DHEA signalling in cell migration

The mechanism of DHEA signalling in cell migration

Abstract

Steroid hormones, including oestrogens, may influence cell migration during both wound healing and metastasis. More recently the adrenal precursor dehydroepiandrosterone (DHEA) has been shown to increase improve wound healing and cell migration1, 2. Using an established cell line and specific inhibitors (aromatase and steroid sulphatase inhibitors), this project will investigate the effect of DHEA, DHEA-S and oestradiol, alone or in combination, using the scratch wound assay. Migration will be measured over different time points. Single point assays in the presence of pertussis toxin or an ER inhibitor will be performed to determine whether changes in migration are modulated via a genomic or non-genomic pathway

Abbreviations

17β-Oestradiol 17β-(E2)

B-Cell CLL/Lymphoma 2 Bcl-2 proteins

Dehydroepiandrosterone DHEA

Dehydroepiandrosterone-sulphates DHEA-s

Dulbecco's Modified Eagle Medium DMEM

Fetal Calf Serum FCS Guanine nucleotide-binding protein G protein

Hormone replacement therapy HRT

Mitogen-Activated Protein Kinase MAPK Phosphate Buffered Saline PBS

Phosphatidylinositol 3 PI3

Introduction

1.1 DHEA and wound healing

Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal steroid hormone with immunomodulatory properties. DHEA and DHEA-sulphates (DHEA-s) are the most abundant steroid hormones in the bloodstream and healthy levels of DHEA allow a balance of the other body hormones. DHEA is known as the “The Mother Hormone”, as the body can convert DHEA into many different steroids it requires or may be lacking, provided the appropriate enzymes are present, to maintain a proper hormonal balance. DHEA and DHEA-S are synthesised in the adrenal cortex and released into the bloodstream in response to the hormone adrenocorticotropin, secreted by the anterior pituitary gland. DHEA and DHEA-S circulate at very high concentrations in the blood of young adults. Plasma DHEA-S levels in adult men and women are some 100-500 times higher than those of testosterone and 1,000-10,000 times higher than those of 17β-oestradiol, thus providing a large reservoir of substrate for conversion to androgens and/or oestrogens in peripheral intracrine tissues including the skin.

Wound healing is delayed and impaired in the elderly, particularly in males who heal wounds more slowly than females with reduced matrix deposition and an increased inflammatory response, which decrease with age. This suggests that circulating levels of sex hormones as well as cell ageing mechanisms contribute to impaired wound healing in the elderly. The significant declining levels of circulating DHEA and oestrogens may account for the delayed healing of cutaneous wounds in aged individuals. The elderly suffer from impaired healing of acute wounds, which are characterized by delayed closure, increased local inflammation, and excessive proteolytic activity, which can lead to chronic non healing wounds. The conversion of DHEA locally to downstream steroid hormones leads to oestrogenic and/or androgenic effects and this may be important in age-related skin homeostasis. Systemic DHEA levels have been reported to be strongly associated with the protection against chronic venous ulceration in humans. DHEA accelerated impaired healing in an impaired healing model (mice rendered hypogonadal) associated with increased matrix deposition and dampens the exaggerated inflammatory response. These effects were mediated by local conversions of DHEA to oestrogen, via the oestrogen receptor. In vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated protein kinase (MAPK) and phosphatidylinositol 3 (PI3) kinase pathways. Studies have shown that local injection of DHEA accelerates impaired healing in an ageing mouse colony. Suggestions have been made that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis and treatment of human impaired wound healing states and this is particularly interesting because mice don't produce DHEA.

Inactive adrenal precursors DHEA and DHEA-sulphate (DHEAS) synthesize a large amount of androgens locally in peripheral target tissues. Androgens regulate hair growth and sebum production and the skin is an important target organ for androgens and there is evidence from previous studies to suggest that androgens influence the wound healing process. From the in vitro studies and limited number of in vivo studies so far undertaken it appears that androgens influence all phases of wound healing from initial clot formation to long-term wound remodelling

Cutaneous wound healing is a complex process involving several overlapping phases. The are a number of steps involved in the re-establishment of the skin barrier leading up to wound healing. Firstly, hemostasis (blood clot formation) and inflammation occurs; the inflammatory response causes the proliferation and migration of dermal and epidermal cells to reestablish the skin barrier and to replace damaged tissue (re-epithelialisation) via wound closure; and finally tissue remodelling and differentiation for the recovery and restoration of the skin tissue. In this study, the secondary stage (re-epithelialisation) will be the main aim as we are interested in the rate of migration of keratinocytes in a mechanical wound in response to DHEA and DHEA-S.

