Abstract
Background:
Nodulocystic acne is a severe type of acne that is known to improve after treatment with isotretinoin. Melnik has hypothesized a unifying concept on the mechanism of acne pathogenesis involving altered expression of Forkhead box O transcription factor (FoxO1) and role of isotretinoin in improving acne via modulating this pathway.
Aim:
To evaluate the pathway proposed by Melnik in acne pathogenesis by analysing the difference in the expression of FoxO1, peroxisome proliferator-activated receptor (PPARγ), and androgen receptor (AR) between acne patients and non-acne controls and the effect of treatment with isotretinoin on change in expression of these genes in acne patients.
Results:
The gene expression of FoxO1 was non significantly higher in acne patients as compared to controls. After treatment with isotretinoin, a significant decrease in FoxO1 expression in acne patients at mRNA (P = 0.05) level was observed. There was a significant decrease in grade 3 positivity of FoxO1 at protein level (P = 0.0009). A decrease in androgen receptor positivity (P = 0.055) at protein level was also observed.
Conclusion:
Reduction in FoxO1 expression appears to be an important mechanism of action of isotretinoin in acne.
Keywords: Acne, androgen receptor, FoxO1, isotretinoin, peroxisome proliferator-activated receptor
Introduction
Acne is an inflammatory skin disease that has the potential for inducing physical and psychological scarring.[1] The pathogenesis of acne is multifactorial and rests on four pathogenic pillars, that is, follicular hyper cornification, sebaceous hyperplasia, Propionibacterium acnes proliferation and inflammation.[2] The prevalence of acne is highest among the adolescents of 14–19 years of age.[3] A surge in growth hormones and their terminal mediator, the insulin like growth factor-1 (IGF-1), occurs around puberty that exerts direct effect on sebocytes[4] and plays a vital role in acne pathogenesis.[5] Isotretinoin, an USA Food and Drug Administration (US FDA) approved drug, is known to improve severe nodulocystic acne.[6] The unifying concept of mechanism of isotretinoin action as hypothesized by Melnik stems from upregulation of expression of Forkhead box O transcription factor (FoxO1).[6] Increased growth factor signalling associated with the pubertal growth spurt activates phosphoinositol-3-kinase (PI3K)/Akt kinase resulting in nuclear deficiency of FoxO1 which in turn increases androgen receptor (AR) transactivation and modifies the activity of important nuclear receptors like peroxisome proliferator-activated receptors (PPARs).[7] It has been shown that PPARs regulate lipogenesis in sebocytes and alteration in the composition of sebum is known to cause acne. However, difference in FoxO1 expression in acne patients and non-acne individuals to support the unifying hypothesis is limited.[8] Therefore, we assessed the difference in expression of FoxO1, AR and PPARγ receptors between acne patients and non-acne controls. In addition, changes in expression of these parameters after treatment of severe or very severe acne patients with isotretinoin that is otherwise approved for use in severe nodulocystic acne was also analysed.
Methodology
This pilot study was conducted after approval from Ethics Committee of our Institution (NK/1509/study/310 dated 09/04/2014). Twenty patients (severe or very severe nodular acne) and an equal number of age and gender matched non-acne controls with lesions of other dermatoses on the face who required a biopsy from the facial skin for diagnosis of their skin disease were included after taking written informed consent. Acne patients were recruited after proper examination by an experienced dermatologist in properly daylight-illuminated outpatient clinical examination room and acne severity was graded by using Global Acne Grading System (GAGS). Patients above 18 years of age with severe or very severe nodulocystic acne and who had not undergone any active acne treatment, topical and/or oral, in the last one month were recruited. The recruited patients were treated with isotretinoin (0.5 mg/kg/day) for a period of four months. From patients, two skin biopsy specimens (2 mm) were taken aseptically under local anaesthesia from skin immediately surrounding a nodular lesion on face at baseline and after four months of treatment with isotretinoin. The biopsy samples were used to study mRNA expression and immunohistochemistry (IHC). For controls, one facial skin biopsy was taken (aseptically, under local anaesthesia) to study mRNA expression. This helped in comparison of expression status between controls and acne patients at baseline. For mRNA study, RNA was isolated from the biopsy samples of cases and controls by TRIzol method. One microgram of the isolated RNA was transcribed into cDNA using high-capacity cDNA reverse transcription kit (applied biosystems) followed by real time-polymerase chain reaction (RT-PCR, Rotor-Gene 6000) using SYBR green (Dynamo Color Flash SYBR Green qPCR kit, Thermo scientific) and primers specific to FoxO1, AR and PPARγ. The following set of primers were used: FoxO1– F: 5′- GCAGATCTACGAGTGGATGGTC-3′, R: 5′- AAACTGTGATCCAGGGCTGTC-3′; PPARγ - F: 5′-CTGAATGTGAAGCCCATTGAA-3′, R: 5′-GTGGAAGAAGGGAAATGTTGG -3′ and AR - F: 5′- GACCAGATGGCTGTCATTCA-3′, R: 5′- GAAGCCATCCAAACTCTTGA-3′. To study the protein expression, the biopsy samples of acne patients were fixed in formalin. The protein expression was analyzed by IHC using primary antibody against FoxO1 (1:100 dilution, Santa Cruz Biotechnology, Inc. California, USA) and AR (1:200 dilution, Santa Cruz Biotechnology, Inc.). The overall staining intensities of the stained slides were scored. Statistical tests were carried out using Graph pad prism (Dr. Harvey Motulsky, Version 5.00, Boston, USA). The data were statistically analysed using unpaired and paired t-tests to study the mean difference of the parameters between controls and cases and before and after treatment with isotretinoin, respectively. The correlation analysis was performed using Pearson’s correlation. The IHC data were analysed using Fisher’s exact test.
