Cellular differentiation and death in a renaissance castle.
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Animals↗
Biomedical subjects
R L Eckert
Publications and source records attributed to R L Eckert.
MEK6 regulates human involucrin gene expression via a p38alpha - and p38delta -dependent mechanism.
A signaling cascade that includes protein kinase C (PKC), Ras, and MEKK1 regulates involucrin (hINV) gene expression in epidermal keratinocytes (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Because signal transfer downstream of MEKK1 may involve several MAPK kinases (MEKs), it is important to evaluate the regulatory role of each MEK isoform. In the present study we evaluate the role of MEK6 in transmitting this signal. Constitutively active MEK6 (caMEK6) increases hINV promoter activity and increases endogenous hINV levels. The caMEK6-dependent increase in gene expression is inhibited by the p38 MAPK inhibitor, SB203580, and is associated with a marked increase in p38alpha MAPK activity; JNK and ERK kinases are not activated. In addition, hINV gene expression is inhibited by dominant-negative p38alpha and increased when caMEK6 and p38alpha are co-expressed. caMEK6 also activates p38delta, but p38delta inhibits the caMEK6-dependent activation. These results suggest that MEK6 increases hINV gene expression by regulating the balance between activation of p38alpha, which increases gene expression, and p38delta, which decreases gene expression.
Base Sequence↗
S100A7, S100A10, and S100A11 are transglutaminase substrates.
S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.
3T3 Cells↗
MEK7-dependent activation of p38 MAP kinase in keratinocytes.
Previous studies suggest that a PKC/Ras/MEKK1 cascade regulates involucrin (hINV) gene expression in human epidermal keratinocytes. MEK7, which is expressed in epidermis, has been identified as a member of this cascade (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). However, the kinase that functions downstream of MEK7 has not been identified. Our present studies show that MEK7 expression in keratinocytes markedly activates p38alpha and modestly activates JNK. Activation of p38 MAPK by MEK7 is a novel finding, as previous reports have assigned MEK7 as a JNK regulator. We also demonstrate that this regulation is physiologically important, as the p38alpha- and JNK-dependent activities regulate hINV promoter activity and expression of the endogenous hINV gene.
Cells, Cultured↗
Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.
Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.
Analysis of Variance↗
Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.
Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.
Base Sequence↗
TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression.
Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors.
CDC2-CDC28 Kinases↗
Regulation of human involucrin promoter activity by novel protein kinase C isoforms.
Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T. , Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.
Acetophenones↗
Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins.
Human papillomavirus (HPV) survives by reactivating DNA replication in post-mitotic cells. In the present study, we describe a mouse model of HPV-dependent disease. In these mice, DNA synthesis is activated in suprabasal keratinocytes, leading to acanthosis, parakeratosis and enhanced desquamation. The full-length E6/E7 transcript and two alternately spliced products are produced and in most lines the predominant product is E6*. In the present study, we examine the effects of E6/E7 on cell cycle regulatory protein expression. E6/E7 expression in mouse epidermis is correlated with increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E regulatory proteins. Hyperproliferation is also observed in the buccal mucosa and the tongue epithelia of E6/E7 mice, and p53 levels are markedly increased in these epithelia. These results suggest that the major changes in cell cycle regulatory protein expression are in response to the presence of E7 and that E6 has a lesser impact.
Animals↗
The carboxy-terminal hydrophobic domain of TIG3, a class II tumor suppressor protein, is required for appropriate cellular localization and optimal biological activity.
