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• W2085280167 abstract "Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by cotransfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5′-flanking region contains the basal promoter activity, while the region between −162 and −96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: −141 TRE, −131 GT, −123 ETS-like, and −111 TRE-like motifs and in the enhanced protections in −141 TRE and −111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both −141 TRE and −111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the −111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex. Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by cotransfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5′-flanking region contains the basal promoter activity, while the region between −162 and −96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: −141 TRE, −131 GT, −123 ETS-like, and −111 TRE-like motifs and in the enhanced protections in −141 TRE and −111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both −141 TRE and −111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the −111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex. The small proline-rich protein (SPR) 1The abbreviations used are: SPRsmall proline-rich proteinTPA12-O-tetradecanoylphorbol-13-acetateTBEtracheobronchial epitheliumTRETPA-responsive elementCREcAMP-responsive elementtkthymidine kinaseCATchloramphenicol acetyltransferaseDMSdimethyl sulfateGTGGTGG motifETSE-26 transformation specificHSVherpes simplex virusPCRpolymerase chain reaction. family with a molecular mass ranging from 10 to 30 kDa was first reported by Kartasova and van de Putte in 1988(1Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar). They demonstrated that the synthesis of SPR proteins is rapidly induced in human keratinocyte cultures after UV irradiation or treatment with TPA. Both of these treatments enhance the cornification of keratinocytes in culture. Two distinct groups of SPR cDNA clones were subsequently isolated using the differential hybridization technique, and their sequences were determined(1Kartasova T. van de Putte P. Mol. Cell. Biol. 1988; 8: 2195-2203Crossref PubMed Scopus (146) Google Scholar, 2Gibbs S. Lohman F. Teubel W. van de Putte P. Backendorf C. Nucleic Acids Res. 1990; 18: 4401-4407Crossref PubMed Scopus (60) Google Scholar). Immunohistochemical studies, using a polyclonal antibody specific to the C-terminal peptides of the SPR1 protein, demonstrate the presence of a SPR1 antigen in the suprabasal cell layer of various human squamous tissues such as the epidermis and esophagus (3Kartasova T. van Muijen G.N.P. van Pelt-Heerschap H. van de Putte P. Mol. Cell. Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (68) Google Scholar). A close association between the expression of SPR genes and squamous epithelial cell differentiation has been further demonstrated by Northern blot analysis (4An G. Huang T.H.M. Tesfaigzi J. Garcia-Heras J. Ledbetter D.H. Carlson D.M. Wu R. Am. J. Respir. Cell and Mol. Biol. 1992; 7: 104-110Crossref PubMed Scopus (37) Google Scholar) and in situ hybridization(5Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (151) Google Scholar). small proline-rich protein 12-O-tetradecanoylphorbol-13-acetate tracheobronchial epithelium TPA-responsive element cAMP-responsive element thymidine kinase chloramphenicol acetyltransferase dimethyl sulfate GGTGG motif E-26 transformation specific herpes simplex virus polymerase chain reaction. A unique feature of the structure of the SPR gene family is that the central segments of the encoded polypeptides are built up from tandemly repeated units of either eight (SPR1 and SPR3) or nine (SPR2) amino acids with the general consensus XKXPEPXX(6Gibbs S. Fijneman R. Wiegant J. van Kessel G. van de Putte P. Backendorf C. Genomics. 1993; 16: 630-637Crossref PubMed Scopus (183) Google Scholar). The function of such a repeated peptide unit is currently unknown. Backendorf and Hohl (7Backendorf C. Hohl D. Nat. Genet. 1992; 2: 91Crossref PubMed Scopus (91) Google Scholar) suggested that the SPR proteins are potential substrates involved in squamous cell cornification based on a comparison of both the N- and C-terminal amino acid sequences of SPR proteins with involucrin and loricrin, the cornified envelope proteins. Marvin et al.(8Marvin K.W. George M.D. Fujimoto W. Saunders N.A. Bernacki S.H. Jetten A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11026-11030Crossref PubMed Scopus (146) Google Scholar) have suggested that, based on Western blot analysis, the SPR proteins are part of the cornified envelope. We have recently demonstrated a similar finding. Unexpectedly, we also observed SPR1-like antigens in the nucleus(9Tesfaigzi J. An G. Wu R. Carlson D.M. J. Cell. Physiol. 1995; 164: 571-578Crossref PubMed Scopus (8) Google Scholar). A similar observation was recently made by Hohl et al.(5Hohl D. de Viragh P.A. Amiguet-Barras F. Gibbs S. Backendorf C. Huber M. J. Invest. Dermatol. 