- Email: [email protected]
The effects of steroid hormone exposure on direct gene regulation
Medical Hypotheses 83 (2014) 436–440
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
The effects of steroid hormone exposure on direct gene regulation q T.S. Wiley a, J.T. Haraldsen b,⇑ a b
Wiley Compounding Systems, Santa Fe, NM 87504, USA Department of Physics and Astronomy, James Madison University, Harrisonburg, VA 22802, USA
a r t i c l e
i n f o
Article history: Received 28 March 2014 Accepted 13 July 2014
a b s t r a c t Steroid hormones have been widely overlooked as controllers of gene expression. Through the mechanisms of gene expression (DNA methylation, histone methylation, and RNAi), we discuss the impact of normal reproductive templates on the pulsatility and amplitude of potential gene-regulating treatment protocols. By examining the interactions of estradiol (E2) and progesterone (P4) in women, we propose that changes in physiologic reproductive hormone templates of exposure and timing can affect fertility and even cancer through the silencing or amplification of gene products; such as P53 and Bcl-2 in women. We suggest that uncontrolled hormone levels, due to aging and/or the environment, may be restored to a normal youthful template of gene expression through the fluctuating exogenous application of E2 and P4 that mimic the normal hormonal milieu of reproductive health. Furthermore, we hypothesize that restoration of normal hormone levels may lead to a lower risk of the chronic illnesses of aging and a better quality of life in patients suffering those conditions. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Steroid hormones are critical to the natural development of multicellular organisms and complex cellular functions for all plants, animals, and insects [1–4]. Through the use of receptors and gene products, steroid hormones constantly control these regulatory systems using iterative hormone interactions [5,6]. Since many diseases can be traced back to the uncontrolled or silenced gene expression [7], understanding the mechanisms by which steroid hormones control or modify gene products is paramount for the medical community. This is evident in the well-known use of corticosteroids to reduce inflammation by silencing inflammatory gene cascades [8,9]. Since the expression of regulatory genes is critical for the sustainability of life through the production of amino acids, enzymes, and proteins [10], if there is an error in how and when specific genes are expressed, steroid hormones can either produce or reduce the levels of functionality down stream of all gene products throughout the body [7,11]. This leads to body-wide disarray in all the cells’ ability to regulate all gene expression, which has been observed in cancer patients as well as other instances of chronic illnesses in aging [12], such as heart disease, diabetes, and neurodegenerative states [13–17].
q
Funding source: Wiley Compounding Systems.
⇑ Corresponding author. Address: 901 Carrier Dr., Harrisonburg, VA 22802, USA. Tel.: +1 540 568 4173; fax: +1 540 568 2800. E-mail address: [email protected] (J.T. Haraldsen). http://dx.doi.org/10.1016/j.mehy.2014.07.010 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
Although gene expression is controlled through many different mechanisms in the body, the three most prominent and recognized mechanisms today are through DNA (deoxyribonucleic acid) methylation [18], histone methylation and acetylation [19], and RNA (ribonucleic acid) interference (RNAi) [20]. These mechanisms are critical for maintenance of gene function during the replication and transcription processes [18–20], where they control gene expression at different levels: DNA methylation controls genes through a direct tagging of specific cytosine bases on the DNA strand with a methyl group (CH3) [18]. Histone methylation and acetylation through chromatin action allows histones to reduce or increase gene expression by literally stopping the transcription process. This acts as a locking mechanism on the DNA strand [19]. RNAi is a completely different mechanism that involves the literal cutting and splicing of individual small pocket DNA strands (sDNA) to turn genes on and off or insert new material [20]. While these mechanisms differ in their methods, we show that all three of these mechanisms have a striking commonality: they are controlled primarily through interactions with steroid hormones. Since steroid hormones (i.e. estradiol (E2), progesterone (P4), testosterone (T), cortisol (C)) control many gene products [21–24], it is important to examine how the loss or disarray of this hormone product can be manifested as illness. By understanding the potentials of gene regulation within a cell, we can discuss the
T.S. Wiley, J.T. Haraldsen / Medical Hypotheses 83 (2014) 436–440
general mechanisms by which changing steroid hormone levels may have a distinct effect on the expression and repression of genes, which can in turn lead to an increased risk of the diseases of aging. In this paper, we examine the connection of hormone production and deterioration, from aging and/or environmental changes, to the gene regulation through the various methods (cited above) of gene silencing and amplification. Since steroid hormones control the cellular regulation of proliferation and apoptosis, it is apparent that the disruption of these hormones can systematically lead to diseases like diabetes, Alzheimer’s, and cancer through an increase or decrease via the methylation of DNA, uncontrolled histone modifications, and/or effects on the transcription of RNAi. Through an examination of specific hormonal processes, we elucidate the effects of aging and environmental cues on the production E2 and P4 that may ultimately disrupt the regulation of actual gene products. Through a detailed examination of the known literature in the field, we hypothesize that the pulsatility (exposure frequency) and amplitude (varying amounts) of steroid hormones will affect the gene expression. It is our intent to show how steroid hormones may be used to control these mechanisms of regulation through changes in exogenous dosing and timing of administration. Below, we provide a description of the steroid hormones, an overview of the gene expression mechanisms, and a discussion of the implications of pulsatility and amplitude in younger female reproductive templates, which varies significantly from the current standard of static nonvarying dosing of synthetic hormone-like pharmaceuticals. It has been long ignored that steroid hormones production is the response to the environment that has a direct affect on all three mechanisms. This makes steroid hormones likely candidates for study in genomics and epigenetics.
