Biomedical subjects

Michael P Philpott

Publications and source records attributed to Michael P Philpott.

11 recordsLinked to original sources

PubMed2007

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 beta-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 beta-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 beta-catenin-independent non-canonical WNT transduction pathway.

Adenosine Triphosphate↗

PubMed2006

Selective modulation of Hedgehog/GLI target gene expression by epidermal growth factor signaling in human keratinocytes.

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.

Binding Sites↗

PubMed2005

Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event.

Basal cell carcinoma is the most common human cancer with increasing incidence reported worldwide. Despite the aberrant signaling role of the Hedgehog pathway, little is known about the genetic mechanisms underlying basal cell carcinomas. Towards a better understanding of global genetic events, we have employed the Affymetrix Mapping 10K single nucleotide polymorphism (SNP) microarray technique for "fingerprinting" genomewide allelic imbalance in 14 basal cell carcinoma-blood pair samples. This rapid high-resolution SNP genotyping technique has revealed a somatic recombination event-uniparental disomy, leading to a loss of heterozygosity (LOH), as a key alternative genetic mechanism to allelic imbalances in basal cell carcinomas. A highly conserved LOH region at 9q21-q31 was found in 13 of 14 (93%) basal cell carcinomas. Further statistical and fluorescence in situ hybridization analyses confirmed that the 9q LOH was a result of uniparental disomy in 5 of 13 (38%) basal cell carcinomas. De novo mutations in the Patched 1 gene (PTCH) were found in 9 of 13 (69%) basal cell carcinomas with 9q LOH. A second important locus, containing LOH at 6q23-q27 was found in 5 of 14 (36%) basal cell carcinomas, suggesting that the presence of an additional putative tumor suppressor gene may be contributing to basal cell carcinoma development. This study shows that the rate of 9q LOH in basal cell carcinomas has been previously underestimated. Furthermore, we provide the first evidence that uniparental disomy due to somatic recombination constitutes one of the mechanisms of LOH in basal cell carcinoma tumorigenesis.

Aged↗

PubMed2004

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.

Base Sequence↗

PubMed2004

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein.

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.

Animals↗

PubMed2004

FOXE1, a new transcriptional target of GLI2 is expressed in human epidermis and basal cell carcinoma.

Sonic hedgehog (Hh) signaling plays a key role in epidermal development and skin cancer. Mutational inactivation of the tumor suppressor gene patched (PTCH) leads to constitutive activation of the Hh signaling pathway, resulting in activation of target gene transcription by the zinc finger transcription factors GLI1 and GLI2. Recent experiments in mice point to GLI2 as the key mediator of Hh signaling in skin. We have concentrated on the identification of candidate mediators of GLI2 function in the human epidermis. We show here that the forkhead/winged-helix domain transcription factor FOXE1 is likely to be a direct GLI2 target gene. The kinetics of FOXE1 induction are similar to the known direct target PTCH, and a 2.5 kb upstream fragment containing five GLI-binding sites activates transcription in a reporter assay. We show by in situ hybridization that FOXE1 is expressed in the outer root sheath of the hair follicle, where murine Gli2 is also expressed. FOXE1 expression is also found in basal keratinocytes of the human epidermis and basal cell carcinoma (BCC). These data point to a putative role of FOXE1 in mediating Hh signaling in the human epidermis downstream of GLI2.

Basal Cell Carcinoma↗

PubMed2003

Loss of protein kinase Calpha expression may enhance the tumorigenic potential of Gli1 in basal cell carcinoma.

Activation of the Sonic hedgehog signaling pathway, primarily through mutational inactivation of the PTCH1 gene, is associated with the development of basal cell carcinoma (BCC). Gli1, a member of the Gli family of transcription factors, is expressed in BCC and in transgenic mice targeted expression of Gli1 in basal keratinocytes leads to BCC development. In addition to BCC, previous studies have shown that Gli1 is expressed in the outer root sheath (ORS) of the hair follicle but is absent in interfollicular epidermis. In this study, we have characterized the expression pattern of two protein kinase C (PKC) isoforms expressed in BCC and hair follicles. We have then used reporter assays to investigate the effects of these isoforms on Gli1 transcriptional activity. We report that in BCC sections, PKCalpha but not PKCdelta was weakly expressed in the epidermis, whereas in the hair follicle, PKCalpha was expressed in the ORS and PKCdelta in the inner root sheath. In contrast, neither PKCalpha nor PKCdelta was expressed in BCC tumor islands, although both isoforms were often expressed in the surrounding stroma. In mammalian 293T cells, coexpression of constitutively active PKCalpha reduced the activity of Gli1 in a dose-dependent manner, whereas constitutively active PKCdelta increased the activity of Gli1, although this required higher expression levels. Regulation of mutant Gli1 protein localized exclusively to the nucleus was similar to that of the wild-type protein, indicating that nuclear-cytoplasmic shuttling is not a determinant of Gli1 control by either PKC isoform. Furthermore, PKC regulation of Gli1 did not involve activation of mitogen-activated protein kinase signaling. Finally, we show that exogenous Gli1 does not alter the expression of PKCalpha in human primary keratinocytes, suggesting that loss of this isoform in BCC is not via Hedgehog signaling. As BCCs have been proposed to originate from the ORS, loss of PKCalpha expression may be relevant to tumor formation; this may, in part, be because of the predicted increase in Gli1 transcriptional activity.

Basal Cell Carcinoma↗

PubMed2003

Defensins and acne.

