Impacts of arsenic on Rad18 and translesion synthesis.

Arsenite interferes with DNA repair protein function resulting in the retention of UV-induced DNA damage. Accumulated DNA damage promotes replication stress which is bypassed by DNA damage tolerance pathways such as translesion synthesis (TLS). Rad18 is an essential factor in initiating TLS through PCNA monoubiquitination and contains two functionally and structurally distinct zinc fingers that are potential targets for arsenite binding. Arsenite treatment displaced zinc from endogenous Rad18 protein and mass spectrometry analysis revealed arsenite binding to both the Rad18 RING finger and UBZ domains. Consequently, arsenite inhibited Rad18 RING finger dependent PCNA monoubiquitination and polymerase eta recruitment to DNA damage in UV exposed keratinocytes, both of which enhance the bypass of cyclobutane pyrimidine dimers during replication. Further analysis demonstrated multiple effects of arsenite, including the reduction in nuclear localization and UV-induced chromatin recruitment of Rad18 and its binding partner Rad6, which may also negatively impact TLS initiation. Arsenite and Rad18 knockdown in UV exposed keratinocytes significantly increased markers of replication stress and DNA strand breaks to a similar degree, suggesting arsenite mediates its effects through Rad18. Comet assay analysis confirmed an increase in both UV-induced single-stranded DNA and DNA double-strand breaks in arsenite treated keratinocytes compared to UV alone. Altogether, this study supports a mechanism by which arsenite inhibits TLS through the altered activity and regulation of Rad18. Arsenite elevated the levels of UV-induced replication stress and consequently, single-stranded DNA gaps and DNA double-strand breaks. These potentially mutagenic outcomes support a role for TLS in the cocarcinogenicity of arsenite.

Melanocytes and keratinocytes have distinct and shared responses to ultraviolet radiation and arsenic.

The rise of melanoma incidence in the United States is a growing public health concern. A limited number of epidemiology studies suggest an association between arsenic levels and melanoma risk. Arsenic acts as a co-carcinogen with ultraviolet radiation (UVR) for the development of squamous cell carcinoma and proposed mechanisms include generation of oxidative stress by arsenic and UVR and inhibition of UVR-induced DNA repair by arsenic. In this study, we investigate similarities and differences in response to arsenic and UVR in keratinocytes and melanocytes. Normal melanocytes are markedly more resistant to UVR-induced cytotoxicity than normal keratinocytes, but both cell types are equally sensitive to arsenite. Melanocytes were more resistant to arsenite and UVR stimulation of superoxide production than keratinocytes, but the concentration of arsenite necessary to inhibit the activity of the DNA repair protein poly(ADP-ribose)polymerase and enhance retention of UVR-induced DNA damage was essentially equivalent in both cell types. These findings suggest that although melanocytes are less sensitive than keratinocytes to initial UVR-mediated DNA damage, both of these important target cells in the skin share a mechanism related to arsenic inhibition of DNA repair. These findings suggest that concurrent chronic arsenic exposure could promote retention of unrepaired DNA damage in melanocytes and act as a co-carcinogen in melanoma.

Tyrosine-phosphorylated plakoglobin is associated with desmogleins but not desmoplakin after epidermal growth factor receptor activation.

Tyrosine phosphorylation of junctional components has been proposed as a mechanism for modulating cell-cell adhesion. Although a correlation exists between the tyrosine phosphorylation of the adherens junction protein beta-catenin and loss of classical cadherin-mediated adhesion, the effects of tyrosine phosphorylation on the function of the adherens junction and desmosome-associated protein plakoglobin is unknown. In the present study, we investigated the effects of epidermal growth factor receptor (EGFR) tyrosine kinase activation on the subcellular distribution of plakoglobin and its association with its junctional binding partners. Long term epidermal growth factor (EGF) treatment of A431 cells revealed a modest decrease in the cytoskeleton-associated pool of plakoglobin (Pg) and a corresponding increase in the cytosolic pool of Pg. After short term EGF treatment, plakoglobin was rapidly phosphorylated, and tyrosine-phosphorylated Pg was distributed predominantly in a membrane-associated Triton X-100-soluble pool, along with a co-precipitating high molecular weight tyrosine-phosphorylated protein identified as desmoglein 2. Analysis of deletion and point mutants defined the primary EGFR-dependent targets as one or more of three C-terminal tyrosine residues. Whereas phosphorylated Pg remained associated with the desmoglein tail after both short and long term EGFR activation, no phosphorylated Pg was found associated with the N-terminal Pg-binding domain (DPNTP) of the intermediate filament-associated protein, desmoplakin. Together these results are consistent with the possibility that EGF-dependent tyrosine phosphorylation of Pg may modulate cell-cell adhesion by compromising the link between desmosomal cadherins and the intermediate filament cytoskeleton.