1995; 104: 902-909Abstract Full Text PDF PubMed Scopus (151) Google Scholar) in epidermis. This finding suggests that the SPR1-like protein may also play a regulatory role in gene expression. In contrast to squamous tissues, the presence of SPR proteins is very low in respiratory tract epithelia that normally express mucociliary functions; however, we have demonstrated a rapid increase of SPR1 gene product in isolated human and monkey airway epithelial cells upon plating on a culture dish(4An G. Huang T.H.M. Tesfaigzi J. Garcia-Heras J. Ledbetter D.H. Carlson D.M. Wu R. Am. J. Respir. Cell and Mol. Biol. 1992; 7: 104-110Crossref PubMed Scopus (37) Google Scholar). This increase can be reduced by supplementing the culture medium with vitamin A or its synthetic retinoid derivatives(4An G. Huang T.H.M. Tesfaigzi J. Garcia-Heras J. Ledbetter D.H. Carlson D.M. Wu R. Am. J. Respir. Cell and Mol. Biol. 1992; 7: 104-110Crossref PubMed Scopus (37) Google Scholar). Another study has demonstrated that vitamin A down-regulates the stability of SPR1 mRNA (10An G. Tesfaigzi J. Carlson D.M. Wu R. J. Cell. Physiol. 1993; 157: 562-568Crossref PubMed Scopus (25) Google Scholar). We have also observed increased expression of SPR1 protein and mRNA in the patchy squamous cell metaplasia of monkey tracheal tissues after maintaining them under a vitamin A-free organ culture condition (data not shown). TPA, a potent squamous cell differentiation inducer, also stimulates SPR1 gene expression at the transcriptional level(11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar). These results establish a close relationship between the up-regulation of SPR1 gene expression and squamous airway epithelial cell differentiation. The expression of squamous cell function in respiratory tract epithelium is a phenomenon that is frequently associated with injury. Squamous cell metaplasia has been implicated in the development of bronchogenic cancer(12Harris C.C. Sporn M.B. Kaufman D.G. Smith J.M. Baker M.S. Saffioti U. J. Natl. Cancer Inst. 1973; 48: 743-761Google Scholar, 13Jetten A.M. Nervi C. Volberg T.M. J. Natl. Cancer Invst. Monogr. 1992; 13: 93-100PubMed Google Scholar). The nature of the induction of squamous cell differentiation in the conducting airway epithelium is still unresolved. Therefore, studies of SPR1 gene expression in non-squamous airway epithelial cells may be different from those carried out in epithelial cells that express only the differentiation of skin-like properties such as keratinization and cornification. Results obtained from studying squamous cell differentiation may provide essential understanding of the mechanism underlying the divergent pathways of cell differentiation in conducting airway epithelium(14Jetten A.M. Environ. Health Perspect. 1989; 80: 149-160Crossref PubMed Scopus (62) Google Scholar). We have isolated the human SPR1 genomic clone and have completed the DNA sequencing of the 5′-flanking region(11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar). The purpose of this communication is to use the transient transfection study, in vivo genomic footprinting, site-directed mutagenesis, and the gel mobility shift assay to elucidate the elements essential for both the basal, uninduced and TPA-inducible promoter activities. We observed that the expression of the human SPR1 gene in conducting airway epithelium is dependent on JUN and TRE motifs located between −141 and −111 of the 5′-flanking region. Furthermore, the expression is cell type-specific, with a decrease in the promoter activity from primary epithelial cells to established cell lines, with no activity in the non-epithelial cell type. Primary TBE cells were isolated from rhesus monkey and human tracheobronchial tissues, which were obtained from the California Regional Primate Research Center and the Medical Center of the University of California at Davis, respectively. All procedures involved in the tissue procurement were approved by the University of California at Davis Animal Protocol Review Committee and the Human Subject Research Review Committee. Epithelial cell isolation and the culture conditions were carried out as described previously(15Wu R. Schiff L.J. In Vitro Models of Respiratory Epithelium. CRC Press, Boca Raton, FL1986: 1-26Google Scholar, 16Robinson C.B. Wu R. J. Tissue Culture Methods. 1991; 13: 95-102Crossref Scopus (39) Google Scholar). Experiments were carried out between 7 and 14 days after the initiation of primary culture. The immortalized normal human TBE cell line, BEAS-2B, subclone S, was obtained from J. F. Lechner (Lovelace Biomedical & Environmental Research Institute, Albuquerque, NM). This cell line was maintained in a serum-free hormone-supplemented medium(15Wu R. Schiff L.J. In Vitro Models of Respiratory Epithelium. CRC Press, Boca Raton, FL1986: 1-26Google Scholar, 16Robinson C.B. Wu R. J. Tissue Culture Methods. 1991; 13: 95-102Crossref Scopus (39) Google Scholar). Other cell lines, HepG2 (ATCC HB8065, a hepatocellular carcinoma), Caco-2 (ATCC HTB37, a colon adenocarcinoma), and A172 (ATCC CRL1629, a glioblastoma), were obtained from the American Type Culture Collection (ATCC), and they were maintained in serum-supplemented culture conditions according to the supplier's data sheet. Plasmid pBL-CAT2 contains the herpes simplex virus (HSV) thymidine kinase (tk) promoter in front of the CAT structural gene. Plasmid pBL-CAT3 is a promoterless construct. The expression plasmids encoding JUN B and JUN D cDNAs are pBR322-based vectors driven by a long terminal repeat promoter of the mouse sarcoma virus. The other expression plasmid encoding c-JUN cDNA was derived from pSVL vector (Pharmacia Biotech. Inc.) driven by the SV40 early promoter. The control plasmid pCH110 encodes β-galactosidase cDNA, which is driven by the SV40 promoter. