Michael P. Philpott

What is the pathogenesis of acne

Abstract:  For a long time, the mantra of acne pathogenesis debates has been that acne vulgaris lesions develop when (supposedly largely androgen‐mediated) increased sebum production, ductal hypercornification, and propionibacteria come together with local inflammatory process in the unlucky affected individual. And yet, the exact sequence, precise interdependence, and choreography of pathogenic events in acne, especially the ‘match that lights the fire’ have remained surprisingly unclear, despite the venerable tradition of acne research over the past century.

Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes†

ABSTRACT Hedgehog (HH)/GLI signaling plays a critical role in epidermal development and basal cell carcinoma. Here, we provide evidence that epidermal growth factor receptor (EGFR) signaling modulates the target gene expression profile of GLI transcription factors in epidermal cells. Using expression profiling and quantitative reverse transcriptase PCR, we identified a set of 19 genes whose transcription is synergistically induced by GLI1 and parallel EGF treatment. Promoter studies of a subset of GLI/EGF-regulated genes, including the genes encoding interleukin-1 antagonist IL1R2, Jagged 2, cyclin D1, S100A7, and S100A9, suggest convergence of EGFR and HH/GLI signaling at the level of promoters of selected direct GLI target genes. Inhibition of EGFR and MEK/ERK but not of phosphatidylinositol 3-kinase/AKT abrogated synergistic activation of GLI/EGF target genes, showing that EGFR can signal via RAF/MEK/ERK to cooperate with GLI proteins in selective target gene regulation. Coexpression of the GLI/EGF target IL1R2, EGFR, and activated ERK1/2 in human anagen hair follicles argues for a cooperative role of EGFR and HH/GLI signaling in specifying the fate of outer root sheath (ORS) cells. We also show that EGF treatment neutralizes GLI-mediated induction of epidermal stem cell marker expression and provide evidence that EGFR signaling is essential for GLI-induced cell cycle progression in epidermal cells. The results suggest that EGFR signaling modulates GLI target gene profiles which may play an important regulatory role in ORS specification, hair growth, and possibly cancer.

Effects of interleukins, colony‐stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin‐1 and tumour necrosis factor‐α in alopecia areata

The immune system may be involved in the regulation of normal hair follicle growth as well as in the pathogenesis of some hair diseases. Immunomodulatory cytokines not only act as mediators of immunity and inflammation but also regulate cell proliferation and differentiation and. as such, may play an important part in regulating hair growth. We have investigated the effects of a number of interleukins (IL). colony stimulating factors and tumour necrosis factors (TNF) on hair follicle growth in vitro. Dose‐response studies showed that IL‐1α. IL‐1ß and TNF‐o were potent inhibitors of hair follicle growth. The histology of hair follicles maintained with inhibitory doses of IL‐1α. IL‐1ß and TNF‐α showed similar changes in hair follicle morphology, resulting in the formation of dystrophic anagen hair follicles. These changes in histology were characterized by the condensation and distortion of the dermal papilla, marked vacuolation of the hair follicle matrix, abnormal keratinization of the follicle bulb and inner root sheath, disruption of follicular melanocytes and the presence of melanin granules witbin the dermal papilla. Moreover, these changes in hair follicle morphology are similar to those reported in alopecia areata and suggest that IL‐1α, IL‐1ß and TNF‐α may play an important part in the pathophysiology of inflammatory hair disease.

Human hair growth in vitro: a model for the study of hair follicle biology

The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. We have developed an in vitro human hair growth model that enables us to maintain isolated human hair follicles for up to 10 days, during which time they continue to grow at an in vivo rate producing a keratinised hair fibre. We have shown that epidermal growth factor (EGF) in our system mimics the in vivo depilatory action of EGF in sheep, and suggest that this occurs as a result of EGF stimulating outer root sheath (ORS) cell proliferation which results in the disruption of normal mechanisms of cell-cell interaction in the hair follicle. We identify transforming growth factor-beta (TGF-beta) as a possible negative regulator of hair follicle growth and show that physiological levels of insulin-like growth factor-I (IGF-I) can support the same rates of hair follicle growth as supraphysiological levels of insulin. Furthermore, in the absence of insulin hair follicles show premature entry into a catagen-like state. This is prevented by physiological levels of IGF-I. Finally we demonstrate that the hair follicle is an aerobic glycolytic, glutaminolytic tissue and discuss the possible implications of this metabolism.

The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells

In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1–S phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR‐574‐3p and miR‐720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63‐mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Characterization of liver X receptor expression and function in human skin and the pilosebaceous unit

Abstract:  The nuclear receptors liver X receptor alpha (LXRα) and liver X‐receptor beta (LXRβ) have a well documented role in cholesterol homeostasis and lipid metabolism within tissues and cells including the liver, small intestine and macrophages. In keratinocytes, LXRs have been shown to up‐regulate differentiation in vitro via increased transcription of proteins of the AP1 complex and to down‐regulate proliferation in vivo. In this study, we provide a detailed description of the location and possible role of LXRs within human skin and its associated glands and appendages. Using RT‐PCR, Western blotting and immunohistochemistry, we have demonstrated expression of LXRα and LXRβ mRNA and proteins in whole human skin as well as within a range of primary and immortalized human cell lines derived from human skin, hair follicle and sebaceous glands. Furthermore, we have shown that synthetic LXR specific agonists GW683965 and TO901317 significantly inhibit cell proliferation in primary epidermal keratinocytes, immortalized N/TERT keratinocytes and the immortalized SZ95 sebocyte line, and significantly increase lipogenesis in SZ95 sebocytes. In addition, we showed that the synthetic agonist TO901317 significantly reduced hair growth, in vitro.

Steroid synthesis by primary human keratinocytes; implications for skin disease

Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3βHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-(3)H]-pregnenolone through each steroid intermediate to [7-(3)H]-cortisol in cultured PHK. Trilostane (a 3βHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.

Role for WNT16B in human epidermal keratinocyte proliferation and differentiation

WNT signalling regulates a variety of cell functions including cell fate, polarity, and differentiation via the canonical or β-catenin stabilisation pathway and/or the planar cell polarity or non-canonical pathway. We have previously demonstrated that two isoforms (A and B) from the WNT16 locus have differential expression in various adult human tissues. In this study we show that WNT16B but not WNT16A isoform was upregulated in basal cell carcinomas compared with normal skin. We further investigated the cellular and molecular functions of WNT16B in primary human epidermal keratinocytes and a keratinocyte cell line. Cellular expression of WNT16B neither stabilised β-catenin nor activated the lymphoid enhancer factor or T-cell factor transcriptional reporter in primary keratinocytes. WNT16B activated the Jun-N-terminal kinase cascade suggesting the activation of a non-canonical WNT signalling pathway. Constitutive expression of WNT16B significantly enhanced the rate of cell proliferation and prolonged clonogenicity in primary keratinocytes. Silencing WNT16B by RNA interference reduced keratinocyte proliferation. Furthermore, overexpression of WNT16B induced a hyperproliferation phenotype in an organotypical culture system. This work presents the first evidence that WNT16B activates human keratinocyte proliferation possibly via a β-catenin-independent non-canonical WNT transduction pathway.