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peri-menopausal state preceding menopause. In this state, literature verifies there are significant increases in a woman’s risk of heart disease, diabetes, reproductive cancers, and osteoporosis [27]. It is the ripening of eggs that creates the standard estradiol cycle in the reproductive template, which produces a large peak of estrogen at approximately the 12th day of a woman’s cycle [32,33]. This surge of estrogen provokes a progesterone receptor from the nucleus of the cell as well as spikes the luteinizing hormone in the brain to instruct the ovary’s release of the egg. It is the ensuing degradation of the ruptured corpus luteum that begins the progesterone cycle (P4) at this time. P4 peaks on day 21 of this menstrual cycle. Should fertilization occur between days 19 and 22, the woman will continue to experience even higher levels of progesterone in the now established pregnancy. However, if fertilization does not occur, apoptosis begins, and the hormonal cues instruct the shedding of the uterine lining. This action of P4 blocks E2 receptors to assure repopulation on day 29/1 of the new cycle to restart the template. To understand how the time and amount of hormonal exposure affects the overall expression of genes, we discuss the cited mechanisms that control gene expression and how steroid hormones, especially estradiol, tie into these mechanisms. DNA methylation The methylation of DNA is a process in which a methyl group (CH3) bonds to the 5 position of cytosine (C) only [34]. This process is critical for the development of organisms due to its control over cellular differentiation and its ability to alter gene expression over evolution.
Steroid hormones Steroid hormones are involved in all genetic, cellular, and bodily processes [2–4]. They are growth and reproduction catalysts in bone, brain, heart, and reproductive organs [25]. The production of estrogen in the body of a young female is critical for the regulation of functional gene control [26]. Since estrogen, as a steroid hormone, affects gene expression [27], it is important to understand the possible methods by which steroid hormones act on these mechanisms. Steroids travel through the blood using lipid carriers. This is critical for protein production within the cell that will, in turn, create hormone receptors for various other steroids [28]. Once in a membrane receptor, the steroid will detach from the carrier and translocate through the cell membrane, where it can either directly access the nuclear promoter regions or bind to a non-nuclear receptor. Once the hormone-receptor complex has phosphorylated to the nucleus [29], it will bind to those appropriate specific hormone response elements and promoter regions of the DNA strand. The interaction between the steroid hormone-receptor complex and binding sites causes the DNA signals form messenger RNA (mRNA) transcription, which results in protein synthesis outside the nucleus, often producing other steroid and/or hormone receptors [30]. All cellular and bodily functions are affected by the production of these proteins, which means that the well being of a host is highly dictated by the amplitude and frequency of steroid hormones. The body is accustomed to a regular schedule for steroid hormone exposure [27]. This is clearly evident in the estrogen and progesterone cycles of females across all species. The female menstrual cycle is a complex interaction of estrogen and progesterone through fluctuating repetitive patterns over 28 days (shown in Fig. 1(a)) [31,32]. As a woman ages and her egg base declines, the normal patterns are disrupted and eroded, leading to an erratic
Fig. 1. (Upper Panel) The normal estradiol (black line) and progesterone (red/gray line) cycles throughout a women’s menstrual cycle [31]. The day 12 peak in estradiol correlates to increase in Bcl-2 and a decrease P53 gene products, while the day 21 peak in progesterone correlates to a decrease in Bcl-2 and an increase P53. These signal the proliferation and apoptosis of cells throughout the cycle. (Lower Panel) The E2 (squares) and P4 (circles) dosing template of the Wiley Protocol proposed to mimic the normal cycle using bio-identical hormone replacement therapy through transdermal application.
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The methylation occurs at cytosine sites that are connected to guanine (G) sites through the phosphate backbone, denoted as CpG [35], in the linear sequence of DNA bases. This is different than the hydrogen bonding that occurs between DNA base pairs (C–G and adenine (A)–thymine (T)). Methylation of non-CpG bases can occur, but has only been found, interestingly, in stem cells. The process of DNA methylation occurs by having DNA methyltransferases (DNMT1 and DNMT3) transfer methyl groups to DNA using S-adenosyl methionine as a donor (illustrated in Fig. 2), which is mitigated through the interactions of steroid hormones [18]. The percentage of methylated DNA in the body is known to vary widely in the first stages of life, but seems to stabilize quickly [35]. Once stabilized, enzymes move along DNA and maintain the level of methylated cytosine sites throughout reproductive life. This is evident through epigenetic silencing and amplification of various genes [36,37]. Over the past decade, it has been discovered that hypermethylation (increased methylation) or hypomethylation (decreased methylation) can occur, specifically in the later stages of life when hormone production and interactions decline in both men (testosterone) and women (estrogen). Hypomethylation has been linked to the over-expression of oncogenes in cancer cells. However, in the literature, the exact controlling mechanism still remains unclear. For example, in a 2001 paper by Hartsough et al. [38], a clear link between DNA methylation and the proliferation of cancer cells was discovered. This is an indication of the distinct importance of the need for understanding these mechanisms. For breast cancer patients, the loss of steroid hormones is critical in the silencing of crucial gene products [39,40]. The fact that the use of synthetic hormones does not alleviate this silencing factor is a note of great importance [41,42]. The question must be asked, do synthetic hormones affect these mechanisms of regulation in any normal way?
