The sebum-suppressive effect of isotretinoin has been related to sebocyte apoptosis [42–45] with isotretinoin-mediated p21-induced cell cycle arrest [44] and upregulation of pro-apoptotic transcription factors including FoxO1 [55] and FoxO3a [55] as well as the apoptosis effector TRAI [45, 62]. The transcription factor p53, known as the guardian of the genome [71], is a key regulator of cell fate decisions including cycle control and induction of apoptosis depending on the magnitude of p53 transcription and activation Notably, p53 promotes the expression of the cell cycle inhibitor p21 (CDKN1A [73] and the pro-apoptotic proteins FoxO1 [23], FoxO3a [23, 25], and TRAIL [26] and inhibits anti-apoptotic pro-survival effectors such as IGF1R [28], AR [27] and survivin (BIRC5) [74],all known p53 target genes involved in acne pathogenesis. It has been demonstrated in primary human keratinocytes and melanocytes that isotretinoin and ATRA increase the expression of p53 [56–61]. According to a recent hypothesis, isotretinoin´s mode of action and its adverse effects are related to enhanced expression of p53 [59, 75]. In fact, our study provides first experimental evidence that isotretinoin significantly upregulates the expression of p53 in the skin and sebaceous glands of acne patients after 6 weeks of oral isotretinoin therapy with the commonly used daily dose of 0.6 mg/kg body weight.
Remarkably, the skin of acne patients compared to acne-free controls exhibits lower levels of p53 expression (Fig. 2), whereas after isotretinoin-treatment p53 levels significantly exceeded p53 levels in healthy skin, pointing to a strong induction of p53 by systemic and prolonged isotretinoin exposure.
It is noteworthy to mention that p53 expression is regulated by endocrine and nutrient signalling. Increased insulin and IGF-1 signalling, which both activate the kinase AKT, results in phosphorylation and activation of E3 ubiquitin ligase mouse double minute 2 (MDM2) promoting the proteasomal degradation of p53 [76, 77]. Western diet with increased insulin/IGF-1/AKT signalling [11] may thus reduce the expression of p53, the key negative regulator of mTORC1 [29–31, which exhibits increased activity in the skin and sebaceous glands of acne patients [13–17]. The fundamental ability of mTORC1 signalling promote cell growth and anabolism [32], may also explain the potential relation between BMI and acne risk [33–36]. In fact, p53 is not only a tumour suppressor but has been appreciated as a crucial player in nutrient sensing pathways serving as a negative regulator of mTORC1 [29–31] and adipogenesis [37].
Importantly, activated mTORC1 is a key suppressor of autophagy [78]. Remarkably, p53 not only induces apoptosis but also stimulates autophagy [79–80]. It has recently been shown in immortalized SZ95 sebocytes, that isotretinoin treatment, partly via activation of FoxO1, increased the expression of ATG5 and induced autophagy resulting in reduced sebaceous lipid accumulation [81]. Notably, p53 can activate the expression of a large set of target genes that are involved in the autophagic programme including ATG5 [82, 83]. Autophagy is required for robust p53-dependent apoptosis. Thus, autophagy and apoptosis are two closely related p53-dependent cellular responses [84, 85]. It is thus conceivable that isotretinoin induces both p53-mediated autophagy as well as p53-induced apoptosis, depending on the dose and duration of isotretinoin exposure and the resulting magnitude of p53 expression.
Moreover, it should be kept in mind that p53 is partially inactivated by simion virus 40 large T antigen in immortalized SZ95 and SEB-1 sebocytes [86, 87], which may thus not be suitable cell lines for studying p53-dependent effects of sebaceous gland regulation leading to paradoxical even acnegenic effects [88] disputed earlier [89].
There is compelling translational evidence that isotretinoin-mediated upregulation of p53 expression explains isotretinoin´s teratogenicity via p53-mediated neural crest cell apoptosis [42, 43]. Isotretinoin also induced apoptosis in primary human keratinocytes [58], melanoma cells [60, 61], rat ovarian granulosa cells [90, 91], hepatoma cells [92] associated with decreased expression of the apoptosis inhibitor survivin [92]. Increased serum levels of survivin have been reported in acne patients compared to controls [93]. Of note, survivin (BIRC5) expression is negatively regulated by p53 [94]. In accordance with these findings and our results, we conclude that isotretinoin-induced expression of p53 not only promotes sebocyte apoptosis in human sebaceous glands as the predominant sebum-suppressive effect bit is also responsible for isotretinoin´s adverse effects.