Prevention of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-10A cell line: correlation with increased transforming growth factor alpha production.

We have recently reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits epidermal growth factor (EGF) withdrawal-induced apoptosis in the human mammary epithelial cell line MCF-10A. We hypothesized that TCDD-mediated inhibition of apoptosis was due to its ability to stimulate the EGF receptor (EGFR) pathway. Indeed, in the present studies, the EGFR inhibitor AG1478 was able to prevent TCDD-, EGF-, and transforming growth factor alpha (TGF-alpha)-dependent cell recovery and inhibition of apoptosis. These effects were specific for an EGFR-mediated pathway because cotreatment with AG825, an erbB2 inhibitor, had little effect on apoptosis. In addition, TCDD was able to mimic the EGF and TGF-alpha signaling as demonstrated by increasing Akt and extracellular signal-regulated kinase 1,2 phosphorylation. These effects were dependent on EGFR activity because AG1478, but not AG825, was able to prevent EGF-, TGF-alpha, or TCDD-mediated Akt and extracellular signal-regulated kinase 1,2 phosphorylation. The ability of TCDD to stimulate the EGFR pathway and inhibit apoptosis may be due to the ability of TCDD to increase expression of TGF-alpha, a ligand for EGFR. Treatment with 10 nM TCDD increased TGF-alpha mRNA at 2 h and TGF-alpha protein at 6 h. These data suggest a mechanism whereby TCDD is able to inhibit apoptosis in human mammary epithelial cells by stimulating TGF-alpha production, resulting in an autocrine effect.

Phosphatidylinositol 3-kinase activity in epidermal growth factor-stimulated matrix metalloproteinase-9 production and cell surface association.

Activation of the epidermal growth factor (EGF) receptor regulates many processes associated with metastasis, including modulation of cell:cell and cell:substrate interactions, production of matrix-degrading proteinases, and cellular migration. We have demonstrated previously that EGF stimulates migration and matrix metalloproteinase (MMP)-9-dependent invasion of ovarian cancer cells. In this study, we compare the roles of EGF-induced phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) activities in regulation of cellular responses associated with ovarian tumor cell metastasis. Inhibition of PI3K and MAPK activity impairs EGF-stimulated cell migration, in vitro invasion, and MMP-9 production. PI3K activity is not required for growth factor disruption of cell:cell junctions, whereas inhibitors of extracellular signal-regulated kinase (ERK)1/ERK2 activation and p38 MAPK activity block EGF-dependent junction dissolution. EGF promotes pro-MMP-9 binding to the cell surface through a mechanism that is independent of extracellular enzyme concentration. Interestingly, inhibition of PI3K activity abolishes EGF-induced cell surface association of pro-MMP-9, whereas inhibitors of MAPKs only partially block the response. These data suggest that EGF receptor activation promotes a PI3K-dependent induction of a cell surface pro-MMP-9 binding component that may facilitate gelatinase-mediated cellular invasion and supports an expanded role for elevated PI3K activity in cellular responses associated with ovarian tumor metastasis. In addition, our findings support the hypothesis that divergent kinase activities regulate distinct cellular events associated with growth factor-induced invasion of ovarian cancer cells.

Elevation of intracellular cAMP inhibits growth factor-mediated matrix metalloproteinase-9 induction and keratinocyte migration.

Receptor tyrosine kinases are regulators of diverse cellular functions including cell growth, cell survival, differentiation, locomotion, and morphogenesis. Activation of the cAMP-dependent protein kinase A inhibits receptor tyrosine kinase-stimulated growth responses in a number of cell types. In this study, we investigated the consequences of elevated cAMP on growth factor-mediated keratinocyte migration and matrix metalloproteinase (MMP)-9 induction in a human keratinocyte cell line. We found that elevation of intracellular cAMP by forskolin abolishes epidermal growth factor (EGF)- or scatter factor/hepatocyte growth factor-dependent colony dispersion. Concentrations of forskolin that inhibit growth factor-induced motility also eliminate EGF- or scatter factor/hepatocyte growth factor-dependent induction of the 92-kDa gelatinase/MMP-9. In contrast to findings obtained in fibroblasts, elevated intracellular cAMP did not interfere with growth factor-dependent activation of the p42/44 extracellular signal-regulated kinases, indicating that cAMP-dependent inhibition of migration and MMP-9 induction does not occur through perturbation of the extracellular signal-regulated kinases/mitogen-activated protein kinase pathway. However, forskolin effectively inhibited EGF-dependent activation of c-Jun N-terminal kinase and p38, demonstrating that cAMP selectively interferes with a different subset of growth factor-induced mitogen-activated protein kinase signaling cascades than reported previously in fibroblasts. These findings illustrate that EGF concurrently activates multiple mitogen-activated protein kinase signaling cascades in keratinocytes and suggests that each pathway contributes to maximal EGF-dependent migration and proteinase induction.

Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration.

Activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is required for ligand-dependent regulation of numerous cellular functions by receptor tyrosine kinases. We have shown previously that although many receptor tyrosine kinase ligands are mitogens for keratinocytes, cell migration and induction of the 92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are selectively regulated by the epidermal growth factor and scatter factor/hepatocyte growth factor receptors. In this report we present evidence of an underlying mechanism to account for these observed differences in receptor tyrosine kinase-mediated response. Ligands that are mitogenic, but do not induce MMP-9 or colony dispersion, transiently activate the p42/p44 ERK/MAP kinases. In contrast, ligands that stimulate MMP-9 induction and colony dispersion induced sustained activation of these kinases. The functional significance of sustained MAPK activation was demonstrated by inhibition of the MAP kinase kinase MEK1. Disruption of the prolonged signal by addition of the MEK1 inhibitor PD 98059 up to 4 h after growth factor stimulation substantially impaired ligand-dependent colony dispersion and MMP-9 induction. These findings support the conclusion that duration of MAPK activation is an important determinant for certain growth factor-mediated functions in keratinocytes.

Induction of differentiation-dependent apoptosis in human esophageal squamous cell carcinoma by adenovirus-mediated p21sdi1 gene transfer.

When keratinocytes withdraw from the cell cycle, they migrate from the basal to the superficial layers of the epidermis and undergo morphological and biochemical changes during the process of terminal differentiation. These differentiation features of keratinocytes are known to be altered or reduced in esophageal cancer cells. Therefore, we examined the effects of transferring the cyclin-dependent kinase inhibitor p21sdi1 gene into human esophageal cancer cell lines as well as normal keratinocytes using an adenovirus vector system. Ectopic expression of p21sdi1 protein resulted in cell cycle arrest at the G1 phase and produced morphological changes, such as enlarged nuclei and a flattened cellular shape, changes specific to the differentiated phenotype. The human involucrin protein is a specific product of keratinocyte differentiation, which is selectively expressed in the suprabasal epidermal layers. Western blot analysis and immunohistochemical staining demonstrated that involucrin expression was 3- to 5-fold enhanced by the forced expression of p21sdi1 in esophageal cancer cells, whereas only a mild up-regulation up to 1.2-fold occurred in normal keratinocytes. We also found that exogenous introduction of the p2sdi1 gene transcriptionally activated the upstream promoter function of the involucrin gene. These stimulatory effects on involucrin expression were not observed when another cyclin-dependent kinase inhibitor gene, p16(INK4a), was transduced. Moreover, p21sdi1 expression in esophageal cancer cells transduced with p21sdi1 led to a rapid apoptotic cell death after a transient dormant phase, although keratinocytes transduced with p21sdi1 survived longer by terminally withdrawing from the cell cycle. These results may have an important implication for understanding the biology of differentiation-dependent apoptosis in human esophageal squamous cell carcinoma.

Contributions of the epidermal growth factor receptor to keratinocyte motility.

The epidermal growth factor (EGF) receptor plays a central role in numerous aspects of keratinocyte biology. In normal epidermis, the EGF receptor is important for autocrine growth of this renewing tissue, suppression of terminal differentiation, promotion of cell survival, and regulation of cell migration during epidermal morphogenesis and wound healing. In wounded skin, the EGF receptor is transiently up-regulated and is an important contributor to the proliferative and migratory aspects of wound reepithelialization. In keratinocytic carcinomas, aberrant expression or activation of the EGF receptor is common and has been proposed to play a role in tumor progression. Many cellular processes such as altered cell adhesion, expression of matrix degrading proteinases, and cell migration are common to keratinocytes during wound healing and in metastatic tumors. The EGF receptor is able to regulate each of these cellular functions and we propose that transient and dynamic elevation of EGF receptor during wound healing, or constitutive overexpression in tumors, provides an important contribution to the migratory and invasive potential of keratinocytes.

Proteinase requirements of epidermal growth factor-induced ovarian cancer cell invasion.