of DNA for transcription controlling the expression of the gene [19]. When DNA needs to be transcribed, the histones unravel, allowing the DNA transcription process to occur. In this way, histones play a critical role in gene regulation and expression. Steroid hormones can control gene activation through methylation of the histone (similar to DNA methylation) or through acetylation of the histone in which an acetyl group (CH3O) is attached (illustrated in Fig. 3). Therefore, the way steroid hormones control histone action is through the methyl and acetyl group [19]. The methylation and acetylation processes can stop the histone’s ability to unwind the DNA for transcription, which in turn stops gene expression. This steroid action effectively silences the histone itself and locks the DNA from transcription. When DNA wraps around a histone, that section of DNA becomes inaccessible and the gene is no longer active. Often, histone regulation is controlled through interactions with peptide hormones as well. However, since histone methylation is dependent on interactions with methyltransferases (similar to DNA methylation), steroid hormone interactions are critical [21].
RNA interference (RNAi)
Histones are proteins that affect the compaction and contraction of DNA using tightly wound bundles called nucleosomes. Histones, through chromatin, control the winding and unwinding
RNA interference (RNAi) is the inactivation of genes during the transcription process [43,44]. This occurs when microRNA (miRNA) and small interfering RNA (siRNA) bind to sections of messenger RNA (mRNA) and stop the production of the gene products, proteins and enzymes. The siRNA, which is can be as small as 22 nucleotides, are cut from longer double stranded RNA (dsRNA) by endoribonuclease enzymes called Dicers. RNA silencing is a critical process in eukaryotic cells (plants, animals, and fungi) [45–47], because it can easily stop or enhance the production of vital gene products for functional or even evolutionary purposes. The silencing of gene products through RNAi is critical for an organism’s protection against viral infections or even cancer. The under- or over-expression of various gene products creates an instability in the cell. For more information on RNAi mechanisms, please refer to Ref. [48].
Fig. 2. General DNA Methylation mechanism. The process of DNA methyltransferases (DNMT1 and DNMT3) transferring methyl groups to DNA using S-adenosyl methionine as a donor, which is mitigated through the interactions of steroid hormones.
Fig. 3. Histones methylation and the activation of DNA. Histones methylate through histone methyltransferases using the S-adenosyl methionine as a donor, which places methyl groups on the H3 and H4 histones. Acetylation typically uses an interaction with acetyl-coenzyme A [19].
Histones
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RNAi, while capable of silencing and voicing genes, is also [48,49], interestingly, instrumental in creating steroid production. These steroids may methylate by histones and regulate gene protein production in endless cascades. Hypothesis It is our hypothesis that gene expression is controlled, not just by the sheer presence of steroid hormones in the blood, but also by the frequency and amplitude of hormone production within the body. Steroid hormones are known to control the cell’s regulatory systems through modification of gene expression. It is also well known that all steroid hormones follow consistent rhythmic patterns of production driven by environmental cues. When these templates are disrupted by aging, pharmaceutical, or environmental interference, they may shutdown or reverse gene expression. We further hypothesize that this may lead to the diseases of cancer, heart disease, diabetes, or osteoporosis in aging. Below, we elucidate by example some of these effects of unregulated hormones, specifically in women (estradiol and progesterone). We also propose a basic genomic experiment that will confirm how the pulsatility and amplitude of hormone dosing can affect the activation of specific regulatory gene products such as Bcl-2 and P53. It is our belief that the deviation from the normal rhythmic template of exposure to steroid hormones in men and women can cause a regulatory chaos on the cellular level via the three mechanisms outlined above, which manifests itself in many ways, including breast cancer and other peri-menopausal symptoms. Because of the nature of steroid hormones, static dosing of synthetic hormone-like drugs (the standard of care in hormone replacement therapy) does not take into account natural rhythmic production, which provide cells with the proper signaling to produce gene products necessary for homeostasis. Discussion: genomic consequences of eroded estrogen cycles Since steroid hormones are critical to gene regulation, we focused on just estrogen as an example of the major consequences of eroded hormone cycles. The menstrual cycle in women produces greatly varying levels (amplitudes) of hormones (specifically estrogen and progesterone) in a pulsating fashion over 28 days. These hormones affect gene products through methylation of DNA or histones or through gene silencing via RNAi (Figs. 2 and 3). Since the onset of peri-menopause signals wild fluctuations in estrogen production, it is reasonable to assume that the ensuing control of gene expression also deeply affected adversely (shown in Fig. 4). The normal cycles of estrogen and progesterone manifest predictable regulatory gene expression through the repression or expression of genes like P53 and Bcl-2 and their gene products, which are timed throughout the cycle as well as initiating cascades of other functional gene products. As shown in Fig. 1(a), the normal cycle consists of an estrogen peak on day 12. This produces an increase in Bcl-2 (") until day 12 and a decrease in P53 (;). On day 21, the progesterone peaks and commands the cells to begin apoptosis and signals a decrease in Bcl-2 (;) by blocking E2 and an increase in P53 ("). This allows for regulation and control of the increase and decrease of cells all over the body. The hormone cycle signals cells to proliferate and when apoptosis should occur for normal functioning. As a woman enters perimenopause, these signals can be interrupted due to the body’s inability to produce normal estrogen and progesterone peaks. Therefore, the body’s clinical response is one of chaos, which produces hot flashes, mood swings, irritability, and loss of libido. However, physically the signals regulating proliferation and apoptosis have also deteriorated, which can lead to osteoporosis or uncontrolled cell growth, known as cancer.