Acne a disease of the pilosebaceous unit is characterised by hypercornification and hyperkeratosis of outer root sheath (ORS) and sebaceous duct and perilesional infiltrate. Lesions may be characterised as "non"-inflammatory versus inflammatory. Hypercornification of the distal ORS and the pilosebaceous duct in concert with increased sebum production and abnormalities of the microbial flora are considered to be major factors in the pathogenesis of acne vulgaris. However, the basic mechanisms involved in the development of inflammation during acne vulgaris remain unclear. We have investigated the expression patterns of two antimicrobial peptides, human beta-defensin 1 (hBD1) and human beta-defensin 2 (hBD2) in healthy human hair follicles as well as in peri- and intralesional skin of acne vulgaris lesions such as comedones, papules and pustules. Strong hBD1 and hBD2 immunoreactivity was found in all suprabasal layers of the epidermis, and all permanent compartments of the hair follicle including the distal ORS of the hair follicle and the pilosebaceous duct. Moreover, marked hBD1 and hBD2 expression was also detected in the hair follicle stem cell compartment. In contrast, the proximal follicle bulb which undergoes apoptotic regression and is also able to regenerate following injury did not express hBD1 or hBD2. The majority of acne biopsies displayed a marked upregulation of hBD2 IR in the lesional and perilesional epithelium; in particular in pustules, and a less marked upregulation of hBD1 IR. The upregulation of beta-defensins expression in acne vulgaris lesions when compared to controls suggests that beta-defensins may be involved in the pathogenesis of acne vulgaris.

Acne Vulgaris↗

PubMed2003

Animal and in vitro models for the study of hair follicles.

Since the way in which the hair follicle functions is not well understood, many hair disorders are poorly controlled. A range of in vitro and in vivo models have therefore been developed to investigate the cell biological and biochemical mechanisms involved in the organization of this complex tissue. These range from cultures of a single cell type, such as those of the regulatory, mesenchyme-derived dermal papilla, through organ culture of isolated follicles to natural or genetically manipulated animal models. Each system has advantages and disadvantages for studying particular aspects of follicular function and some are potentially useful for the development of novel treatments for hair disorders.

Animals↗

PubMed2002

FOXM1 is a downstream target of Gli1 in basal cell carcinomas.

Forkhead box (FOX) proteins have been shown to play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, longevity, and transformation. The functional importance of this gene family in normal human skin physiology and disease processes is not well understood. Activation of Sonic Hedgehog (Shh) signaling plays a key role in the development of basal cell carcinomas (BCCs) of the skin in humans. Recent studies have established that some FOX genes are downstream targets of Shh signaling. We have investigated the role of FOX proteins in transducing Shh effects in human skin by using degenerate PCR to identify FOX genes differentially expressed in BCCs. All three known FOXM1 isoforms (a, b, and c) were detected in human skin and cultured keratinocytes, and the transcriptionally active FOXM1b isoform was found to be up-regulated in BCCs. Real-time quantitative RT-PCR showed that the increase in FOXM1 mRNA levels was specific for BCCs and not a reflection of increased cell proliferation in that no up-regulation was seen in squamous cell carcinomas or proliferating primary human keratinocyte cultures. Immunostaining studies showed intense nuclear and cytoplasmic staining throughout BCC tumor islands and not confined to the periphery regions of the tumor where proliferating Ki-67-immunopositive cells are predominantly localized. Expression of the Shh target glioma transcription factor-1 (Gli1) in primary keratinocytes and other cell lines caused a significant elevation of FOXM1 mRNA level and transcriptional activity, indicating that FOXM1 is a downstream target of Gli1. Our data provide the first evidence that activation of Shh signaling via Gli1 is an important determinant of FOXM1 expression in mammalian cells. Given the role of FOXM1 in cell proliferation, the up-regulation of FOXM1 in BCCs may be one of the mechanisms whereby Shh signaling exerts its mitogenic effect on basal keratinocytes, leading to the development of this common human cancer.

Animals↗

PubMed2002

Contrasting expression patterns of CCAAT/enhancer-binding protein transcription factors in the hair follicle and at different stages of the hair growth cycle.

Hair follicles undergo repeated cycles of growth and regression, throughout the entire life of the organism. These dynamic changes require closely co-ordinated regulation of gene expression. The CCAAT/enhancer-binding proteins are a family of basic region/leucine zipper transcription factors that regulate gene transcription in various tissues. They have been implicated in epidermal differentiation and may therefore play an important role in the hair follicle. We have investigated the localization of four members of this family--CCAAT/enhancer-binding protein-alpha, -beta, and -delta, and Gadd153--in both human and murine hair follicles by immunohistochemistry. Furthermore, we examined CCAAT/enhancer-binding protein-alpha, -beta, and -delta immunoreactivity at different stages of the depilation-induced murine hair growth cycle. Distinct immunoreactivity patterns for CCAAT/enhancer-binding protein-alpha, -beta, and -delta, and Gadd153 were observed in the outer root sheath, sebaceous gland, dermal papilla, and connective tissue sheath of human anagen hair follicles. In murine follicles, CCAAT/enhancer-binding protein-alpha was expressed in the outer root sheath, sebaceous gland, and dermal papilla, whereas CCAAT/enhancer-binding protein-beta expression was confined to the matrix, sebaceous gland, and inner and outer root sheaths. Both CCAAT/enhancer-binding protein-alpha and -beta were upregulated during anagen, then downregulated in catagen follicles. In contrast, CCAAT/enhancer-binding protein-delta showed no hair cycle-dependent variation in immunoreactivity. These data suggests that the expression of CCAAT/enhancer-binding protein-alpha and -beta may, in turn, play a part in regulating hair cycle-dependent gene expression. Moreover, as CCAAT/enhancer-binding protein-alpha, -beta, and -delta are crucial in the regulation of adipocyte differentiation and lipid metabolism, their expression in sebocytes suggests they may also play a similar role in differentiation and lipid metabolism of the sebaceous gland.

Animals↗