Beyond the blot: cutting edge tools for genomics, proteomics and metabolomics analyses and previous successes.

The skin has an amazing array of complex interacting biological processes. Recent advances in investigational techniques now allow evaluation of these processes at the level of the gene, protein and metabolite. Sometimes collectively known as the omics, these fields of inquiry, known as genomics, proteomics and metabolomics, respectively, are yielding new and important insights into skin structure and processes, its responses to injury and age, and the mechanisms by which new interventions and compounds may work to improve the health and integrity of this crucial organ.

Dandruff/seborrhoeic dermatitis is characterized by an inflammatory genomic signature and possible immune dysfunction: transcriptional analysis of the condition and treatment effects of zinc pyrithione.

BACKGROUND: Dandruff/seborrhoeic dermatitis is a common scalp condition that is characterized by flakes, pruritus and sometimes mild erythema. These symptoms reflect tissue level events that are poorly understood at the molecular level. OBJECTIVES: The purpose of this work was: (i) to compare gene expression profiles in subjects with dandruff vs. those of subjects without dandruff to determine the key physiological disruptions manifest in the condition; and (ii) to determine the effect on this profile of treatment with a shampoo containing potentiated zinc pyrithione (ZPT). METHODS: In study 1, scalp biopsies were taken from 16 normal subjects and from involved and uninvolved sites in 15 subjects with dandruff. In study 2, 30 subjects with dandruff were treated for 3 weeks with a commercial ZPT shampoo (n = 15) or a vehicle (n = 15), and scalp lesional biopsies were collected at baseline and end of study for transcriptomic analysis. RNA was extracted from all biopsies and Affymetrix gene chips were used to analyse transcriptomic profiles, followed by bioinformatic analysis. RESULTS: Analysis of study 1 biopsies revealed more than 7000 individual probes differentially regulated in dandruff lesional skin relative to normal. Enriched Gene Ontology categories included: lipid metabolism, immune response, response to stimulus, apoptosis, cell proliferation, and epidermal development. The most striking feature of lesional skin relative to normal was the reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. Induced inflammatory genes were also enriched in dandruff uninvolved skin, suggesting the existence of predisposing factors associated with inflammation. Many genes increased in lesional skin were increased at the level of protein in stratum corneum samples (e.g. IL-1RA, S100A8, S100A9, S100A11, IL-8). Under conditions known to improve overall scalp condition, the ZPT shampoo treatment in study 2 produced a transcriptomic profile resembling that of normal scalp skin. CONCLUSIONS: These data provide novel insights into the nature of dandruff and the therapeutic action of potentiated ZPT-containing shampoo, and provide a basis to explore many new mechanistic questions related to these topics.

Xenobiotic metabolism gene expression in the EpiDermin vitro 3D human epidermis model compared to human skin.

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

Human keratinocytes are a major source of cutaneous platelet-derived growth factor.

PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma.

We have discovered a soluble form of the platelet-derived growth factor (PDGF) alpha receptor, designated sPDGF-R alpha, that is produced by and secreted into the conditioned medium of the human osteosarcoma cell line, MG-63. Additionally, sPDGF-R alpha activity has been detected in normal human blood plasma and serum. We have achieved partial purification of this protein by column chromatography using three different affinity matrices: anti-PDGF-R alpha monoclonal antibody (mAb) 292.15-Sepharose, PDGF-BB-Sepharose, and wheat germ agglutinin-agarose. All three matrices have been shown to purify a 90-kDa protein that is recognized by mAbs specific for the PDGF-R alpha extracellular domain. sPDGF-R alpha is capable of binding PDGF ligand in solution and can compete with cell-associated PDGF receptors for ligand binding. We provide three pieces of data suggesting that the sPDGF-R alpha is generated by proteolytic clipping of the full-length PDGF-R alpha protein. First, the conditioned medium of an expression cell line transfected with a cDNA construct designed to produce only full-length PDGF-R alpha exhibits sPDGF-R alpha activity. Second, a truncated intracellular fragment of the PDGF-R alpha, presumably representing the intracellular counterpart of the clipped sPDGF-R alpha, can be immunoprecipitated from the MG-63 osteosarcoma cell extracts using antiserum raised against an intracellular portion of PDGF-R alpha. Finally, we have been unable to detect alternative splicing in the PDGF-R alpha transcript using reverse transcription-polymerase chain reaction.

Transcriptional regulation of the murine k-FGF gene in embryonic cell lines.

Previous studies have shown that embryonal carcinoma (EC) cells express the fibroblast growth factor k-FGF; however, there is a large decrease in the expression of this gene when EC cells differentiate. In addition, it has been shown that differentiation of mouse F9 EC cells reduces the expression of a reporter gene under the control of both the putative human k-FGF promoter and an enhancer-like element that is located in the third exon of the k-FGF gene. Given the low degree of sequence similarity between the human k-FGF gene and the murine k-FGF gene upstream of the transcription start site, it was unclear whether human sequences mimic fully the regulation of the k-FGF gene in mouse cells. To address this question, we have examined the expression of gene constructs containing various regions of the murine k-FGF gene in two mouse EC cell lines and one mouse embryonic stem (ES) cell line. Our results demonstrate that the mouse 5' flanking region, like the human 5' flanking region, cannot support expression of the reporter gene. In both EC cell lines and the ES cell line, expression of the reporter gene is elevated 10- to 100-fold by the addition of a 316-bp region taken from the third exon of the murine k-FGF gene. In addition, we provide evidence that octamer binding proteins are involved in the regulation of the k-FGF gene. Last, this study has identified regions upstream of the transcription start site that appear to regulate the expression of the murine k-FGF gene in EC cells and in ES cells.