Fig. 4. An illustration of the estrogen in urine (black solid line) and relative cancer rate (red/gray dashed line) as a function of age [52]. As age increases and a woman’s estrogen level begins to decrease, her relative cancer risk begins to increase as gene expression becomes irregular.
Studies show that static replacement of hormones is unsuccessful in the long term in helping women with symptoms, as well as ameliorating disease in aging. These studies have also shown that static dosing can acutely increase cardiac risk. Therefore, we conjecture that by following the body’s normal and evolved rhythmic production template with bio-mimetic, bio-identical therapy, the body may regain the normal expression of gene products that may defend against cell abnormalities. We refer to the only bio-mimetic, bio-identical hormone replacement therapy available (The Wiley ProtocolÒ) [50], because it is such a regimen that takes these normal templates of reproductive fitness into account. This re-establishment of hormone production should control gene expression as well. Shown in Fig. 1(b), the Wiley ProtocolÒ provides women with transdermal hormone therapy that consists of estrogen and progesterone provided topically. Recent unpublished studies of this regimen have demonstrated that women experience an increase in sleep, energy, bone growth, libido, and overall quality of life with no increase in cancer incidence [51]. Further clinical investigation is warranted. Proposed clinical experiment To examine how hormone regulation will control gene expression, we intend to examine the activation of gene products occur in various groups of women (young, peri-menopausal, post-menopausal) on different degrees of hormone replacement therapies (static, bio-mimetic, and none). The examination of various groups of women divided into youth, post-menopausal, and Wiley ProtocolÒ will be subject to blood spot gene identification on days 12 and 21 of the normal cycle for young and Wiley ProtocolÒ women. Post-menopausal women will follow chosen cycle days 12 and 21. Using the blood spot gene identification, we can examine the expression of P53 and Bcl-2 for comparison to the expression in normally reproductive subjects. In particular, we expect that one should specifically look at the expression and repression of P53 and Bcl-2. Conclusion Overall, we propose a hypothesis that steroid hormones affect gene expression through changes in both amplitude and frequency of dosing or exposure. A key example of this would be through the changing of hormones in women through menopause. Advances in hormone replacement therapy provide a wide range of testable
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parameters for this claim. We suggest the investigation of the expression of P53 and Bcl-2 in both peri- and post-menopausal women. Conflicts of interest statement The authors declare no conflicts of interest. All work was sponsored by Wiley Compounding Systems. Acknowledgments We would like to thank A. Raden for her assistance on the biomimetic hormone levels and J. Raden for useful discussions. References [1] Geuns JMC. Steroid hormones and plant growth and development. Phytochemistry 1978;17(1):1–14. [2] Beyer C. Estrogen and the developing mammalian brain. Anat Embryol 1999;199(5):379–90. [3] Harris GW. Hormonal differentiation of the developing central nervous system with respect to patterns of endocrine function. Philos Trans R Soc Lond B Biol Sci 1970;259:165–77. [4] Dubrovsky EB. Hormonal cross talk in insect development. Trends Endocrinol Metab 2005 Jan;16(1):6–11. [5] Formby B, Wiley TS. Progesterone inhibits growth and induces apoptosis in breast cancer cells: inverse effects on Bcl-2 and p53. Ann Clin Lab Sci 1998;28(6):360–9. [6] Evans RM. The steroid and thyroid hormone receptor superfamily. Science 1988 May 13;240(4854):889–95. [7] Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 2006;313(5795):1929–35. [8] Stellato C. Post-transcriptional and nongenomic effects of glucocorticoids. Proc Am Thorac Soc 2004;1:255–63. [9] Barnes PJ. How corticosteroids control inflammation: quintiles prize lecture 2005. Br J Pharmacol 2006;148(3):245–54. [10] Zhang X, Ho S-M. Epigenetics meets endocrinology. J Mol Endocrinol 2011;46(1):R11–32. [11] Ringold GM. Steroid hormone regulation of gene expression. Annu Rev Pharmacol Toxicol 1985;25(1):529–66. [12] Münzer T, Harman SM, Sorkin JD, Blackman MR. Growth hormone and sex steroid effects on serum glucose, insulin, and lipid concentrations in healthy older women and men. J Clin Endocrinol Metab 2009;94(10):3833–41. [13] Hansen