Non-epidermal Cells Research Articles

Single-cell transcriptome sequencing revealing the difference in photosynthesis and carbohydrate metabolism between epidermal cells and non-epidermal cells of Gracilariopsis lemaneiformis (Rhodophyta).

The allocation of photoassimilates is considered as a key factor for determining plant productivity. The difference in photosynthesis and carbohydrate metabolism between source and sink cells provide the driven force for photoassimilates' allocation. However, photosynthesis and carbohydrate metabolism of different cells and the carbon allocation between these cells have not been elucidated in Gracilariopsis lemaneiformis. In the present study, transcriptome analysis of epidermal cells (EC) and non-epidermal cells (NEC) of G. lemaneiformis under normal light conditions was carried out. There were 3436 differentially expressed genes (DEGs) identified, and most of these DEGs were related to photosynthesis and metabolism. Based on a comprehensive analysis both at physiological and transcriptional level, the activity of photosynthesis and carbohydrate metabolism of EC and NEC were revealed. Photosynthesis activity and the synthesis activity of many low molecular weight carbohydrates (floridoside, sucrose, and others) in EC were significantly higher than those in NEC. However, the main carbon sink, floridean starch and agar, had higher levels in NEC. Moreover, the DEGs related to transportation of photoassimilates were found in this study. These results suggested that photoassimilates of EC could be transported to NEC. This study will contribute to our understanding of the source and sink relationship between the cells in G. lemaneiformis.

Induction of epidermal cell fate in Arabidopsis shoots

Land plants have evolved a cuticle-bearing epidermis to protect themselves from environmental stress and pathogen attack. Despite its important role, little is known about the molecular mechanisms regulating shoot epidermal cell identity. In a recent study, we found that the Arabidopsis thaliana ATML1 gene is possibly a master regulator of shoot epidermal cell fate. We revealed that ATML1 has the ability to confer shoot epidermis-related traits to non-epidermal cells of the seedlings. These data are consistent with the previous loss-of-function mutant analyses, which implied a positive role of ATML1 in epidermal cell differentiation. Importantly, ectopic epidermal cells induced in ATML1-overexpressing lines provide a novel tool to assess the intrinsic properties of epidermal cells and to study epistatic interactions among genes involved in epidermal/mesophyll differentiation. Using this system, we obtained data revealing that ATML1 negatively influenced mesophyll cell fate. In addition, we provided a working model of how division planes in epidermal cells are determined.

Profiling and metaanalysis of epidermal keratinocytes responses to epidermal growth factor.

BackgroundOne challenge of systems biology is the integration of new data into the preexisting, and then re-interpretation of the integrated data. Here we use readily available metaanalysis computational methods to integrate new data on the transcriptomic effects of EGF in primary human epidermal keratinocytes with preexisting transcriptomics data in keratinocytes and in EGF-treated non-epidermal cell types.ResultsWe find that EGF promotes keratinocyte proliferation, attachment and motility and, surprisingly, induces DUSPs that attenuate the EGF signal. Our metaanalysis identified overlapping effects of EGF with those of IL-1 and IFNγ, activators of keratinocyte in inflammation and wound healing. We also identified the genes and pathways suppressed by EGF but induced by agents promoting epidermal differentiation. Metaanalysis comparison with the EGF effects in other cell types identified extensive similarities between responses in keratinocytes and in other epithelial cell types, but specific differences with the EGF effects in endothelial cells, and in transformed, oncogenic epithelial cell lines.ConclusionsThis work defines the specific transcriptional effects of EGF on human epidermal keratinocytes. Our approach can serve as a suitable paradigm for integration of new omics data into preexisting databases and re-analysis of the integrated data sets.

VZV infection of keratinocytes: production of cell-free infectious virions in vivo.