Nucleotide sequence of the 5'-flanking region of the mouse k-FGF oncogene exhibits an alternating purine:pyrimidine motif with the potential to form Z-DNA.

The nucleotide sequence of the 5'-flanking region of the mouse k-FGF oncogene has been determined. This sequence extends 2.1 kb upstream from the transcriptional start point (tsp) and includes two Sp1 and two AP-2 consensus binding sequences immediately 5' of the TATA box. In addition, the sequence contains an alternating purine:pyrimidine motif that lies approx. 1 kb upstream from the tsp.

Regulation and expression of transforming growth factor type-beta during early mammalian development.

We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-beta) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-beta activity increases approximately 25-100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-beta activity reflects the induction of TGF-beta 2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-beta 2 as well as a distant member of the TGF-beta family, Vgr-1. Transcripts for TGF-beta 2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC-13 but not in their undifferentiated counterparts. Moreover, TGF-beta 2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-beta 2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-beta 2 and Vgr-1 is closely associated with the induction of differentiation during early development.

Expression and developmental regulation of the k-FGF oncogene in human and murine embryonal carcinoma cells.

Embryonal carcinoma (EC) cells provide an effective model system for studying growth factor production and regulation during mammalian embryogenesis. Our earlier data indicated that the mouse EC cell lines F9 and PC-13 and the human EC cell line NT2/D1 produce a factor with properties similar to those ascribed to members of the fibroblast growth factor (FGF) family and that production of this FGF-related factor is suppressed when all three EC cell lines are induced to differentiate. Subsequent studies suggested that NT2/D1 EC cells express transcripts for basic FGF (bFGF). The current study confirms and extends these findings using a combination of reverse transcription and polymerase chain reaction (RT-PCR). In this study, the expression of bFGF and other members of the FGF family have been examined in F9 and PC-13 cells in addition to NT2/D1 EC cells. In contrast to NT2/D1 EC cells, bFGF expression could not be detected in F9 and PC-13 EC cells. Additionally, expression of four other members of the FGF family (acidic FGF, int-2, FGF-5, and FGF-6) were not detected in NT2/D1, F9, or PC-13 EC cells. However, expression of another member of the FGF family, the k-FGF oncogene, was detected in NT2/D1, F9, and PC-13 EC cells. Moreover, the expression of this transcript is reduced dramatically when each of the three EC cell lines is induced to differentiate. Taken together, our findings argue that expression of the k-FGF oncogene is predominantly responsible for the FGF-related activity detected in EC cells and that differentiation of these EC cells results in suppression of this oncogene.

Recent developments in the structure, function and regulation of platelet-derived growth factor and its receptors.

Recent evidence strongly suggests that production of platelet-derived growth factor (PDGF) is regulated by several mechanisms, including noncoordinate expression of the PDGF A- and B-chain genes, alternative transcript splicing, differential mRNA stability, and translational control. Considerable progress has also been made in our understanding of PDGF receptors. Recent studies demonstrate that there are two distinct PDGF receptor genes. PDGF receptors can undergo dimerization and it has been postulated that dimerization can lead to the formation of three different receptor dimers. In addition, it appears that dimerization is required for activation of PDGF receptors and the biological activity of PDGF. Although relatively little is known about the regulation of PDGF receptors, PDGF receptors are known to be down regulated by PDGF, and expression of PDGF receptors begins relatively early during embryogenesis. Furthermore, the binding of PDGF is regulated by cell density. Unexpectedly, recent evidence suggests that PDGF can localize to the nucleus, which suggests an intranuclear function for PDGF may exist. Together, these findings imply a complex and multi-leveled regulatory scheme for controlling the production of PDGF and its receptors.

Production of growth factors related to fibroblast growth factor and platelet-derived growth factor by human embryonal carcinoma cells.

Previous studies have demonstrated that mouse embryonal carcinoma (EC) cells produce at least two growth factors: one related to platelet-derived growth factor (PDGF) and another related to basic fibroblast growth factor (FGFb). Since human EC cell lines are being used with increased frequency, the current study examined whether human EC cells produce growth factors, in particular those produced by mouse EC cells. In this study, it was determined that the human EC cell line NT2/D1 produces a heat-labile heparin-binding growth factor that behaves like FGF in a bioassay. Three additional criteria suggest that this factor is closely related or identical to FGFb. The factor from NT2/D1 EC cells, bovine FGFb and FGFb produced by the human hepatoma cell line SK-HEP-1 elute from heparin at similar salt concentrations. The factor produced by NT2/D1 EC cells exhibits a thermal stability curve that is nearly identical to those for bovine FGFb and FGFb from SK-HEP-1 cells. Lastly, NT2/D1 and SK-HEP-1 cells express transcripts of the same size that hybridize with a cDNA probe for human FGFb. In the course of these studies it was determined that NT2/D1 EC cells also express several transcripts that hybridize with a cDNA probe for the human PDGF A-chain. Thus, our findings suggest that the pattern of growth factor production by human and mouse EC cells is evolutionarily conserved.

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells.

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.