M, Hsu A-L, Dillin A, Kenyon C. New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLoS Genet 2005 Jul 25;1(1):e17. [14] Jiang N, Du G, Tobias E, Wood JG, Whitaker R, Neretti N, et al. Dietary and genetic effects on age-related loss of gene silencing reveal epigenetic plasticity of chromatin repression during aging. Aging 2013;5(11):813–24. [15] Iacopino AM, Christakos S. Specific reduction of calcium-binding protein (28kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. PNAS 1990 Jun 1;87(11):4078–82. [16] Selvi RB, Kundu TK. Reversible acetylation of chromatin: Implication in regulation of gene expression, disease and therapeutics. Biotechnol J 2009;4(3):375–90. [17] Rahman K. Studies on free radicals, antioxidants, and co-factors. Clin Interv Aging 2007;2(2):219–36. [18] Das PM, Singal R. DNA Methylation and cancer. JCO 2004;22(22):4632–42. [19] Karlic´ R, Chung H-R, Lasserre J, Vlahovicˇek K, Vingron M. Histone modification levels are predictive for gene expression. PNAS 2010 Feb 16;107(7):2926–31. [20] Hannon GJ. RNA interference. Nature 2002 Jul 11;418(6894):244–51. [21] Ceschin DG, Walia M, Wenk SS, Duboé C, Gaudon C, Xiao Y, et al. Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin. Genes Dev 2011 Jun 1;25(11):1132–46. [22] Liao X, Tang S, Thrasher JB, Griebling TL, Li B. Small-interfering RNA–induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther 2005;4(4):505–15.
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Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
The effects of steroid hormone exposure on direct gene regulation q T.S. Wiley a, J.T. Haraldsen b,⇑ a b
Wiley Compounding Systems, Santa Fe, NM 87504, USA Department of Physics and Astronomy, James Madison University, Harrisonburg, VA 22802, USA
a r t i c l e
i n f o
Article history: Received 28 March 2014 Accepted 13 July 2014
a b s t r a c t Steroid hormones have been widely overlooked as controllers of gene expression. Through the mechanisms of gene expression (DNA methylation, histone methylation, and RNAi), we discuss the impact of normal reproductive templates on the pulsatility and amplitude of potential gene-regulating treatment protocols. By examining the interactions of estradiol (E2) and progesterone (P4) in women, we propose that changes in physiologic reproductive hormone templates of exposure and timing can affect fertility and even cancer through the silencing or amplification of gene products; such as P53 and Bcl-2 in women. We suggest that uncontrolled hormone levels, due to aging and/or the environment, may be restored to a normal youthful template of gene expression through the fluctuating exogenous application of E2 and P4 that mimic the normal hormonal milieu of reproductive health. Furthermore, we hypothesize that restoration of normal hormone levels may lead to a lower risk of the chronic illnesses of aging and a better quality of life in patients suffering those conditions. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Steroid hormones are critical to the natural development of multicellular organisms and complex cellular functions for all plants, animals, and insects [1–4]. Through the use of receptors and gene products, steroid hormones constantly control these regulatory systems using iterative hormone interactions [5,6]. Since many diseases can be traced back to the uncontrolled or silenced gene expression [7], understanding the mechanisms by which steroid hormones control or modify gene products is paramount for the medical community. This is evident in the well-known use of corticosteroids to reduce inflammation by silencing inflammatory gene cascades [8,9]. Since the expression of regulatory genes is critical for the sustainability of life through the production of amino acids, enzymes, and proteins [10], if there is an error in how and when specific genes are expressed, steroid hormones can either produce or reduce the levels of functionality down stream of all gene products throughout the body [7,11]. This leads to body-wide disarray in all the cells’ ability to regulate all gene expression, which has been observed in cancer patients as well as other instances of chronic illnesses in aging [12], such as heart disease, diabetes, and neurodegenerative states [13–17].
q
Funding source: Wiley Compounding Systems.
⇑ Corresponding author. Address: 901 Carrier Dr., Harrisonburg, VA 22802, USA. Tel.: +1 540 568 4173; fax: +1 540 568 2800. E-mail address: [email protected] (J.T. Haraldsen). http://dx.doi.org/10.1016/j.mehy.2014.07.010 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.
Although gene expression is controlled through many different mechanisms in the body, the three most prominent and recognized mechanisms today are through DNA (deoxyribonucleic acid) methylation [18], histone methylation and acetylation [19], and RNA (ribonucleic acid) interference (RNAi) [20]. These mechanisms are critical for maintenance of gene function during the replication and transcription processes [18–20], where they control gene expression at different levels: DNA methylation controls genes through a direct tagging of specific cytosine bases on the DNA strand with a methyl group (CH3) [18]. Histone methylation and acetylation through chromatin action allows histones to reduce or increase gene expression by literally stopping the transcription process. This acts as a locking mechanism on the DNA strand [19]. RNAi is a completely different mechanism that involves the literal cutting and splicing of individual small pocket DNA strands (sDNA) to turn genes on and off or insert new material [20]. While these mechanisms differ in their methods, we show that all three of these mechanisms have a striking commonality: they are controlled primarily through interactions with steroid hormones. Since steroid hormones (i.e. estradiol (E2), progesterone (P4), testosterone (T), cortisol (C)) control many gene products [21–24], it is important to examine how the loss or disarray of this hormone product can be manifested as illness. By understanding the potentials of gene regulation within a cell, we can discuss the
T.S. Wiley, J.T. Haraldsen / Medical Hypotheses 83 (2014) 436–440
general mechanisms by which changing steroid hormone levels may have a distinct effect on the expression and repression of genes, which can in turn lead to an increased risk of the diseases of aging. In this paper, we examine the connection of hormone production and deterioration, from aging and/or environmental changes, to the gene regulation through the various methods (cited above) of gene silencing and amplification. Since steroid hormones control the cellular regulation of proliferation and apoptosis, it is apparent that the disruption of these hormones can systematically lead to diseases like diabetes, Alzheimer’s, and cancer through an increase or decrease via the methylation of DNA, uncontrolled histone modifications, and/or effects on the transcription of RNAi. Through an examination of specific hormonal processes, we elucidate the effects of aging and environmental cues on the production E2 and P4 that may ultimately disrupt the regulation of actual gene products. Through a detailed examination of the known literature in the field, we hypothesize that the pulsatility (exposure frequency) and amplitude (varying amounts) of steroid hormones will affect the gene expression. It is our intent to show how steroid hormones may be used to control these mechanisms of regulation through changes in exogenous dosing and timing of administration. Below, we provide a description of the steroid hormones, an overview of the gene expression mechanisms, and a discussion of the implications of pulsatility and amplitude in younger female reproductive templates, which varies significantly from the current standard of static nonvarying dosing of synthetic hormone-like pharmaceuticals. It has been long ignored that steroid hormones production is the response to the environment that has a direct affect on all three mechanisms. This makes steroid hormones likely candidates for study in genomics and epigenetics.
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peri-menopausal state preceding menopause. In this state, literature verifies there are significant increases in a woman’s risk of heart disease, diabetes, reproductive cancers, and osteoporosis [27]. It is the ripening of eggs that creates the standard estradiol cycle in the reproductive template, which produces a large peak of estrogen at approximately the 12th day of a woman’s cycle [32,33]. This surge of estrogen provokes a progesterone receptor from the nucleus of the cell as well as spikes the luteinizing hormone in the brain to instruct the ovary’s release of the egg. It is the ensuing degradation of the ruptured corpus luteum that begins the progesterone cycle (P4) at this time. P4 peaks on day 21 of this menstrual cycle. Should fertilization occur between days 19 and 22, the woman will continue to experience even higher levels of progesterone in the now established pregnancy. However, if fertilization does not occur, apoptosis begins, and the hormonal cues instruct the shedding of the uterine lining. This action of P4 blocks E2 receptors to assure repopulation on day 29/1 of the new cycle to restart the template. To understand how the time and amount of hormonal exposure affects the overall expression of genes, we discuss the cited mechanisms that control gene expression and how steroid hormones, especially estradiol, tie into these mechanisms. DNA methylation The methylation of DNA is a process in which a methyl group (CH3) bonds to the 5 position of cytosine (C) only [34]. This process is critical for the development of organisms due to its control over cellular differentiation and its ability to alter gene expression over evolution.
Steroid hormones Steroid hormones are involved in all genetic, cellular, and bodily processes [2–4]. They are growth and reproduction catalysts in bone, brain, heart, and reproductive organs [25]. The production of estrogen in the body of a young female is critical for the regulation of functional gene control [26]. Since estrogen, as a steroid hormone, affects gene expression [27], it is important to understand the possible methods by which steroid hormones act on these mechanisms. Steroids travel through the blood using lipid carriers. This is critical for protein production within the cell that will, in turn, create hormone receptors for various other steroids [28]. Once in a membrane receptor, the steroid will detach from the carrier and translocate through the cell membrane, where it can either directly access the nuclear promoter regions or bind to a non-nuclear receptor. Once the hormone-receptor complex has phosphorylated to the nucleus [29], it will bind to those appropriate specific hormone response elements and promoter regions of the DNA strand. The interaction between the steroid hormone-receptor complex and binding sites causes the DNA signals form messenger RNA (mRNA) transcription, which results in protein synthesis outside the nucleus, often producing other steroid and/or hormone receptors [30]. All cellular and bodily functions are affected by the production of these proteins, which means that the well being of a host is highly dictated by the amplitude and frequency of steroid hormones. The body is accustomed to a regular schedule for steroid hormone exposure [27]. This is clearly evident in the estrogen and progesterone cycles of females across all species. The female menstrual cycle is a complex interaction of estrogen and progesterone through fluctuating repetitive patterns over 28 days (shown in Fig. 1(a)) [31,32]. As a woman ages and her egg base declines, the normal patterns are disrupted and eroded, leading to an erratic
Fig. 1. (Upper Panel) The normal estradiol (black line) and progesterone (red/gray line) cycles throughout a women’s menstrual cycle [31]. The day 12 peak in estradiol correlates to increase in Bcl-2 and a decrease P53 gene products, while the day 21 peak in progesterone correlates to a decrease in Bcl-2 and an increase P53. These signal the proliferation and apoptosis of cells throughout the cycle. (Lower Panel) The E2 (squares) and P4 (circles) dosing template of the Wiley Protocol proposed to mimic the normal cycle using bio-identical hormone replacement therapy through transdermal application.