TIG3 is a recently discovered class II tumor suppressor protein, originally isolated from retinoid-treated cultured epidermal keratinocytes, that suppresses the proliferation of a variety of epithelial cell types. In the present study, we examine the ability of this protein to reduce CHO, T47D and HaCaT cell proliferation, and the role of the carboxy-terminal hydrophobic domain in this regulation. Vector-mediated expression of the full length TIG3 protein, TIG31-164, results in a 50-70% reduction colony formation efficiency. Expression of a truncated mutant, TIG31-134, that lacks the putative carboxy-terminal membrane-anchoring domain, results in a partial loss of ability to suppress colony formation. The fact that the truncated protein remains partially active suggests that both the amino- and carboxy-terminal regions of TIG3 are required for optimal growth suppression. The full-length protein is distributed in a perinuclear location, and is not present in the nucleus. TIG31-134, in contrast, is distributed in the cytoplasm. Thus, a change in location is associated with the partial loss of activity. We also monitored the distribution of green fluorescent protein (GFP)-TIG3 fusion proteins. GFP-TIG31-164 was localized in a pattern similar to that observed for TIG31-164, while GFP-TIG31-134 displayed a distribution pattern similar to GFP. This suggests that the c-terminal hydrophobic domain has an important role in determining the intracellular localization of TIG3. In addition, GFP-TIG31-164 retains the ability to inhibit cell function, while GFP-TIG31-134 is inactive.
Amino Acid Sequence↗
Transgenic animal models of human papillomavirus-dependent disease (Review).
Human papillomaviruses (HPVs) are DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia. A wide variety of studies implicate the viral E6 and E7 oncoproteins as cell immortalizing agents, and show that these proteins work, respectively, by interfering with the function of the p53 and pRb tumor suppressor genes. Most of these studies have been performed using cell culture models. However, recently, a variety of in vivo mouse model systems have been developed for the study of HPV-dependent disease. These models use tissue-specific promoters to deliver HPV oncoprotein expression to specific body sites. Using this strategy, mouse models have been designed for the study of cancer progression in epithelia, and additional models have been designed to use E6 and E7, respectively, to probe the role of p53 and pRb on tissue differentiation and function. In the present report, we summarize the literature describing these systems, and highlight some of the important findings derived from these studies.
Animals↗
Expression of a retinoid-inducible tumor suppressor, Tazarotene-inducible gene-3, is decreased in psoriasis and skin cancer.
Tazarotene-induced gene-3 (TIG-3), isolated from human keratinocytes treated with the retinoic acid receptor-selective retinoid Tazarotene, is homologous to H-rev, a class II tumor suppressor. TIG-3 gene localized to chromosome 11q23, a site of loss of heterozygosity in several malignancies. Retinoids influence epidermal differentiation and are used to treat and prevent skin cancer. Therefore, we studied TIG-3 mRNA expression in psoriasis and in basal and SCCs by in situ hybridization and a quantitative QT-RT-PCR assay. Psoriasis lesions had significantly lower staining (median, 3) than paired normal control skin (median, 4; P = 0.012). TIG-3 mRNA was significantly higher in normal control skin (P = 0.001), in paired adjacent skin (median, 3; P = 0.007), and in overlying epidermis (median, 3.0; P = 0.0001) than in 21 SCC specimens as a group (median, 1.5).
Aged↗
CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.
The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of keratinocyte differentiation and of involucrin gene expression. In the present study we show that a CCAAT/enhancer-binding protein (C/EBP) site in the proximal regulatory region is required for the phorbol ester response. Mutation of the C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis shows that C/EBPalpha binding to this site is increased by TPA treatment. Moreover, cotransfection of the human involucrin reporter plasmid with C/EBPalpha increases promoter activity to an extent comparable with TPA treatment. Mutation of the C/EBP-binding site eliminates these responses. Transfection experiments using GADD153 to create C/EBP-null conditions confirm that C/EBP factors are absolutely required for promoter activity and TPA responsiveness. C/EBPbeta and C/EBPdelta inhibit both TPA- and C/EBPalpha-dependent promoter activation, indicating functional differences among C/EBP family members. These results suggest that C/EBP transcription factor activity is necessary for basal promoter activity and TPA response of the involucrin gene.
CCAAT-Enhancer-Binding Proteins↗
Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types.
