ABSTRACT
Epidermal growth factor (EGF) enhances the expression of the keratinocyte terminal differentiation marker SPRR2A, when added to monolayers of basal keratinocytes, induced to stratify by increasing the extracellular calcium concentration. A similar stimulation is found during suspension-induced differentiation in methylcellulose. This effect, which is observed after several hours of EGF addition, is restricted to terminally differentiating keratinocytes and is dependent on PKC signaling. EGF also transiently activates the Ras signaling pathway, with a maximum induction after 10 min (Medema et al., 1994, Mol. Cell. Biol. 14, 7078-7085). The cellular effects of activated Ras were determined by transient transfection of Ha-ras(Leu-61) into normal human keratinocytes. Activated Ras completely inhibited PKC-mediated expression of SPRR2A. This inhibition is mediated via c-Jun as it is reversed by a dominant-negative c-Jun mutant (cJunDelta6/194) and c-Jun can substitute for activated Ras. The inhibitory effect is targeted to a 150-bp minimal promoter region, which is essential and sufficient for SPRR2A expression during keratinocyte terminal differentiation. This indicates that the Ras and PKC pathways, which both can be triggered by EGF, although at different time points, have opposite effects on SPRR2A gene expression.
Subject(s)
Gene Expression Regulation , Keratinocytes/metabolism , Membrane Proteins/genetics , Oncogene Protein p21(ras)/metabolism , Protein Kinase C/metabolism , Protein Precursors/genetics , Calcium/antagonists & inhibitors , Calcium/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Cornified Envelope Proline-Rich Proteins , Enzyme Activation , Epidermal Growth Factor/pharmacology , Gene Expression Regulation/drug effects , Genes, fos/genetics , Humans , Indoles/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/enzymology , Maleimides/pharmacology , Membrane Proteins/metabolism , Mutation , Oncogene Protein p21(ras)/genetics , Promoter Regions, Genetic/genetics , Protein Kinase C/antagonists & inhibitors , Protein Precursors/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effectsABSTRACT
SPRR3, a member of the SPRR family of cornified envelope precursor proteins, is expressed in oral and esophageal epithelia, where it is strictly linked to keratinocyte terminal differentiation. This gene is characterized by intragenic duplications that have created the characteristic proline-rich repeats in the coding sequence, an alternative noncoding exon, and a 200-bp polypyrimidine tract in the promoter region. Mutational analysis of the promoter region and transient transfection in normal human keratinocytes showed that in addition to the polypyrimidine tract, multiple regulatory elements are involved in differentiation-specific expression. These elements include a high-affinity Ets binding site bound by ESE-1, an AP-1 site (TRE) recognized by the Jun/Fos family of transcription factors, and an ATF/CRE bound by Jun/Fos and ATF factors. The repositioning of the SPRR3 Ets binding site during evolution has a major effect on the relative contribution of this site to promoter activity.
Subject(s)
DNA-Binding Proteins , Evolution, Molecular , Gene Expression Regulation , Peptides , Proteins/genetics , Activating Transcription Factor 2 , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cells, Cultured , Chromosome Mapping , Cornified Envelope Proline-Rich Proteins , Cyclic AMP Response Element-Binding Protein/metabolism , DNA , Exons , Humans , Introns , Male , Molecular Sequence Data , Proline-Rich Protein Domains , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , Regulatory Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Trans-Activators/metabolism , Transcription Factor AP-1/metabolism , Transcription Factors/metabolismABSTRACT
The 173-base pair proximal promoter of SPRR1A is necessary and sufficient for regulated expression in primary keratinocytes induced to differentiate either by increasing extracellular calcium or by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Whereas calcium-induced expression depends both on an AP-1 and an Ets binding site in this region, responsiveness to TPA resides mainly (but not exclusively) on the Ets element, indicating that Ets factors are important targets for protein kinase C signaling during keratinocyte terminal differentiation. This conclusion is further substantiated by the finding that expression of ESE-1, an Ets transcription factor involved in SPRR regulation, is also induced by TPA, with kinetics similar to SPRR1A. The strict AP-1 requirement in SPRR1A for calcium-induced differentiation is not found for SPRR2A, despite the presence of an identical AP-1 consensus binding site in this gene. Binding site swapping indicates that both the nucleotides flanking the TGAGTCA core sequence and the global promoter context are essential in determining the contribution of AP-1 factors in gene expression during keratinocyte terminal differentiation. In the distal SPRR1A promoter region, a complex arrangement of positive and negative regulatory elements, which are only conditionally needed for promoter activity, are likely involved in gene-specific fine-tuning of the expression of this member of the SPRR gene family.
