Biography:

In the past Richard L. Eckert has collaborated on articles with Benita S. Katzenellenbogen and Lakshmi Kasturi. One of their most recent publications is Mechanisms of estrogen and antiestrogen action in mammary cancer. Which was published in journal Journal of Steroid Biochemistry.

More information about Richard L. Eckert research including statistics on their citations can be found on their Copernicus Academic profile page.

Richard L. Eckert's Articles: (21)

Mechanisms of estrogen and antiestrogen action in mammary cancer

AbstractWe have evaluated the estrogen antagonist and agonist actions of antiestrogens in ovarian-dependent and autonomous rat mammary tumors, and in human MCF-7 breast cancer cells. Two of the antiestrogens (CI628 and U23,469), which we have prepared in high specific activity radiolabeled form, are metabolized during their action in vivo to demethylated forms that are preferentially localized in the nucleus and have a higher receptor affinity.Antiestrogens elicit regression of 90% of DMBA-induced tumors and during tumor regression, estrogen receptors are mainly localized in the nucleus and the level of prolactin receptors is diminished. Antiestrogens also markedly diminish the growth rate of the ovarian-autonomous but estrogen-sensitive R3230AC mammary tumor. Our studies indicate differences in the responses of tumor enzymes (glucose-6-phosphate dehydrogenase, malic enzyme, peroxidase) to estradiol and antiestrogenUtilizing the high affinity tritium-labeled CI628 metabolite, CI628M, we find the antiestrogen-receptor complex to be similar to that of the estradiol-receptor complex in many ways, but to differ in its sedimentation properties and in its binding to DNA-cellulose. These may be suggestive of differences in chromatin interaction that may underlie the antagonist character of antiestrogens.

ArticleStructure and evolution of the human involucrin gene

AbstractInvolucrin is a keratinocyte protein that first appears in the cell cytosol, but ultimately becomes cross-linked to membrane proteins by transglutaminase. The gene for human involucrin has now been cloned and sequenced. The central segment of the coding region contains 39 repeats of a 30 nucleotide sequence whose ten encoded amino acids include three glutamines and two glutamic acids. This segment must have originated by successive duplications. Later duplications of modified sequences within the central segment can also be identified. Flanking the central segment lie shorter coding segments, a part of which must have given rise to the central segment. The flanking segments also show homology to a simpler 30 nucleotide sequence from which they likely originated. The evolution of involucrin as a substrate of transglutaminase and an envelope precursor was evidently made possible by this process of repeated mutation and duplication.

Regular ArticleCalcium Modulates Cornified Envelope Formation, Involucrin Content, and Transglutaminase Activity in Cultured Human Ectocervical Epithelial Cells

AbstractThe entrance to the cervical canal, the ectocervix, is covered by a stratified squamous epithelium. In the present study we demonstrate that the extracellular calcium levels regulate the differentiation of cultured human ectocervical epithelial cells (ECE cells). ECE cells growing in serum-free medium containing 0.09 mM calcium remain as individual cells that do not spread on the culture dish or form cornified envelopes. Increasing the extracellular calcium concentration results in a dose-dependent increase in ECE cell differentiation that is manifest by an increase in cornified envelope (superficial cell) formation. These morphological changes are accompanied by a twofold increase in the levels of involucrin, a precursor of the cornified envelope. The extent of increase in involucrin mRNA is similar to that of involucrin protein, suggesting that the regulation is at the level of involucrin mRNA synthesis and/or stability. Transglutaminase, a calcium-activated enzyme responsible for assembly of the covalent cross-links that form the cornified envelope, increases to a similar extent to the increase in cornified envelope formation, suggesting that in ECE cells, activation of transglutaminase may be the rate-limiting step in envelope assembly. These results and the finding of a calcium gradient in other stratifying epithelia in vivo , indicate that the calcium is likely to be an important regulator of ectocervical epithelial cell differentiation in vivo.