The gene for human involucrin (hINV) is selectively expressed in stratifying epithelial cells lining external body surfaces. Previously, we characterized the hINV promoter 5' distal regulatory region (DRR) located between nt -2473 and -2088 upstream of the transcription start site. This region is required for optimal hINV gene expression. The DRR contains weak and strong activator elements. The strong activator comprises AP1- and Sp1-binding sites that combine to drive high-level promoter expression in human keratinocytes. Here we show that the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements that are partly responsible for cell-type-specific expression of hINV. hINV promoter activity is barely detectable in 3T3 fibroblasts or HEK-293 human embryonic kidney cells. Reporter plasmids containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and HEK-293 cells by co-transfection with a plasmid encoding the transcription factor Sp1. Consistently with the lower hINV promoter activity, immunoblotting studies indicate that Sp1 protein levels are lower in 3T3 and HEK-293 cells than in human epidermal keratinocytes. Increased Sp1 protein in transfected 3T3 cells and HEK-293 cells correlates with increased promoter activity. In addition, Sp1 transfection activates the expression of the endogenous gene for hINV in HEK-293 cells. These studies suggest that Sp1 might have a role in cell-specific expression of hINV.
3T3 Cells↗
Vitamin D regulates human ectocervical epithelial cell proliferation and insulin-like growth factor-binding protein-3 level.
The differentiation status of the cervical epithelial cell has an important influence on responsiveness to estrogens and progestins. Several agents, including glucocorticoids and retinoids, are known to influence cervical cell differentiation. However, the effects of vitamin D have not been examined. Vitamin D is known to regulate cell proliferation and gene expression in a variety of epithelial cells. In the present study we investigated the ability of 1alpha25-dihydroxyvitamin D3 (D3) to regulate cell proliferation and expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in human ectocervical epithelial cells. ECE16-1, a non-tumorigenic cervical cell line, was growth inhibited by D3 with maximal inhibition at 1000 nM. IGFBP-3 levels increased in parallel with the growth inhibition. IGFBP-3 levels were half-maximally increased at approximately 10-100 nM and maximally increased (10- to 30-fold) at 1000 nM D3. These studies show that vitamin D regulates cervical epithelial cell gene regulation and cell proliferation and that IGFBP-3 may be an in vivo marker of vitamin D action in the cervix.
Calcitriol↗
Insulin-like growth factor binding protein-3 does not mediate the interferon-dependent suppression of human ectocervical epithelial cell proliferation.
Interferon is a potential therapeutic agent for the treatment of cervical cancer. In the present study we examine the role of IFNgamma as a regulator of proliferation and production of IGFBP-3 expression in ectocervical epithelial cells. ECE16-1 cells are a model for studying early human papillomavirus-dependent cervical disease. IFNgamma produces a concentration-dependent inhibition of ECE16-1 cell proliferation that is associated with an increase in insulin-like growth factor binding protein-3 level. Growth suppression and IGFBP-3 increase is maximal at concentrations of IFNgamma >/=0.75 ng/ml. The increased IGFBP-3 expression is mediated via an increase in IGFBP-3 encoding mRNA. In contrast, IFNgamma inhibits proliferation of CaSki and SiHa cells, but IGFBP-3 is barely detectable and levels are not regulated by IFNgamma. These results suggest that the IFNgamma-dependent suppression of CaSki and SiHa cell proliferation is not mediated by secreted IGFBP-3. This result was confirmed when vector-mediated overexpression of immunoreactive IGFBP-3 in SiHa and CaSki cells did not consistently result in reduced cell proliferation rate.
Animals↗
Cell type and gene-specific activity of the retinoid inverse agonist AGN 193109: divergent effects from agonist at retinoic acid receptor gamma in human keratinocytes.
Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RARalpha-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RARgamma using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression.
Calcium-Binding Proteins↗
Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene.
Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display-PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.
Amino Acid Sequence↗