Varicella-zoster virus (VZV) is the cause of varicella (chickenpox) and zoster (shingles). Varicella is a primary infection that spreads rapidly in epidemics while zoster is a secondary infection that occurs sporadically as a result of the reactivation of previously acquired VZV. Reactivation is made possible by the establishment of latency during the initial episode of varicella. The signature lesions of both varicella and zoster are cutaneous vesicles, which are filled with a clear fluid that is rich in infectious viral particles. It has been postulated that the skin is the critical organ in which both host-to-host transmission of VZV and the infection of neurons to establish latency occur. This hypothesis is built on evidence that the large cation-independent mannose 6-phosphate receptor (MPR(ci)) interacts with VZV in virtually all infected cells, except those of the suprabasal epidermis, in a way that prevents the release of infectious viral particles. Specifically, the virus is diverted in an MPR(ci)-dependent manner from the secretory pathway to late endosomes where VZV is degraded. Because nonepidermal cells are thus prevented from releasing infectious VZV, a slow process, possibly involving fusion of infected cells with their neighbors, becomes the means by which VZV is disseminated. In the epidermis, however, the maturation of keratinocytes to give rise to corneocytes in the suprabasal epidermis is associated uniquely with a downregulation of the MPR(ci). As a result, the diversion of VZV to late endosomes does not occur in the suprabasal epidermis where vesicular lesions occur. The formation of the waterproof, chemically resistant barrier of the epidermis, however, requires that constitutive secretion outlast the downregulation of the endosomal pathway. Infectious VZV is therefore secreted by default, accounting for the presence of infectious virions in vesicular fluid. Sloughing of corneocytes, aided by scratching, then aerosolizes the virus, which can float with dust to be inhaled by susceptible hosts. Infectious virions also bathe the terminals of those sensory neurons that innervate the epidermis. These terminals become infected with VZV and provide a route, retrograde transport, which can conduct VZV to cranial nerve (CNG), dorsal root ganglia (DRG), and enteric ganglia (EG) to establish latency. Reactivation returns VZV to the skin, now via anterograde transport in axons, to cause the lesions of zoster. Evidence in support of these hypotheses includes observations of the VZV-infected human epidermis and studies of guinea pig neurons in an in vitro model system.

The widely expressed extracellular matrix protein SMOC-2 promotes keratinocyte attachment and migration

SMOC-2 is a recently discovered member of the BM-40/SPARC/osteonectin family of extracellular multidomain proteins of so far unknown function. While we have shown earlier that the homologous protein SMOC-1 is associated with basement membranes, in this study we demonstrate that, in the mouse, SMOC-2 could be detected in a large number of non-basement membrane localizations, often showing a diffuse tissue distribution. A more distinct expression pattern was seen in skin where SMOC-2 is mainly present in the basal layers of the epidermis. Functionally, recombinant SMOC-2 stimulated attachment of primary epidermal cells as well as several epidermal-derived cell lines but had no effect on the attachment of non-epidermal cells. Inhibition experiments using blocking antibodies against individual integrin subunits allowed the identification of αvβ6 and αvβ1 integrins as important cellular receptors for SMOC-2. Cell attachment as well as the formation of focal adhesions could be attributed to the extracellular calcium-binding domain. The calcium-binding domain also stimulated migration, but not proliferation of keratinocyte-like HaCaT cells. We conclude that SMOC-2, like other members of the BM40/SPARC family, acts as a regulator of cell–matrix interactions.

Viewpoint 3

Viewpoint 3

The combination of ubiquitous transcription factors AP‐1 and Sp1 directs keratinocyte‐specific and differentiation‐specific gene expression in vitro

Previous studies of epidermal-specific gene promoters suggested that a limited set of transcription factors regulate keratinocyte-specific and differentiation-specific gene expression in the epidermis. In the present study, we investigated the functional importance of AP-1- and Sp1-binding elements in the determination of cell type-specific and differentiation-specific gene expression by transient transfection into undifferentiated and differentiated keratinocytes as well as into various non-epidermal cell lines. Synthesized short AP-1- and/or Sp1-binding elements were inserted into a minimal reporter vector, and the artificial promoter containing both AP-1 and Sp1 elements showed high levels of transcriptional activity only when transfected into differentiated keratinocytes. Promoters containing either the AP-1 or the Sp1 motif alone showed little activity in any of the cells examined. We also found that close proximity of the Sp1 and AP-1 sites is essential for transcriptional activity, suggesting that the physical interaction between Sp1 and AP-1 factors is important for functional activity. These results clearly demonstrate that the combination of ubiquitously expressed transcription factors AP-1 and Sp1 confers keratinocyte specificity and differentiation specificity on the gene expression. Our findings also provide a simple model of the mechanisms underlying regulation of cell type-specific and cell differentiation-specific gene expression by ubiquitously expressed transcription factors.