Regular ArticleTranscriptional Regulation of the EGF Receptor Promoter by HPV16 and Retinoic Acid in Human Ectocervical Epithelial Cells

AbstractWe have previously demonstrated that human papillomavirus 16 (HPV16)-immortalized human ectocervical epithelial cells and cells derived from tumors which express HPV16 oncogenes express high levels of epidermal growth factor receptor (EGFR) compared to normal cervical cells. We have also shown that proliferation of these cells is inhibited by retinoid treatment. We have hypothesized that the retinoid inhibition of cell proliferation may be due to the retinoid-dependent reduction in EGFR level. In this study we examine the regulation of EGFR expression in cervical cells with emphasis on two aspects: (1) the mechanism of retinoid-dependent suppression of EGFR levels in HPV16-positive cells and (2) the mechanism of EGFR upregulation by HPV16. EGFR levels were found to be elevated 5-, 3.7-, and 1.25-fold in the HPV16-immortalized ECE16-1, ECE16-D1, and ECE16-D2 cells, respectively, compared to normal cervical cells. Treatment of ECE16-1 and ECE16-D1 cells with retinoic acid suppresses proliferation, EGFR level, EGFR mRNA level, and EGFR promoter activity. The reduction in EGFR promoter activity appears to account for the reduction in EGFR protein and mRNA levels. In contrast, retinoic acid does not affect cell growth or EGFR level in ECE16-D2 cells or normal cervical cells. To study the mechanisms regulating EGFR expression in HPV16-positive cells, normal ECE cells were cotransfected with an EGFR promoter reporter plasmid and an expression plasmid encoding the HPV16 E6/E7 open reading frames. In the presence of E6/E7, EGFR promoter activity was increased by 2- to 3-fold, suggesting that the E6/E7 proteins are directly or indirectly responsible for the increased EGFR level and that the EGFR promoter contains the DNA elements necessary to mediate this response. Nevertheless expression of E6/E7 proteins did not confer retinoic acid regulation, as EGFR promoter activity remained elevated in normal cells cotransfected with pHPVE6/E7 and treated with retinoic acid. These results suggest that human papillomavirus and retinoic acid regulate EGFR levels by independent effects on the EGFR promoter.

Selected papers from Pacific Coast Fertility SocietyIdentification of insulin-like growth factor binding proteins in human oviduct*†

ObjectiveTo determine if human oviduct expresses messenger ribonucleic acids (mRNAs) encoding insulin-like growth factor binding proteins (IGFBPs), if oviductal epithelium secretes IGFBPs into conditioned medium (CM), and if IGFBP secretion is regulated by steroid hormones.DesignNorthern blots of RNA, isolated from late proliferative phase human fimbria and oviductal isthmus, were probed with complementary deoxyribonucleic acids encoding IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. In addition, oviductal ampullary epithelial cells were cultured with and without estrogen and/or progesterone (P), and IGFBPs were examined in CM by Western ligand blot analysis and identified using specific antisera.SettingTissue was obtained from hysterectomy specimens at Stanford University Hospital, a private teaching institution.Patients, ParticipantsPatients undergoing hysterectomy for benign disease.InterventionsNone.Main Outcome MeasuresTranscripts of IGFBP mRNA and IGFBPs secreted into CM were detected by autoradiography of Northern and Western ligand blots, respectively.ResultsMessenger RNA transcripts encoding IGFBP-2, -3, and -4 were detected, whereas IGFBP-1 mRNA was barely detectable in oviductal tissue. In CM, IGFBP-2 and IGFBP-3 were detected, as was a unique 24–kd IGFBP, although IGFBP-1 was not observed. Estrogen and/or P did not regulate the secretion of these IGFBPs by cultured oviductal epithelium.ConclusionsHuman oviduct expresses mRNAs encoding IGFBP-2, IGFBP-3, and IGFBP-4, and in vitro oviductal epithelium secretes IGFBP-2, IGFBP-3, and a unique binding protein of 24 kd, which may be the recently identified IGFBP-4.