Aberrant expression or activity of the epidermal growth factor (EGF) receptor family of tyrosine kinases has been associated with tumor progression and an invasive phenotype. In this study, we utilized 4 ovarian cancer cell lines, OVCA 432, DOV 13, OVEA6 and OVCA 429, to determine the effects of EGF on the regulation of proteolytic enzymes and their inhibitors, cellular migration and in vitro invasion. Induction of urinary-type plasminogen activator (u-PA) activity and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was observed in all 4 cell lines. OVCA 432 cells showed strong PAI-1 induction; however, the other 3 lines displayed substantial baseline PAI-1 expression that was not induced by EGF. EGF-dependent stimulation of migration and induction of matrix metalloproteinase (MMP)-9 (gelatinase B) was observed in OVEA6 and OVCA 429 cells only. Upon EGF receptor activation, DOV 13, OVEA6 and OVCA 429 cells were induced to invade through an artificial basement membrane (Matrigel); however, no invasion was detected in OVCA 432 cells. Cell lines displaying induction of migration and MMP-9 (OVEA6 and OVCA 429) demonstrated robust EGF-induced invasion (5- to 20-fold), and cell invasion was substantially reduced in the presence of anti-catalytic MMP-9 antibody. Addition of anti-catalytic u-PA antibody inhibited the modest (<2-fold) EGF-induced invasion in a cell line that did not express MMP-9 (DOV 13) and in OVEA6 cells that displayed the highest baseline u-PA activity. Together, our findings indicate that multiple proteinases are important in ovarian cell invasion and implicate EGF induction of MMP-9 and migration as key components of more aggressive ligand-induced invasion.

Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)- mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9.

Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion.

Functional consequences of reduced retinoic acid receptor gamma expression in a human squamous cell carcinoma line.

Nuclear receptors for retinoic acid are important modulators of epidermal cell proliferation and terminal differentiation. Aberrant expression of retinoic acid receptors (RARs) and retinoid X receptors in the epidermis has been associated with altered differentiation capacity and tumor progression. In this study, we describe a human squamous cell carcinoma line, SCC 12F, which displays reduced RARgamma expression and diminished responsiveness to retinoic acid. When compared with normal keratinocytes or other squamous cell carcinoma lines that display normal levels of RARgamma, several measures of cellular response to retinoic acid are altered in SCC 12F cells, including inhibition of cornified envelope formation, reduction of involucrin mRNA expression, and transcriptional regulation of the involucrin gene. Normal patterns of ligand-dependent transcriptional response were restored upon co-transfection of an expression vector containing either RARalpha or RARgamma. Our findings demonstrate that reduced expression of RAR may have direct functional consequences with regard to keratinocyte differentiation and that the defect may be alleviated by reintroduction of functional receptor.

Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines.

In keratinocytes, epidermal growth factor (EGF) promotes cell motility in addition to proliferation. As EGF receptor expression is elevated during wound healing and in many epithelial tumors, we wanted to investigate whether there is a direct relationship between EGF receptor expression and ligand-mediated cellular locomotion. EGF receptor activation induced cell migration in normal keratinocytes and their tumorigenic counterparts; however, the rate of colony dispersion and in vitro reepithelialization was more rapid in the squamous cell carcinoma (SCC) lines that exhibited elevated (> or = 5-fold) EGF receptor levels. Within a single SCC line, submaximal concentrations of EGF or reduction of EGF receptor activity by an anti-EGF receptor neutralizing antibody resulted in delayed kinetics of in vitro reepithelialization. Thus, suppression of EGF receptor activity in an overexpressing SCC line restores a migratory response that more closely resembles that of normal keratinocytes. Conversely, ligand-induced colony dispersion was augmented in stable clonal cell lines in which EGF receptor expression was elevated after introduction of an EGF receptor complementary DNA construct. Collectively, these findings suggest that the migratory potential of keratinocytes is modulated at the level of both receptor expression and ligand concentration, with a positive correlation between EGF receptor levels and ligand-induced cell motility.

Regulation of involucrin gene expression by retinoic acid and glucocorticoids.

Involucrin is a major component of the cornified envelope and a marker for terminal differentiation in keratinocytes. We examined hormone regulation of involucrin mRNA expression and transcriptional modulation of the involucrin gene in human keratinocyte cell lines. Dexamethasone enhanced, and retinoic acid decreased, endogenous involucrin mRNA expression in cells treated with these ligands. Functional interactions between each ligand were observed; all-trans or 9-cis retinoic acid reduced dexamethasone enhancement of involucrin expression. Transcriptional control of the involucrin promoter reflected the responses observed for the endogenous gene. All-trans or 9-cis retinoic acid inhibited involucrin expression, and both retinoids antagonized glucocorticoid-mediated induction of reporter gene activity equally well. In contrast, a control promoter was unaffected by hormone treatment, indicating that stabilization of reporter gene mRNA or protein did not account for the observed differences in activity. These results demonstrate that transcriptional control plays a role in regulation of the endogenous involucrin gene by glucocorticoids and retinoids and that DNA sequences within the isolated promoter region confer hormone responsiveness. Hormone-mediated responses mapped to a proximal promoter region that contained a functional AP1 site. Stimulation of AP1 activity by phorbol ester was suppressed by retinoic acid, and cotransfection with a c-jun expression vector reversed the retinoic acid response. Based on these data, we suggest that suppression of involucrin promoter activity by retinoic acid may be mediated through interaction with the AP1 transcriptional complex.