Immunological characterization of a newly developed antibody for localization of a beta‐keratin in turtle epidermis

Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.

Immunological characterization and fine localization of a lizard beta‐keratin

Scales of lizards contain beta-keratin of poorly known composition. In the present study, a rat polyclonal serum against a lizard beta-keratin of 14-15 kDa has been produced and the relative protein has been immunolocalized in the epidermis. The observations for the first time show that the isolated protein band derives from the extraction of a protein component of the beta-keratin filaments of lizard epidermis. In immunoblots and immunocytochemistry, the antiserum recognizes most lizard beta-keratins, but produces a variable cross-reactivity with snake beta-keratins, and weak or no reactivity with beta-keratins isolated from tuatara, turtles, alligator and birds. In bidimensional immunoblots of lizard epidermis, three main spots at 15-16 kDa with isoelectric point at 7.0, 7.6 and 8.0, and an unresolved large spot at 29-30 kDa and with pI at 7.5-8.0, are obtained, may be derived from the aggregation of smaller beta-keratin proteins. The ultrastructural immunolocalization with the antibody against lizard beta-keratin shows that only small and large beta-keratin filaments of beta-cells of lizard epidermis are labeled. Keratin bundles in oberhautchen cells are less immunolabeled. Beta-keratin is rapidly polymerized into beta-packets that merge into larger beta-keratin filaments. No labeling is present over other cell organelles or cell layers of lizard epidermis, and is absent in non-epidermal cells. The antiserum recognizes epitope(s) characteristics for lizard beta-keratins, partially recognized in snakes and absent in non-lepidosaurian species. This result indicates that beta-keratins among different reptilian groups posses different immunoreactive regions.

Demonstration of characteristic skin surface contours of extramammary Paget's disease and parapsoriasis en plaque by image analysis of negative impression replicas

The surface contours of lesional skin of certain skin diseases, such as parapsoriasis en plaque (PEP) and extramammary Paget's disease (EMPD), in which there is a massive infiltration by non-epidermal cells, looks somewhat different from that of the adjacent normal skin, needless to state that they are apparently different from that of ordinary chronic inflammatory dermatoses where we found acanthotic epidermis accompanied by hyperkeratosis. We attempted to objectively characterize these unique skin surface changes qualitatively and quantitatively using non-invasive methods. Negative impression replicas were taken from the lesional skin of patients with EMPD or PEP as well as from the adjacent uninvolved skin. The findings were confirmed histologically. The replicas were examined by using computerized image analysis. Several parameters were analyzed that correlate with the changes in the anisotropy of the skin furrows (VC1), average skin roughness (KSD), average length of skin furrows (LEN), and number of skin furrows (NUM). There were significant decreases in KSD and NUM in EMPD, indicating a smoother skin surface in the lesional skin than in the adjacent normal skin. In contrast, the PEP lesion had an increase in VC1 and LEN and a decrease in NUM, which suggests larger skin ridges in the lesional skin than in the uninvolved skin. Thus the unique skin surface of the cutaneous disorders accompanied by epidermal invasion by non-epidermal cells, such as EMPD and PEP, was characterized both qualitatively and quantitatively using computerized image analysis of negative impression replicas.

Variation in epidermal housekeeping gene expression in different pathological states.