The most common mucocutaneous side effects of isotretinoin therapy, dry skin, has been related to increased expression of keratinocyte aquaporin 3 (AQP3), which damages the skin barrier and enhances trans epidermal water loss causing skin dryness [95]. Notably, AQP3 is a p53 target gene [96]. Other members of the aquaporin family, AQP1 and AQP4 [97, 98] have been linked to intracranial hypertension (pseudotumor cerebri), a potential adverse effect of isotretinoin [99], and appear as well to be related to upregulated p53 [100, 101]. In addition, isotretinoin-induced hypertriglyceridemia [102] is associated with increased plasma levels of apolipoprotein B100 in very low-density lipoprotein (VLDL) and low density lipoproteins (LDL) [103]. The gene encoding apoB100 (APOB) has been identified as p53 target gene [104].
It is important to remember that retinoids induce primary and secondary transcriptional responses depending on dose and duration of retinoid exposure [52]. Sufficient nuclear transport of ATRA via CRABP2 is mandatory for ATRA-induced transcriptomic changes [52] including isotretinoin/ATRA-induced transcriptional modification resulting in sufficient sebum suppression [47]. Increased expression of CRABP2 in isotretinoin-treated sebaceous glands of patients with acne has been observed after weeks of oral isotretinoin exposure [47], whereas short term (6h, 24h) isotretinoin exposure of immortalized p53-inactivated SZ95 sebocytes did neither exhibit increased CRABP2 nor upregulated p53 or FoxO1 expression [105]. This is in contrast to our in vivo findings under clinical conditions observed in patients treated with isotretinoin for 6 weeks, whose sebaceous glands are not p53-inactivated by SV40 viral transfection [55].
Notably, p53 maintains baseline expression of common tumour suppressor genes including FoxO1 [23]. Over the last 10 years, decreased FoxO1 expression has been linked to acne pathogenesis [11, 16, 55, 106], whereas isotretinoin treatment increases FoxO1 expression in sebaceous glands of acne patients [55, 107, 108]. The p53 target gene FoxO1 is a nuclear co-suppressor of multiple transcription factors critically involved in acne pathogenesis such as AR [19], SREBF1 [109], PPARA [110] and is a crucial promoter of genes involved in apoptosis [54]. Recent evidence indicates that FoxO1 is involved in the induction of autophagy in isotretinoin-treated SZ95 sebocytes [81]. Furthermore, FoxO1 promotes the expression of GATA6, a critical transcription factor maintaining appropriate keratinocyte proliferation and differentiation of the infundibulum of the human sebaceous follicle [111], which is deficiently expressed in sebaceous follicles of acne patients linking p53-FoxO1-GATA6 deficiency to comedogenesis.
Reduced baseline expression of the tumour suppressor p53 in acne patients compared to acne-free controls may also explain the increased risk of acne patients for common p53-related malignancies such as prostate cancer [112, 113], and breast cancer [114]. Notably, there is no observed acne and very low cancer incidence in IGF-1-deficient patients with Laron syndrome [115], who exhibit higher p53-FoxO1 signalling [116], In contrast, Western diet with high glycemic load and milk/dairy consumption increases insulin/IGF-1 signalling promoting AKT/MDM2-mediated proteasomal degradation of p53 [51, 117, 119], whereas forced upregulation of p53-FoxO1 signalling may contribute to the tumour suppressing effect of isotretinoin in neuroblastoma [120, 121] and retinoid chemoprevention of non-melanoma skin cancer [122].
Taken together, our study provides experimental evidence for increased nuclear expression of p53 in sebaceous glands and skin of acne patients after oral isotretinoin treatment and substantiates that enforced p53-FoxO signalling causes all desired and adverse effects of systemic isotretinoin therapy.