, F Constabel and IK Vasil, eds, 4 New York: Academic Press (1987) pp 17–42
3. Kurz WGW, Semicontinuous metabolite production through repeated elicitation of plant cell cultures: A novel process, in Plant Biotechnology, TJ Mabry, ed, Austin: IC2 Institute (1988) pp 93–103
4. R Alonso, S Elvira, FJ Castillo and BS Gimeno, Interactive effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis, Plant Cell Envirn 24(9) 905-916 (2001)
5. JE Cropley, CM Suter, KB Beckman and DI Martin, Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation, Proc Natl Acad Sci USA 103 17308–17312 (2006)
6. IC Weaver, MJ Meaney and M Szyf, Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood, Proc Natl Acad Sci USA 103 3480–3485 (2006)
7. VK Rakyan, S Chong, ME Champ, PC Cuthbert and HD  Morgan, Transgenerational inheritance of epigenetic states at the murine Axin (Fu) allele occurs after maternal and paternal transmission, Proc Natl Acad Sci USA 100 2538–2543 (2003)
8. R Metivier, R Gallais, C Tiffoche, PC Le and RZ Jurkowska, Cyclical DNA methylation of a transcriptionally active promoter, Nature 452 45–50 (2008)
9. A Bird, DNA methylation patterns and epigenetic memory, Genes Dev 16 6–21 (2002)
10. BC Christensen, JJ Godleski, CJ  Marsit, EA Houseman and CY Lopez-Fagundo, Asbestos exposure predicts cell cycle control gene promoter methylation in pleural mesothelioma, Carcinogenesis 29: 1555–1559 (2008)
11. CM Lyon, DM Klinge, KC Liechty, FD Gentry and TH March, Radiation-induced lung denocarcinoma is associated with increased frequency of genes inactivated by promoter hypermethylation, Radiat Res 168 409–414 (2007)
12. CJ Marsit, EA Houseman, AR Schned, MR Karagas and KT Kelsey, Promoter hypermethylation is associated with current smoking, age, gender and survival in bladder cancer, Carcinogenesis 28 1745–1751 (2007)
13. S Toyooka, R Maruyama, KO Toyooka, D McLerran and Z Feng,  Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer, Int J Cancer 103 153–160 (2003)
14. CJ Marsit, MD McClean, CS Furniss and KT Kelsey, Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma, Int J Cancer 119 1761–1766 (2006)
15. EG Forbes, AD Cronshaw, JR MacBeath and DJ Hulmes, Tyrosine-rich acidic matrix protein (TRAMP) is a tyrosine-sulphated and widely distributed protein of the extracellular matrix, FEBS Lett 351(3) 433-436 (1994)
16. O Okamoto, K Hozumi, F Katagiri, N Takahashi, H Sumiyoshi, N Matsuo, H Yoshioka, M Nomizu and S Fujiwara, Dermatopontin promotes epidermal keratinocyte adhesion via α3β1 integrin and a proteoglycan receptor, Biochemistry 49 147-155 (2010)

 

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Figure 1. Harnessing the power of undifferentiated plant cells through elicitation

Harnessing the power of undifferentiated plant cells through elicitation

ReGeniStem™ Red Rice (INCI: Ozonized Oryza Sativa (Rice) Callus Culture Extract) was produced by harnessing the power of undifferentiated plant cells through elicitation, as shown below.

Figure 2. Method of culturing meristem cells from Himalayan red rice

Figure 2. Method of culturing meristem cells from Hilalayan red rice

Meristems are taken from the shoot of the plant and transferred to solid culture media for callus growth. Calli of a certain size are then transferred to liquid culture media.

Figure 3. ReGeniStem™ Red Rice without ozone stress

Figure 3. ReGeniStem Red Rice without ozone stress

Although only one peak has been identified at this time as biotin, multiple peaks were observed in the extract, demonstrating that exposure to ozone causes the production of secondary metabolites by the rice meristem culture. These differences are clearly seen in the chromatograms in Figure 3 and Figure 4.

Figure 4. ReGeniStem™ Red Rice with ozone elicitation

Fiugre 4. ReGeniStem Red Rice with ozone elicitation

Although only one peak has been identified at this time as biotin, multiple peaks were observed in the extract, demonstrating that exposure to ozone causes the production of secondary metabolites by the rice meristem culture. These differences are clearly seen in the chromatograms in Figure 3 and Figure 4.

Figure 5. Young cells can be thought of as having their “youth switch” turned on

Figure 5. Young cells can be thought of as having their “youth switch” turned on

Young cells have low levels of CpG methylation in the promoter region. As cells age, the promoters become progressively more methylated resulting in diminishment of gene transcription.

Figure 6. Methylation in aging skin cells

Figure 6. Methylation in aging skin cells

As cells age, there is an increase in promoter methylation, leading to a decrease in gene transcription and protein expression. The application of elicited undifferentiated red rice meristem cultures may contain various cellular factors that can help modulate promoter DNA methylation and rejuvenate the cells.

Figure 7. ReGeniStem™ Red Rice on DNA methylation reduction

Figure 7. ReGeniStem Red Rice on DNA methylation reduction

Schematic diagram of the in vitro test methodology used to analyze ReGeniStem™ Red Rice’s (R3) effect on DNA methylation reduction

Graph 1. Average Global CpG methylation at the promoter region with ReGeniStem™ Red Rice treatment

Average Global CpG methylation at the promoter region with ReGeniStem Red Rice treatment

The resulting data consisted of three sets of approximately 250,000 CpG sites each. Since interest was in the change of methylation at the promoter level, all CpG sites within all promoters were analyzed, as shown below.

Graph 2. Average CpG methylation at the COL1A1 gene promoter

Graph 2. Average CpG methylation at the COL1A1 gene promoter

When COL1A1 was examined for its promoter methylation levels, the resulting data showed that treatment with 2% ReGeniStem™ Red Rice also reduces CpG methylation at the COL1A1 promoter region on treated aged cells, as depicted below.

Graph 3. Protein assay showing the effect of ReGeniStem™ Red Rice on collagen synthesis

Graph 3. Protein assay showing the effect of ReGeniStem Red Rice on collagen synthesis

The results, shown below, confirm that treatment with 2% ReGeniStem™ Red Rice increases collagen synthesis in cells when compared to old untreated cells.

Graph 4. Protein assay showing the effect of ReGeniStem™ Red Rice on dermatopontin synthesis

Graph 4. Protein assay showing the effect of ReGeniStem Red Rice on dermatopontin synthesis

The microarray assay conducted uncovered that this gene was upregulated in keratinocytes treated with ReGeniStem™ Red Rice, as shown below.

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