Identification of a functional determinant of differentiation-dependent expression in the involucrin gene.

Involucrin is an integral component of the cornified envelope which is a characteristic feature of the differentiated keratinocyte. Involucrin expression is tightly linked to the onset of differentiation and first expressed in the immediate suprabasal layers of the epidermis. We have identified a transcriptional response element within the distal 5'-flanking region of the involucrin gene which contributes to differentiation-dependent expression. Deletion of this site impairs differentiation-dependent promoter activity in transient transfection analysis, and conversely, this region imparts differentiation-dependent expression to a heterologous promoter. The identified site bears sequence similarity to several AP2-like response elements identified in keratinocyte-specific genes and binds a protein complex (keratinocyte differentiation factor, KDF-1) which is distinct from AP2 by several criteria. The migration of KDF-1 is distinct from AP2 in electrophoretic mobility shift assays, KDF-1 is antigenically unrelated to AP2 since AP2 specific antibodies do not supershift the KDF-1-DNA complex and KDF-1 is poorly competed by oligonucleotides representing consensus AP2 recognition sequences. In addition, the KDF-1 complex is not detected in nuclear extracts derived from human dermal fibroblasts or an enriched population of basal keratinocytes. These findings provide insights to the underlying basis of differentiation-dependent expression of a keratinocyte specific gene.

Suppression of cornified envelope formation and type 1 transglutaminase by epidermal growth factor in neoplastic keratinocytes.

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Enhanced modulation of keratinocyte motility by transforming growth factor-alpha (TGF-alpha) relative to epidermal growth factor (EGF).

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are high-affinity polypeptide ligands for the EGF receptor, which mediates their biologic activities. In this study, we directly compared the actions of both ligands in promoting keratinocyte motility. We found that normal and tumorigenic human keratinocytes responded to activation of the EGF receptor by either EGF or TGF-alpha; however, the two ligands did not elicit identical responses with regard to cell locomotion. TGF-alpha was more effective than EGF at promoting colony dispersion (cell scattering), in vitro wound closure, and single-cell migration as assessed by phagokinetic track analysis. In contrast, EGF and TGF-alpha evoked identical profiles for DNA synthesis with regard to concentration dependence and magnitude of response in normal keratinocytes and in a squamous cell carcinoma line. The overall pattern of tyrosine phosphorylation of intracellular substrates was similar when cells were stimulated with either growth factor; however, a limited number of differences in the kinetics or magnitude of protein phosphorylation were detected in subcellular fractions. These findings demonstrate that two growth factors implicated in promoting mitogenesis and locomotion may elicit divergent responses with regard to one biologic activity while retaining similar responses for other activities. This suggests that ligand-mediated mitogenic responses may not be tightly coupled to motogenic activity and further illustrates the multifunctional roles of polypeptide growth factors.

T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites.

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Sp1 and for the T3R.retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Sp1 inhibited binding of the T3R complex to this 36-base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Sp1 and T3R, function of the EGFR promoter was strongly dependent on Sp1. Sp1-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. Sp1 enhanced activity of the 36-bp element placed 5' to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Sp1 and T3R functions. However, addition of a second half-site 5' to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited Sp1 stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Sp1-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Sp1-binding site inhibits Sp1 function but is unable to direct positive function.

Identification and characterization of a regulated promoter element in the epidermal growth factor receptor gene.

We have identified a 36-base-pair proximal element (-112 to -77 relative to the AUG translation initiation codon) in the epidermal growth factor receptor 5' region that functions as a promoter; mediates inductive responses to epidermal growth factor, phorbol 12-myristate 13-acetate, and cyclic AMP; and acts in an orientation-independent manner. This region functions as an enhancer when transferred to a heterologous promoter containing a TATA box. Mutations within the 36-base-pair region alter function as assayed by reporter gene expression in recipient cells. A protein has been identified that demonstrates appropriate binding specificity to mutant DNA sequences that correlates with promoter activity observed in vivo. On the basis of DNA binding characteristics and size, the identified protein appears distinct from several previously identified transcription factors known to bind to G+C-rich regions.