Subject(s)
Gene Expression Regulation , Keratinocytes/cytology , Keratinocytes/metabolism , Promoter Regions, Genetic , Proteins/genetics , Proto-Oncogene Proteins/metabolism , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Base Sequence , Biomarkers , Cell Differentiation , Cells, Cultured , Cornified Envelope Proline-Rich Proteins , Humans , Infant, Newborn , Keratinocytes/drug effects , Male , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Molecular Sequence Data , Multigene Family , Protein Biosynthesis , Protein Precursors/biosynthesis , Protein Precursors/genetics , Proto-Oncogene Proteins c-ets , Recombinant Fusion Proteins/biosynthesis , Sequence Alignment , Sequence Deletion , Sequence Homology, Nucleic Acid , Skin/cytology , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels of the cell lines correlated with CDDP concentrations inhibiting cell survival by 50% (IC50); total cellular sulphydryl content (TSH) was unexpectedly inversely correlated with IC50. IC50 correlated neither with glutathione S-transferase (GST) nor with GST pi expression, topoisomerase I or II activity. Immediately after 4 h incubation with CDDP, platinum (Pt) accumulation and Pt bound to DNA were not correlated, but after another 24 h drug-free culture, Pt binding to DNA in germ cell tumour but not in colon carcinoma cell lines correlated with IC50. With the exception of in vitro sensitivity and TSH, none of the parameters studied discriminated between the two groups of cell lines. Correction of CDDP sensitivity parameters for phenotypical differences did not influence statistical correlations. Analysis of variance revealed a correlation between IC50 and the combination of glutathione, GST activity and Pt bound to DNA. But at other CDDP cytotoxicity levels sensitivity was also correlated with Pt accumulation, topoisomerase II activity and TSH in various combinations. This model of intrinsic CDDP resistance showed that multiple parameters ought to be studied to explain CDDP resistance, but did not elucidate the cause of the unique sensitivity of germ cell carcinoma, although the unexpected values of TSH deserve further attention.
Subject(s)
Cisplatin/metabolism , Cisplatin/toxicity , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type I/metabolism , Glutathione Transferase/metabolism , Glutathione/metabolism , Adenocarcinoma , Cell Line , Cell Survival/drug effects , Colonic Neoplasms , DNA, Neoplasm/metabolism , Dose-Response Relationship, Drug , Germinoma , Glutathione Transferase/biosynthesis , Humans , Isoenzymes/biosynthesis , Isoenzymes/metabolism , Tumor Cells, CulturedABSTRACT
Human primary keratinocytes are an elegant model system to study the balance between proliferation and differentiation. Both epidermal growth factor (EGF) and extracellular calcium have been implicated to function in the control of this balance, although the molecular mechanism underlying this process is poorly understood. In this study, we measured the effect of both EGF and calcium treatment on activation of p21ras and ERK2. We found that addition of EGF stimulated the activity of ERK2. This stimulation was dependent on p21ras activity, since it was completely abolished by expression of a dominant negative mutant of p21ras (p21ras(Asn-17)). Raising the level of extracellular calcium (1.8 mM) did not result in activation of ERK2. On the contrary, calcium treatment inhibited EGF-induced stimulation of ERK2 activity. In order to determine the site at which calcium treatment interferes in EGF-induced signaling, we analyzed the effect of calcium on the various steps that are involved in EGF-induced, p21ras-dependent activation of ERK2. We observed that calcium treatment inhibited EGF-induced p21ras activation. Calcium treatment, however, did not interfere with EGF-induced EGF receptor autophosphorylation or association of mammalian SOS with the EGF receptor and Shc. This, together with the observation that calcium treatment alone decreased the basal level of p21ras activity, indicates that calcium treatment interferes in EGF-mediated signaling at the level of p21ras. This type of cross talk may play a role in the decision between proliferation and differentiation in human primary keratinocytes.
