Figure 5.9. E2 and Subsequent TGF-β1 Treatment Restores Differentiation in Aged Fibroblasts. Aged fibroblasts were grown to confluence; growth arrested and
incubated with either serum-free media alone, media containing 10ng/ml TGF-β1, or media containing 100nM E2 for 72 hours. An additional treatment with media containing 10ng/ml TGF-β1 was made to E2 treated cells for a further 72 hours. The expression of A. EGFR mRNA, B. HAS2 mRNA, C. miR-7, D. α-SMA mRNA and E. EDA-FN mRNA, was analysed using QPCR. Results are shown as the mean ± s.e.m. of 3 independent experiments. F. Immunocytochemistry was used for 70% confluent
5.3 Discussion
The data reported in this Chapter provide an important mechanistic insight into the regulation of miR-7 by STAT1 activation; and unveils novel interactions between the cytokines that directly or indirectly modulate miR-7 expression and the phenotype of fibroblasts. The role of miR-7 in the regulation of EGFR and its pathway has been reported in many instances and in different species [332-334], highlighting its conserved function. However, this Chapter and the previous results Chapter examined miR-7 expression in the context of cellular ageing and wound healing, specific studies that have not been performed before.
As shown in the previous Chapter, the activity of the EGFR promoter was not modulated by cellular ageing and a candidate for the suppression of EGFR expression, miR-7, was determined to suppress the production of EGFR protein at the mRNA level, pre-translation. Therefore, understanding the principal factor(s) in the upregulation of miR-7 transcription was an important first step of this investigation and it was determined, through the use of cell surface receptor inhibitors, that the EGFR signalling branch (HA-CD44-mediated) of the differentiation pathway was responsible for the upregulation of miR-7 expression and the suppression of EGFR mRNA; a self-regulatory loop involving the EGFR upregulation of miR-7, with miR-7 in turn, causing the downregulation of EGFR. Examination of the miR-7 promoter was ideal for furthering the investigation, as isolating transcription factors that enhance the miR-7 transcription would provide targets for interventional measures. The promoter region of miR-7 was elucidated using previous studies [332] and in silico analysis of TSS associated tags. Determining which transcription factors were binding to the miR-7 promoter and enhancing miR-7 transcription was the next step of the investigation; and it was noted that there was an abundance of interferon regulated elements, specifically STATs, in the putative promoter region.
To further investigate the involvement of STATs and whether the EGFR pathway was responsible for increased miR-7 promoter activity, the promoter region was ligated into a luciferase reporter plasmid. The data shown suggested that EGFR was capable of mediating the promoter activity of miR-7; and the induction by IFNγ treatment supported the hypothesis that STATs were the responsible transcription factors. These data led to the hypothesis of a potential mechanism involving EGFR activation of the JAK/STAT pathway, which has been reported previously [1, 3]; and was further supported by the EGFR-mediated phosphorylation of STAT1 as shown in Figure 5.4. However, the fluctuations in STAT1 mRNA expression did not seem to impact on the total STAT1 protein present from whole cell lysates, a change that may be seen at longer time-points. The inhibition of JAK did not alter TGF-β1 upregulation of α-SMA, providing evidence that the differentiation pathway was independent in function from the EGFR-mediated JAK/STAT pathway. However, when JAK was inhibited, EGFR mRNA did not down-regulate and HAS2 mRNA increased in the presence of TGF-β1 or IFNγ, suggesting multiple functions of STATs on the regulation of differentiation pathway components. Suppression of EGFR through miR-7 upregulation may be separate from HAS2 suppression (possibly through an alternative miRNA), a mechanism that should be investigated further.
The effects of E2 on wound healing have been an area of research that has generated much interest over recent years [346, 380, 386, 387, 395]. Although previous research has shown the changes in fibroblasts treated with E2, exploring the links between E2 and its effect on the proteins necessary for myofibroblast differentiation has had limited attention. The results in this Chapter illustrated the changes of EGFR, HAS2 and miR-7 expression in young and aged fibroblasts, by E2 treatment. Whether E2 also affects translational or post-translational changes in EGFR, HAS2 and HA synthesis is not known and warrants further investigation. The data that is shown here revealed a means by which incubation of aged cells with E2
“prepared” the cells for the subsequent TGF-β1 treatment by increasing the expression of the mRNA for key components (EGFR and HAS2), whilst decreasing the expression of the pathway limiter (miR-7). Additionally, it was shown that E2 followed by TGF-β1 treatment was able to restore the ability of aged fibroblasts to form α-SMA stress-fibres.
The in vitro benefits of E2 on fibroblasts described here indicate a mechanism of action that downregulates miR-7 and thereby relieves the suppression placed on EGFR mRNA, increasing its expression. Therefore, further investigation is required to elucidate the specific interactions or functional and mechanistic outcomes of direct actions by E2 treatment.
Two areas of investigation warrant further analysis, the first is to assess the direct action of E2 on the miR-7 promoter. In silico analysis identified a sequence motif within the promoter region used, for estrogen response elements (IR3) and estrogen receptor (ER) binding, potentially indicative of a transcriptional suppression role. Through experiments exploring E2 stimulation using either site deletions or ER inhibition, the effect of E2 on the miR-7 promoter could be explored. Additionally, it was observed that the phosphorylation of STAT1 was reduced the longer cells were incubated with E2. The second area of investigation would be examination of whether E2 has direct effects on other STAT targets (e.g. STAT3), the production of activators of STATs, such as IFNγ, the previously reported downregulation of IL-6 [105, 404-407]; and their corresponding cell surface receptors, could help detail a full mechanism of action for E2 and explain its impact on miR-7, EGFR, HAS2 and the differentiation pathway in fibroblasts.
Specifically, analysis of IL-6 would be of primary interest as the correlation between cellular ageing and IL-6 has been previously reported in a plethora of studies [408, 409]; and the serum concentrations of IL-6 have been reported to increase with progressing age [410], although one study did not observe this correlation [411]. IL-6 is also a potent activator of
STATs and stimulates increased production of STAT mRNA [412, 413], which would go some way to supporting the observations made in this results Chapter. Furthermore, a broader analysis of changes in miRNAs under TGF-β1 and E2 treatments in young and aged fibroblasts, using microarrays, could indicate other miRNAs that may be present and actively inhibiting the differentiation pathway components.
Although with direct E2 treatment in vitro, the fibroblasts established beneficial outcomes, the effects of circulating serum levels of E2 in vivo may have a limited effect on the fibroblasts within the dermis or in other organ systems. Additionally, when combined with the low levels of estrogen in men and in post-menopausal women, E2 application may only be of therapeutic significance in terms of wound healing, when used to upregulate factors lost from cellular ageing. It can be concluded from the data presented in this results Chapter that a combinational treatment of E2 and TGF-β1 to aged fibroblasts was beneficial in reversing the age-associated loss of differentiation. The initial E2 treatments had a profound effect on fibroblasts, upregulating the key components needed for successful differentiation (EGFR, HAS2) and downregulating the limiting factors (p-STAT1, miR-7). The subsequent incubation with TGF-β1 was, therefore, able to drive myofibroblast differentiation, as shown by the production of myofibroblastic markers (α-SMA, EDA-FN). However, the treatment regime of 72 hours of E2 followed by 72 hours of TGF-β1 incubation may require further optimisation in order to achieve the best results.