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The methylation occurs at cytosine sites that are connected to guanine (G) sites through the phosphate backbone, denoted as CpG [35], in the linear sequence of DNA bases. This is different than the hydrogen bonding that occurs between DNA base pairs (C–G and adenine (A)–thymine (T)). Methylation of non-CpG bases can occur, but has only been found, interestingly, in stem cells. The process of DNA methylation occurs by having DNA methyltransferases (DNMT1 and DNMT3) transfer methyl groups to DNA using S-adenosyl methionine as a donor (illustrated in Fig. 2), which is mitigated through the interactions of steroid hormones [18]. The percentage of methylated DNA in the body is known to vary widely in the first stages of life, but seems to stabilize quickly [35]. Once stabilized, enzymes move along DNA and maintain the level of methylated cytosine sites throughout reproductive life. This is evident through epigenetic silencing and amplification of various genes [36,37]. Over the past decade, it has been discovered that hypermethylation (increased methylation) or hypomethylation (decreased methylation) can occur, specifically in the later stages of life when hormone production and interactions decline in both men (testosterone) and women (estrogen). Hypomethylation has been linked to the over-expression of oncogenes in cancer cells. However, in the literature, the exact controlling mechanism still remains unclear. For example, in a 2001 paper by Hartsough et al. [38], a clear link between DNA methylation and the proliferation of cancer cells was discovered. This is an indication of the distinct importance of the need for understanding these mechanisms. For breast cancer patients, the loss of steroid hormones is critical in the silencing of crucial gene products [39,40]. The fact that the use of synthetic hormones does not alleviate this silencing factor is a note of great importance [41,42]. The question must be asked, do synthetic hormones affect these mechanisms of regulation in any normal way?
of DNA for transcription controlling the expression of the gene [19]. When DNA needs to be transcribed, the histones unravel, allowing the DNA transcription process to occur. In this way, histones play a critical role in gene regulation and expression. Steroid hormones can control gene activation through methylation of the histone (similar to DNA methylation) or through acetylation of the histone in which an acetyl group (CH3O) is attached (illustrated in Fig. 3). Therefore, the way steroid hormones control histone action is through the methyl and acetyl group [19]. The methylation and acetylation processes can stop the histone’s ability to unwind the DNA for transcription, which in turn stops gene expression. This steroid action effectively silences the histone itself and locks the DNA from transcription. When DNA wraps around a histone, that section of DNA becomes inaccessible and the gene is no longer active. Often, histone regulation is controlled through interactions with peptide hormones as well. However, since histone methylation is dependent on interactions with methyltransferases (similar to DNA methylation), steroid hormone interactions are critical [21].
RNA interference (RNAi)
Histones are proteins that affect the compaction and contraction of DNA using tightly wound bundles called nucleosomes. Histones, through chromatin, control the winding and unwinding
RNA interference (RNAi) is the inactivation of genes during the transcription process [43,44]. This occurs when microRNA (miRNA) and small interfering RNA (siRNA) bind to sections of messenger RNA (mRNA) and stop the production of the gene products, proteins and enzymes. The siRNA, which is can be as small as 22 nucleotides, are cut from longer double stranded RNA (dsRNA) by endoribonuclease enzymes called Dicers. RNA silencing is a critical process in eukaryotic cells (plants, animals, and fungi) [45–47], because it can easily stop or enhance the production of vital gene products for functional or even evolutionary purposes. The silencing of gene products through RNAi is critical for an organism’s protection against viral infections or even cancer. The under- or over-expression of various gene products creates an instability in the cell. For more information on RNAi mechanisms, please refer to Ref. [48].
Fig. 2. General DNA Methylation mechanism. The process of DNA methyltransferases (DNMT1 and DNMT3) transferring methyl groups to DNA using S-adenosyl methionine as a donor, which is mitigated through the interactions of steroid hormones.
Fig. 3. Histones methylation and the activation of DNA. Histones methylate through histone methyltransferases using the S-adenosyl methionine as a donor, which places methyl groups on the H3 and H4 histones. Acetylation typically uses an interaction with acetyl-coenzyme A [19].