Results
Out of 20 cases, expression of two samples could not be assessed due to insufficient RNA. The mean mRNA expression of FoxO1 was non-significantly (P = 0.47) higher in cases in comparison to controls [Table 1]. However, treatment with isotretinoin decreased mean FoxO1 expression (P = 0.05). The difference in mean expression between cases (before treatment) and controls of AR and PPARγ was not significantly different. Isotretinoin treatment did not alter the expression of AR and PPARγ [Table 1]. Further, the correlation between expression of FoxO1, AR and PPARγ was analysed using Pearson’s correlation (Graph pad prism, V5.00). It was observed that before isotretinoin treatment, the mRNA expression of FoxO1 is positively correlated with expression of AR (r2 = 0.395, P = 0.012, 95% CI 0.17–0.86) but not with PPARγ (r2 = 0.067, P = 0.33, 95% CI 0.67–0.27). However, after isotretinoin treatment no correlation between the expressions of the three genes was observed. In cases, the expression of FoxO1 and AR at protein level before and after isotretinoin treatment was analysed using Fisher’s exact test but the expression of PPARγ at protein level could not be determined. A significant decrease in the frequency of grade 3 positivity of FoxO1 was observed with treatment (38.88%–16.66%, P = 0.0009). There was a significant increase in frequency of grade 1 (P = 0.0249) and a marginal increase in grade 2 positivity (P = 0.146). Also, a significant decrease in grade 1 and grade 2 positivity of AR was observed (44.44%–27.77%, P = 0.027 and 22.22%–11.11%, P = 0.055 respectively) with isotretinoin treatment.
Table 1.
Changes in the mRNA expression of various genes between control and cases and after isotretinoin treatment
| Control | Cases (Before treatment) | After treatment | Statistical test used, P | |
|---|---|---|---|---|
| FoxO1 | 0.11 (0.14), 95% CI 0.036-0.19 | 0.45 (0.75), 95% CI 0.06-0.83 | 0.078 (0.056), 95% CI 0.05-0.12 | P=0.47#, P=0.05$ |
| Androgen receptor | 0.033 (0.034), 95% CI 0.013-0.053 | 0.18 (0.54), 95% CI 0.10-0.48 | 0.10 (0.94), 95% CI 0.053-0.151 | P=0.95#, P=0.52$ |
| PPARγ | 0.51 (0.65), 95% CI 0.18-0.85 | 0.54 (0.73), 95% CI 0.16-0.92 | 0.38 (0.94), 95% CI 0.16-0.59 | P=0.94#, P=1.00$ |
#Denotes P value of comparison between controls and cases (before treatment) using unpaired t-test; $denotes P value of comparison of cases before and after treatment with isotretinoin using paired t-test
Discussion
Acne starts to appear around puberty, and it has been proven that androgens along with an elevation in the levels of IGF-1 play a very important role in initiation of the disease process.[5,9,10] Insulin like growth factor-1 (IGF-1) plays a vital role in acne pathogenesis.[5] It initiates signalling cascade resulting in increased transactivation of AR and modifies the activity of important nuclear receptors like PPARs. PPARs are known to regulate lipogenesis and alter sebum synthesis.[7] One of the most effective drugs that is known to act on all the four pillars of acne is isotretinoin. Several mechanisms for how isotretinoin acts on these pillars have been known. Recently, a unifying concept hypothesized by Melnik suggests an important role of FoxO1 in acne pathogenesis. During puberty, growth factors and IGF-1 initiate PI-3K/Akt signaling pathway that phosphorylates FoxO1 leading to its translocation from nucleus to cytoplasm. As a result, it is no longer available to suppress AR transactivation[7] and thus increases the synthesis of androgens. PPARγ, a transrepressor of FoxO1, is essential for the development and functioning of sebaceous glands and serves as a co-regulator of lipogenesis.
In our study, the observed higher mRNA expression of FoxO1 in cases as compared to controls represents the increased cytoplasmic expression that may suggest its nuclear deficiency thereby leading to transactivation of AR and PPARγ. This was further supported by the positive correlation observed between FoxO1 and AR mRNA expression. Furthermore, isotretinoin treatment decreased mRNA expression and protein expression of FoxO1, thereby suggesting that FoxO1 is not translocated from the nucleus to the cytoplasm and hence prevents acne pathogenesis. Hence, it can be inferred that one mechanism of isotretinoin action in treatment of acne is by normalization of the expression of FoxO1. However, our observations need to be validated in a larger study population.
Financial support and sponsorship
Intramural Research Grant of Postgraduate Institute of Medical Education and Research, Chandigarh, India (Grant approval: 71/6-Edu/13/1931 dated: 01/09/2014).
Conflicts of interest
There are no conflicts of interest.
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