Original Articlep38 Mitogen-Activated Protein Kinases on the Body Surface – A Function for p38δ

The p38 family of mitogen-activated protein kinases includes p38α (SAPK2a, CSBP), p38β (SAPK2b), p38δ (SAPK4), and p38γ (SAPK3/ERK6). p38α and p38β are widely expressed p38 isoforms that are involved in regulation of cell proliferation, differentiation, development, and response to stress. Relatively less is known regarding the function of the p38δ isoform. In this review, we discuss the role of the p38α, p38β, and p38γ isoforms and then present recent findings that define a role for p38δ as a regulator of differentiation-dependent gene expression in keratinocytes.

Review: Dermatology FoundationRegulation of Involucrin Gene Expression

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38δ–extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

Original ArticleThe Distal and Proximal Regulatory Regions of the Involucrin Gene Promoter have Distinct Functions and Are Required for In Vivo Involucrin Expression

Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions – the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia.

Original ArticleS100A7 (Psoriasin) – Mechanism of Antibacterial Action in Wounds

S100A7, also called psoriasin, is a member of the S100 multigene family that is encoded in the epidermal differentiation complex on chromosome 1q21. S100A7 is highly expressed in epidermal hyperproliferative disease; however, its function is not well understood. These studies show high levels of monomer and covalently crosslinked high molecular weight S100A7 in human wound exudate and granulation tissue. Immunohistological studies suggest that this S100A7 is produced by keratinocytes surrounding the wound and is released into the wound exudate. S100A7 is also detected in keratinocyte-conditioned cell culture medium. Studies using recombinant S100A7 indicate that it adheres to and reduces E. coli survival. Mutation of the conserved carboxyl-terminal EF-hand calcium-binding motif or heat denaturation slightly reduces S100A7 antibacterial activity; however, the antibacterial activity is destroyed by protease treatment. Mutation of the zinc-binding motif, located at the C-terminus, reduces antibacterial activity; however, this reduction can be reversed by simultaneous removal of the amino terminus. This indicates the surprising finding that the central region of S100A7, including only amino acids 35–80, is sufficient for full antibacterial activity. These studies also indicate that reduced S100A7 association with bacteria is associated with reduced antibacterial activity.

Original ArticleExpression of Bmi-1 in Epidermis Enhances Cell Survival by Altering Cell Cycle Regulatory Protein Expression and Inhibiting Apoptosis

The polycomb group (PcG) genes are epigenetic suppressors of gene expression that play an important role in development. In this study, we examine the role of Bmi-1 (B-cell-specific Moloney murine leukemia virus integration site 1) as a regulator of human epidermal keratinocyte survival. We identify Bmi-1 mRNA and protein expression in epidermis and in cultured human keratinocytes. Bmi-1 is located in the nucleus in cultured keratinocytes, and in epidermis it is expressed in the basal and suprabasal layers. Adenovirus-delivered Bmi-1 promotes keratinocyte survival and protects keratinocytes from stress agent-mediated cell death. This is associated with increased levels of cyclin D1 and selected cyclin-dependent kinases, and reduced caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage. Bmi-1 may be involved in the maintenance of disease state, as Bmi-1 levels are elevated in transformed keratinocytes, skin tumors, and psoriasis. The presence of Bmi-1 in suprabasal non-proliferative cells of the epidermis and within a high percentage of cells within skin tumors suggests a non-stem cell pro-survival role for Bmi-1 in this tissue. Based on the suprabasal distribution of Bmi-1 in epidermis, we propose that Bmi-1 may promote maintenance of suprabasal keratinocyte survival to prevent premature death during differentiation. Such a function would help assure proper formation of the stratified epidermis.

Original ArticleSuppressing AP1 Factor Signaling in the Suprabasal Epidermis Produces a Keratoderma Phenotype