1.2 DHEA in the presence of aromatase inhibitors (Arimidex)

Aromatase inhibitors (such as Arimidex) may block the beneficial effects DHEA has on cutaneous wound healing and this has been demonstrated on a mouse model but to date there have been no investigations into the effects of aromatase inhibitors on the cutaneous wound healing process in humans. Therefore, Post menopausal patients who take aromatase inhibitors as an adjunct to breast cancer therapy may be at increased risk of delayed wound healing. Administration of oestradiol and DHEA reverses age-related chronic wound healing impairment via hormone replacement therapy (HRT) in postmenopausal women significantly. The administration of 17β-oestradiol, either systemically or topically, has been shown to reverse the fundamental repair defects observed in postmenopausal women. By contrast, androgenic species retard repair and interfere with the accumulation of the structural proteins that reconstitute the damaged dermis.

1.3 DHEA and the skin

There are a number of layers of specialised epithelial cells, keratinocytes, that make up the human epidermis. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. DHEA has been shown to have a protective role against apoptosis in keratinocytes using non-cancerous immortalized human HaCaT cells. It does so by transmitting signals via specific G protein-coupled, membrane binding sites and inhibiting apoptosis, through prevention of mitochondrial disruption and altered balance of Bcl-2 proteins.

Human keratinocytes are the primary cells and is a major component of the epidermis, comprising 95% of the cells, they are specialised cells that synthesise keratin. The keratinocytes proliferation and migration can be mediated by the activation of the non-genomic pathway via the 17β-oestradiol isoforms.

The skin fibroblast tissues and human embryonic stem cells can also express both 17β-oestradiol receptors. Studies have suggested treatment with oestradiol can control differentiation of human embryonic stem cells into a series of cell types to induce gene expressions of those involved in differentiation. Two 17β-oestradiol pathways mechanisms exist. Firstly the classical (i.e genomic) pathway which is induced through ligand activated intracellular receptors in order to regulate transcription of genes via protein to protein directly interacting on the DNA and binding to specific co activators or repressors. Secondly the non-genomic pathway is mediated by signaling responses binding directly to oestradiol.

1.4 Cell migration and wound assay

Embryonic development, homeostasis and wound healing, immune responses, and cancer metastasis are examples of biological processes in which cell migration has a vital role. The wound healing assay is a simple, inexpensive method which allows studies of directional cell migration in vitro as this method mimics cell migration during wound healing in vivo. The in vitro method is only 2 dimensional and therefore 3 dimensional methods in vivo would be required to minimize any drawbacks to the method. This method involves creating a "wound" in a cell monolayer, in this case the keratinocyte monolayer, and capturing images at the beginning and at regular intervals during cell migration until the wound closes. These images are then compared to allow quantification of the migration rate of the cells.

1.5 Aims

The aim of this study was to investigate the effects of DHEA, DHEA-S and oestradiol, alone and in combination with specific inhibitors (aromatase and steroid sulphatase inhibitors) on the migration of cultured adult human keratinocyte cell line (NCTC), following in vitro mechanical wounding.

Materials and Methods

All materials and equipment used, together with the supplier and manufacturer information can be found in tables shown in Appendix 1.

2.1 The effect of DHEA on the migration of established human transformed cell line keratinocyte; NCTC 12544, following mechanical wounding in vitro

A scratch wound assay was used to assess established human transformed cell line keratinocyte; NCTC 12544 cell migration. Human keratinocytes; NCTC 12544 derived from the skin and provided by Dr. M.J Thornton (n=2x25cm2) were passaged and trypsinised EDTA to establish subcultures. Once the cells were confluent, the media was removed and fresh media was added to the flask. Haemocytometry was used to estimate the number of cells per suspension and the total number of viable cells present in the suspension was calculated as shown in Appendix 2.

The estimation of cell number was carried out by haemocytometry. The total number of viable cells present in the suspension was calculated; as shown in Appendix 2, table 2.