Using non-radioactive in situ hybridization we studied the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PolyA+ RNA in psoriasis and normal skin, and GAPDH in epidermis following application of a range of noxious stimuli, including ultraviolet radiation, Sellotape stripping and various irritants. In keeping with what might have been expected from previous results on cell culture of various non-epidermal cell types, expression of these putative control gene products is not constant. We suggest that the use of GAPDH, and possibly other control genes, may lead to error in interpreting experiments using Northern blotting or even RT-PCR of epidermal samples.

Activity regulation of a Hox protein and a role for the homeodomain in inhibiting transcriptional activation.

Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). We present results supporting a novel mechanism for Hox cofactor function: regulation of transcriptional activation function. First, we provide evidence that the Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. Secondly, either Dfd or a Dfd-VP16 hybrid mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independently of Exd enhancement of Dfd-binding affinity on the composite site. Thirdly, transfection assays confirmed that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family.

Anti-tumor promoting action of phthalic acid mono-n-butyl ester cupric salt, a biomimetic superoxide dismutase

Skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by a concurrent and topical application of phthalic acid mono-n-butyl ester cupric salt (PAMBCu) in CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene. PAMBCu inhibited TPA-caused epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. skin inflammation. However, neither PAMBCu nor superoxide dismutase (SOD) inhibited TPA-caused ODC induction in primary cultured mouse epidermal cells. 7-Bromomethylbenz[a]anthracene (BrMBA) is known to be a non-TPA type of tumor promoting agent. Epidermal ODC induction and inflammation caused by BrMBA were not inhibited by a concurrent application of PAMBCu. When mice were topically treated twice with PAMBCu, i.e. concurrently with and 7 h after BrMBA treatment, BrMBA-caused ODC induction was markedly suppressed. The same dose regimen of PAMBCu, however, failed to inhibit tumor promotion and inflammation caused by BrMBA. PAMBCu showed SOD-mimetic activity in superoxide generating systems, i.e. xanthine-xanthine oxidase reaction and TPA-stimulated polymorphonuclear leukocytes (PMN). Mono-n-butyl phthalate, which lacks SOD-mimetic activity, failed to inhibit TPA-caused ODC induction and skin inflammation. Therefore, inhibition by PAMBCu of TPA-caused tumor promotion, epidermal ODC induction and inflammation may be attributable to its SOD-mimetic activity. The results also support the contention that a superoxide anion of non-epidermal cell origin, such as PMN and macrophages, plays a role (probably some enhancing role) in in vivo ODC induction and tumor promotion caused by TPA. Failure of PAMBCu to inhibit BrMBA-caused tumor promotion suggests that superoxide anion generation is not involved in the tumor promoting action of this agent and that the anti-tumor promoting action of PAMBCu is dependent on the nature of the tumor promoting agents.

Developmental changes in keratin patterns during epidermal maturation

The biochemical maturation of the epidermis of Xenopus laevis was examined through an identification of the keratins expressed at selected stages of development. The keratin patterns obtained were compared to those observed in the adult epidermis and two Xenopus non-epidermal, epithelial cell lines. The keratins expressed during development can be grouped into three classes: (1) keratins which are restricted to the embryonic epidermis (58 and 59 kDa); (2) keratins which are prominent during development, but become minor components of the adult epidermis (47, 48, and 60 kDa); and (3) keratins which accumulate during development to become the major keratins of the adult epidermis (49, 53, 56, and 63 kDa). The embryo-specific keratins are present at all developmental stages prior to metamorphosis which we have investigated, but disappear when the epidermis keratinizes during metamorphosis. Both class 1 and 2 keratins, while undetectable or minor components of the adult skin, are present in the two non-epidermal cell lines. In contrast, the class 3 keratins show little overlap with the keratins of these cell lines. All of the class 3 keratins appear after hatching with the exception of the 53-kDa keratin which is present at the earliest developmental stage which we have examined. All of the major keratins of the adult epidermis accumulate as metamorphosis proceeds, while the embryo-restricted keratins are gradually lost.