Keratodermas comprise a heterogeneous group of highly debilitating and painful disorders characterized by thickening of the skin with marked hyperkeratosis. Some of these diseases are caused by genetic mutation, whereas other forms are acquired in response to environmental factors. Our understanding of signaling changes that underlie these diseases is limited. In the present study, we describe a keratoderma phenotype in mice in response to suprabasal epidermis-specific inhibition of activator protein 1 transcription factor signaling. These mice develop a severe phenotype characterized by hyperplasia, hyperkeratosis, parakeratosis, and impaired epidermal barrier function. The skin is scaled, constricting bands encircle the tail and digits, the footpads are thickened and scaled, and loricrin staining is markedly reduced in the cornified layers and increased in the nucleus. Features of this phenotype, including nuclear loricrin localization and pseudoainhum (autoamputation), are characteristic of the Vohwinkel syndrome. We confirm that the phenotype develops in a loricrin-null genetic background, indicating that suppressed suprabasal AP1 factor function is sufficient to drive this disease. We also show that the phenotype regresses when suprabasal AP1 factor signaling is restored. Our findings suggest that suppression of AP1 factor signaling in the suprabasal epidermis is a key event in the pathogenesis of keratoderma.

Original ArticleInvolucrin Is a Covalently Crosslinked Constituent of Highly Purified Epidermal Corneocytes: Evidence for a Common Pattern of Involucrin Crosslinking in Vivo and in Vitro

Involucrin (hINV) is an important structural component of the keratinocyte confined envelope that is expressed early in the keratinocyte differentiation process and is thought to be a component of the initial envelope scaffolding. We have previously shown that cyanogen bromide (CNBr) cleavage of cornified envelopes isolated from cultured foreskin keratinocytes releases several discrete involucrin-immunoreactive peptides. In this study, we compare the pattern of release of immunoreactive hINV fragments from envelopes prepared from human breast skin and foreskin, and from spontaneous and induced envelopes prepared from cultured keratinocytes. We also identify one of the released products. Envelopes prepared from human breast skin or foreskin, or spontaneous or induced envelopes prepared from cultured cells differ significantly in structure. The envelopes isolated from epidermis appear to be structurally complete, whereas spontaneous envelopes appear less complete and the induced envelopes appear to be the least complete. In spite of these structural differences, CNBr cleavage releases an identical quartet of hINV-immunoreactive peptides migrating between 68 and 81 kDa from each preparation. Immunoblots indicate that the quantity of hINV-immunoreactive material released per μg of envelope protein is as follows: induced >  spontaneous >  foreskin >  breast skin. The fastest migrating peptide (68 kDa) comigrates with a peptide that is released after CNBr cleavage of bacterially produced recombinant hINV. Amino-terminal amino acid sequencing of this peptide from recombinant hINV and from the cornified envelopes yields the sequence G-Q-L-K-H-L-E-Q-Q-E-G-Q-P-K-H. These results suggest that this fragment is the 275-amino acid segment of hINV beginning at G311 and extending to K585 and that this peptide is not crosslinked to another protein. These results indicate that a population of the enveloped-associated hINV present in cultured and in vivo keratinocytes is crosslinked in the amino-terminal half. It is possible that this species represents an early intermediate in the involucrin crosslinking process.

Original ArticleGeneticsEmbryonic AP1 Transcription Factor Deficiency Causes a Collodion Baby-Like Phenotype

AP1 transcription factors are important controllers of gene expression in the epidermis, and altered AP1 factor function can perturb keratinocyte proliferation and differentiation. However, our understanding of how AP1 signaling changes may underlie or exacerbate skin disease is limited. We have shown that inhibiting AP1 factor function in suprabasal adult epidermis leads to reduced filaggrin levels and to a phenotype that resembles the genetic disorder ichthyosis vulgaris. We now show that inhibiting AP1 factor function during development in embryonic epidermis produces marked phenotypic changes including reduced filaggrin mRNA and protein levels, compromised barrier function, marked ultrastructural change, and enhanced dehydration susceptibility that resembles the phenotype observed in the flaky tail mouse, a model for ichthyosis vulgaris. In addition, the AP1 factor-deficient newborn mice display a collodion membrane phenotype that is not observed in flaky tail mice or in newborn individuals with ichthyosis vulgaris but is present in other forms of ichthyosis. This mixed phenotype suggests the need for a better understanding of the possible role of filaggrin loss and AP1 transcription factor deficiency in ichthyoses and collodion membrane formation.