Histones
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RNAi, while capable of silencing and voicing genes, is also [48,49], interestingly, instrumental in creating steroid production. These steroids may methylate by histones and regulate gene protein production in endless cascades. Hypothesis It is our hypothesis that gene expression is controlled, not just by the sheer presence of steroid hormones in the blood, but also by the frequency and amplitude of hormone production within the body. Steroid hormones are known to control the cell’s regulatory systems through modification of gene expression. It is also well known that all steroid hormones follow consistent rhythmic patterns of production driven by environmental cues. When these templates are disrupted by aging, pharmaceutical, or environmental interference, they may shutdown or reverse gene expression. We further hypothesize that this may lead to the diseases of cancer, heart disease, diabetes, or osteoporosis in aging. Below, we elucidate by example some of these effects of unregulated hormones, specifically in women (estradiol and progesterone). We also propose a basic genomic experiment that will confirm how the pulsatility and amplitude of hormone dosing can affect the activation of specific regulatory gene products such as Bcl-2 and P53. It is our belief that the deviation from the normal rhythmic template of exposure to steroid hormones in men and women can cause a regulatory chaos on the cellular level via the three mechanisms outlined above, which manifests itself in many ways, including breast cancer and other peri-menopausal symptoms. Because of the nature of steroid hormones, static dosing of synthetic hormone-like drugs (the standard of care in hormone replacement therapy) does not take into account natural rhythmic production, which provide cells with the proper signaling to produce gene products necessary for homeostasis. Discussion: genomic consequences of eroded estrogen cycles Since steroid hormones are critical to gene regulation, we focused on just estrogen as an example of the major consequences of eroded hormone cycles. The menstrual cycle in women produces greatly varying levels (amplitudes) of hormones (specifically estrogen and progesterone) in a pulsating fashion over 28 days. These hormones affect gene products through methylation of DNA or histones or through gene silencing via RNAi (Figs. 2 and 3). Since the onset of peri-menopause signals wild fluctuations in estrogen production, it is reasonable to assume that the ensuing control of gene expression also deeply affected adversely (shown in Fig. 4). The normal cycles of estrogen and progesterone manifest predictable regulatory gene expression through the repression or expression of genes like P53 and Bcl-2 and their gene products, which are timed throughout the cycle as well as initiating cascades of other functional gene products. As shown in Fig. 1(a), the normal cycle consists of an estrogen peak on day 12. This produces an increase in Bcl-2 (") until day 12 and a decrease in P53 (;). On day 21, the progesterone peaks and commands the cells to begin apoptosis and signals a decrease in Bcl-2 (;) by blocking E2 and an increase in P53 ("). This allows for regulation and control of the increase and decrease of cells all over the body. The hormone cycle signals cells to proliferate and when apoptosis should occur for normal functioning. As a woman enters perimenopause, these signals can be interrupted due to the body’s inability to produce normal estrogen and progesterone peaks. Therefore, the body’s clinical response is one of chaos, which produces hot flashes, mood swings, irritability, and loss of libido. However, physically the signals regulating proliferation and apoptosis have also deteriorated, which can lead to osteoporosis or uncontrolled cell growth, known as cancer.
Fig. 4. An illustration of the estrogen in urine (black solid line) and relative cancer rate (red/gray dashed line) as a function of age [52]. As age increases and a woman’s estrogen level begins to decrease, her relative cancer risk begins to increase as gene expression becomes irregular.
Studies show that static replacement of hormones is unsuccessful in the long term in helping women with symptoms, as well as ameliorating disease in aging. These studies have also shown that static dosing can acutely increase cardiac risk. Therefore, we conjecture that by following the body’s normal and evolved rhythmic production template with bio-mimetic, bio-identical therapy, the body may regain the normal expression of gene products that may defend against cell abnormalities. We refer to the only bio-mimetic, bio-identical hormone replacement therapy available (The Wiley ProtocolÒ) [50], because it is such a regimen that takes these normal templates of reproductive fitness into account. This re-establishment of hormone production should control gene expression as well. Shown in Fig. 1(b), the Wiley ProtocolÒ provides women with transdermal hormone therapy that consists of estrogen and progesterone provided topically. Recent unpublished studies of this regimen have demonstrated that women experience an increase in sleep, energy, bone growth, libido, and overall quality of life with no increase in cancer incidence [51]. Further clinical investigation is warranted. Proposed clinical experiment To examine how hormone regulation will control gene expression, we intend to examine the activation of gene products occur in various groups of women (young, peri-menopausal, post-menopausal) on different degrees of hormone replacement therapies (static, bio-mimetic, and none). The examination of various groups of women divided into youth, post-menopausal, and Wiley ProtocolÒ will be subject to blood spot gene identification on days 12 and 21 of the normal cycle for young and Wiley ProtocolÒ women. Post-menopausal women will follow chosen cycle days 12 and 21. Using the blood spot gene identification, we can examine the expression of P53 and Bcl-2 for comparison to the expression in normally reproductive subjects. In particular, we expect that one should specifically look at the expression and repression of P53 and Bcl-2. Conclusion Overall, we propose a hypothesis that steroid hormones affect gene expression through changes in both amplitude and frequency of dosing or exposure. A key example of this would be through the changing of hormones in women through menopause. Advances in hormone replacement therapy provide a wide range of testable
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