Commentary: Cyclic Adenosine Monophosphate Regulation of Normal and Psoriatic Epidermis

An article previously published in theArchives1proposed decreased activity in the cyclic adenosine monophosphate (cAMP) cascade as one potential molecular explanation of the following three characteristics of lesional epidermis in psoriasis: (1) increased cellular proliferation, (2) increased glycogen, and (3) decreased cellular differentiation. In nonepidermal cells, cAMP was known to decrease proliferation, decrease glycogen, and increase differentiation. The cAMP hypothesis assumed that, at the level of molecular regulation, epidermal and nonepidermal cells would be similar rather than dissimilar. If so, decreased cascade activity in different epidermal layers would be one explanation for three lesional characteristics. EPIDERMAL HOMEOSTASIS If the cAMP hypothesis is a tenable explanation for misregulated epidermal proliferation in psoriasis, it must be compatible with our current understanding of proliferative homeostasis. This is the mechanism used to maintain low-level cell proliferation that is sufficient to provide new cells to replace those sloughed from the surface of relatively

Epidermal nuclear IgG deposition in normal skin: characterization by anti-ribonucleoprotein IgG-Fab fragments

Epidermal nuclear IgG deposition in clinically normal skin may occur in patients with scleroderma or scleroderma-like features. In order to evaluate the mechanisms of the fixation, Fab fragments of anti-RNP IgG antibodies, obtained after papain digestion, were incubated for increasing times with various substrates: human skin, human mononuclear cells, cultured human fibroblasts and rabbit lip. Our results showed that anit-RNP IgG-Fab fragments could penetrate most of the living cells of human skin and rabbit lip and, to a lesser degree, mononuclear cells and poorly cultured fibroblasts. No ability to fix was found either with anti-RNP IgG-Fe fragments or with anti-nDNA/DNP IgG-Fab. It was concluded that anti-RNP IgG could penetrate viable epidermal and non-epidermal cells and that surface Fe receptors must play a minor role in the cellular penetration of antibodies.

The Culture of Small Aggregates of Amphibian Embryonic Cells in vitro

ABSTRACT Methods are described for the short term culture of disaggregated amphibian embryonic cells in a medium consisting of about 0·1 per cent protein in Steinberg’s physiological salt solution. In a medium containing whole serum or serum protein cells differentiate according to their presumptive fate in the embryo. The use of a particle-free extract of mouse bone marrow causes ectoderm cells to develop as mesoderm. Gastrula ectoderm cells will differentiate ciliated epidermis providing they are permitted to reaggregate into balls of about thirty cells and upwards. Gastrula mesoderm cells and induced ectoderm cells likewise do not differentiate unless aggregated, but the majority of aggregates fail to differentiate. This seems to be because these cells have exacting requirements for precise concentrations of the protein component in the medium. There is some variation between species in this respect, and requirements vary between ovulations in the case of Rana pipiens and Xenopus laevis. Ciliated epidermal cells differentiate in cohesive balls that detach from the glass. All other cell types differentiate as thin outgrowths of cells attached to the glass by cytoplasmic fringes or pseudopodia. The first visible event in the differentiation of non-epidermal cells is their attachment to the glass by pseudopodia or cytoplasmic fringes, and the rapid expansion of these contributes to the deformation of the cell from the spherical embryonic form to that approximating to the form of the differentiated cell. This rapid change in form and activity is termed germination. It appears to be a necessary preliminary for cytodifferentiation, the rapid utilization of yolk and the build up of large mitochondrial populations. Germination precedes the formation of striated muscle fibres, vacuoles in chordal cells and other indications of cytodifferentiation by a time interval that is characteristic for each cell type within a species. The time and order in which cells differentiate in culture is related to the behaviour of comparable cells in the embryo. It is concluded that, by the neurula stage, the control of the time of germination is vested in the individual cells. It is concluded that the development of neural and mesodermal cells involves two necessary periods of decisive change, one at the end of gastrulation unconnected with any gross morphological changes in the cells, the second, closely following neural closure and involving the in vivo counterpart of germination in culture, by which the potentialities of the cells are mobilized in the interests of cytodifferentiation. Both these important epigenetic events coincide with changes in the adhesive properties of cells.