Original ArticleGlutathione S-Transferases in Human and Rodent Skin: Multiple Forms and Species-Specific Expression

The glutathione S-transferases (GST) are a family of widely distributed multifunctional detoxification enzymes that catalyze the reaction between reduced glutathione and a variety of electrophiles. Of interest is the fact that several extracutaneous tissues exhibit a distinct spectrum of isozymes that are expressed in a highly controlled fashion. Despite the fact that the skin is continuously exposed to numerous injurious agents, little is known about the expression of GST isozymes and their role in metabolism of physiologic and xenobiotic substrates in cutaneous tissue. Using specific polyclonal antibodies to the Alpha, Mu, and Pi classes of GST, we identified their expression in rat, mouse, and human skin cytosol. In each species, GST isozymes expressed activities towards 1- chloro-2,4-dinitrobenzene, benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, leukotriene A4, and ethacrynic acid, but not towards bromosulfophthalein and cumene hydroperoxide. Western blot analysis indicated the predominant expression of Pi isozyme in all three species. Alpha class of isozyme(s) was present only in human skin, whereas Mu class of isozyme(s) was detected only in rat and mouse skin. Similarly, in normal and transformed cultured human keratinocytes Pi was the predominant isozyme. In situ localization studies using immunohistochemical techniques confirmed the observations of Western blotting. In mouse skin, Pi and Mu isozyme(s) were found to be predominantly localized in sebaceous glands, whereas no reactivity was observed with the Alpha class of isozymes. Our data show that multiple forms of GST exist in rodent and human skin and that GST Pi is the predominant isozyme in each species. Furthermore, cutaneous GST can metabolize both endogenous substrates and foreign compounds.

ReviewInvolucrin—Structure and Role in Envelope Assembly

Recent findings have revealed much about the structure of involucrin. These findings make it possible to propose specific models regarding the role of involucrin and the mechanism of its cross-linking as an envelope precursor. These models provide clearly testable hypotheses that are expected to provide additional insights into the mechanism of cornified envelope assembly.

ReviewKeratinocyte proliferation, differentiation, and apoptosis—Differential mechanisms of regulation by curcumin, EGCG and apigenin

AbstractWe have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (−)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulates keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38δ-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents.

Retinoid-dependent transcriptional suppression of cytokeratin gene expression in human epidermal squamous cell carcinoma cells

AbstractWe have previously demonstrated that cytokeratin levels are coordinately regulated in normal cultured human keratinocytes. In the present study we examine the mechanism of this regulation using human squamous cell carcinoma (SCC) cells. Treatment of SCC-13 cells with 20 or 200 nM trans-retinoic acid results in nearly complete suppression of cytokeratin K5 and K6 expression. This change is accompanied by a simultaneous reduction (> 20-fold) in the level of the mRNAs encoding K5 and K6. Transcriptional analysis indicates that the transcription rate of the K5 and K6 genes drops by approximately four to fivefold in retinoid treated nuclei. Retinol (2000 nM) also promotes this change. In contrast, cytokeratin K19 does not increase in the presence of retinoic acid, thus the normal coordinate regulation of keratin gene expression by retinoids appears to be uncoupled in SCC-13 cells. However, this does not represent a general defect in positive regulation of gene expression by retinoids, since in a transient transfection assay trans-retinoic acid positively regulates a reporter plasmid containing the retinoid response element from the retinoic acid receptor-β gene.The synthetic retinoids Ro 13–6298 (ethyl ester) and its metabolic derivative Ro 13–7410 (free acid) are both active in modulating the differentiation of normal keratinocytes. In contrast, only Ro 13–7410 is active in SCC-13 cells. As Ro 13–6298 binds poorly to the retinoic acid receptors, this suggests that SCC-13 cells, unlike normal keratinocytes, lack the ability to convert Ro 13–6298 to the active Ro 13–7410. These results further suggest that an inability to metabolize retinoid ethyl esters may be a property of transformed epidermal keratinocytes.

Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype

AbstractInvolucrin is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of the epidermis and other stratifying squamous epithelia. To study involucrin gene expression and the function of involucrin, we expressed a 6 kb DNA fragment of the human involucrin gene, containing approximately 2.5 kb of upstream sequence and 0.5 kb of downstream sequence, in transgenic mice. The transgene produces a 68 kDa protein that is detected by a human involucrin-specific antibody, and is expressed in a tissuespecific and differentiation-appropriate manner (i.e., expression is confined to the suprabasal layers of the epidermis, extocervix, trachea, esophagus and conjunctiva).Soluble involucrin levels are two to four times higher in transgenic epidermal keratinocytes compared to human foreskin keratinocytes. Newborn heterozygous animals have a normal birth weight and a normal appearing epidermis and hair growth begins at 4 to 5 days of age (i.e., the same time as hair growth in non-transgenic animals). In a subpopulation of the newborn homozygous animals birth weight is reduced, the epidermis is scaly and hair growth begins late, at around 9 to 10 days of age. In addition, the hair tends to stand erect on both heterozygous and homozygous adult animals giving the appearance of diffuse alopecia.Immunofluorescent and electron microscopy localize involucrin in the hair follicle and cornified envelope, respectively. These results suggest that overexpression of involucrin may cause abnormalities in hair follicle structure/function and cornified envelope structure. These animals provide a new model for the study of cornified envelope structure and function.

THE JOURNAL OF THE INTERNATIONAL SOCIETY OF DIFFERENTIATIONHuman uterine cervical epithelial cells grown on permeable support – a new model for the study of differentiation

AbstractThe purpose of the present study was to establish culture conditions for human uterine cervical epithelial cells on permeable support and to determine how it affects cervical cell differentiation. Human ectocervical epithelial cells (hECE), HPV-16 immortalized hECE cells (ECE16-1) and Caski cells were grown on collagen-coated filters. Culture conditions, density of cells in culture and expression of epithelial and cervical-cell phenotypic markers were determined and compared in cells grown on filter and on solid support. Compared with the latter, cultures on filter had a higher cell density, hECE cells stratified to 5–12 cell layers compared to 1–3 on solid support, and cells of all three types expressed intercellular tight junctions. The cytokeratin profiles revealed differences between the three cell types as well as differences within the same cell species when grown on filter, compared to solid support. Of particular importance was the finding of a higher expression of K-13 in hECE grown on filter compared to solid support; K-13 is a marker of ectocervical cell differentiation. The cytokeratin profiles of the cultured hECE, ECE16-1 and Caski cells resembled those of ectocervical, squamous metaplastic and endocervical epithelia, respectively. hECE and ECE16-1 expressed involucrin protein, the level of which in both was higher in cells grown on filter compared to solid support.Polarization of the cultures was determined by morphology (stratification of hECE cells, expression of pseudomicrovilli in the apical cell membrane), selective apical vs. basolateral secretion of [35S]methionine- and [35S]cys-teine-, [3H]fucose- and [14C]glucosamine-labeled mole-cules, and positive short-circuit current (Isc) under voltage-clamp conditions.Confluency of the cultures was determined by measuring transepithelial unidirectional fluxes of inert molecules with different molecular weights (MWs) through the paracellular pathway, and by measuring transepithelial conductance. The results indicated transepithelial permeability of 7–22 · 10−6 cm · sec−1, which was 5–100 fold smaller compared to blank inserts, with a cut-off MW of 40–70 kDa for hECE and Caski cells. Transepithelial conductance ranged 18.5 to 51.5 mS · cm−2, indicating a leaky but confluent epithelia.Collectively the results indicate the epithelial nature of the cells and their improved differentiation when grown on filter support; hECE is a model for ectocervical epithelium while ECE16-1 and Caski express phenotypic characteristics of squamous metaplastic cervical epithelium and endocervical epithelium, respectively.

ReviewBiochemistry of epidermal stem cells☆

AbstractBackgroundThe epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue.Scope of reviewA great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer.Major conclusionsAn assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis.General significanceUnderstanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Back | Next (Page 1 of 2)
Advertisement
Join Copernicus Academic and get access to over 12 million papers authored by 7+ million academics.
Join for free!

Contact us