Cells were seeded into 35mm dishes at a density of 35,000 cells per dish. 12ml DMEM growth medium supplemented with 10% FBS (50 ml FBS with 500 ml DMEM), Penicillin and glutamine was added to each dish (2ml per well). The cells were incubated at 5% CO2 and 37°C until confluent, which took approximately 3 to 4 days, with a media change of 2% low serum media (10 ml FBS with 500 ml DMEM) every 2 to 3 days. The medium was then removed once the cells were ≥ 95 % confluent (check under microscope at *10 magnification) and the cells were washed two times with PBS to remove any debris. Once washed, the cells were mechanically wounded using a standard paperclip (Figure ). A template of the dish (Figure 13) was designed prior to wounding, and this template was used to scratch the cells along a standard diameter of the wells. The use of a paperclip was preferred over a pipette tip as the paperclips had a standard width which helped keep the wounds a constant size and they were firmer preventing any bending.

At time point 0 (t=0hr) the scratches were photographed using an inverted microscope. At time points 0, 3, 6, 24, 36, 48 and 72 hours and at varying concentration of 1nM, 10nM, 100nM and 1000nM, photographs of the distance between the wounded cell edges (migrating cells) were taken. Photographs of the central part of the wound were taken at each time point. The distance between the wound edges per well was measured using a standard template ( Figure ) and measuring how much of the wound had closed at 6 fixed points (1 cm apart) along the length of the wound. The average distance between the wound edges for each individual group at varying concentrations (derived from a total of 24 measurements) was calculated for individual time points and from this the mean migratory distance for each time point was calculated (Appendix 1). The mean migratory distance was then converted into a distance in micrometres (mm) as shown in Appendix 1. A graticule on the microscope would have been more convenient, unfortunately this could not be arranged by the laboratory staff.

2.2 Migration of human cell line keratinocytes NCTC 12544 , in the presence of 17-oestradiol

2.2.1 Dose response effects

Initial experiments (n=4) were carried out on human cell line keratinocytes NCTC 12544 derived from skin to compare the effects of a variety of concentrations of 17b-oestradiol (1nM, 10nM, 100nM and 1000nM). 2ml of phenol red-free, serum-free DMEM supplemented with either absolute ethanol vehicle control (0.0001%), 1nM 17b-oestradiol, 10nM 17b-oestradiol, 100nM 17b-oestradiol or 1000nM 17b-oestradiol was added to individual dishes in triplicate. Cell migration was assessed after fixed time points, as discussed in 2.1. A stock solution of 17b-oestradiol was required to be serial diluted to achieve the varying concentrations. Migrating cells were photographed as described in section 5.1.

5.4. Migration of epidermal fibroblasts, cells in the presence of serum and phenol red

In order to perform a quantitative analysis, the relative effects of serum-free, phenol red-free growth medium on the migration of Established cell line Keratinocytes; NCTC 12544 (n=2x25cm2), cells were prepared and wounded as described in section 5.1. Naturally 10% serum treatment on cells would influence wound healing response, so were cultured in serum deprived (serum-free) cells for 72 hours, phenol red-free growth medium or phenol red-free growth medium containing 10% FBS, or regular growth medium containing phenol red and 10% FBS. The migratory distance was measured at fixed time points following 0, 8, 24, 32, 48 and 72 hours following as described in section 5.1; and ranging concentrations of 17-oestradiol (0.5nM, 1nM, 10nM and 100nM), as described in section 5.2.1.

5.5. Migration of dermal fibroblasts in the presence of specific ER and ER agonists,

Dermal fibroblasts derived from human keratinocyte NCTC (n=4) between passage 3 and 5 were prepared and wounded for migration as described in sections 5.1. 1.5ml; 0.5% serum in DMEM growth medium supplemented with ≥10% FBS of experimental growth medium; containing each agonists hormone, were added to each dish in quadruplicate;

- Absolute ethanol vehicle control (0.0001%)

- 10nM 17-oestradiol

- 10nM PPT (ER-agonist)

10nM DPN (ER-agonist)

The concentration of the specific ER agonists was chosen following review of the data sheets, with consideration of their relative binding affinities (Tocis Bioscience). The cells were then cultured for 72 hours (3 days) and the migratory distance was measured daily and photographs were taken following fixed time points, as described in section 5.2.2.

5.6 Imaging Migrating cells

Following these two condition experiments, dose response and time points, all photographs of migrating cells under specific conditions were obtained using an inverted Leitz microscope, as described in section 5.1

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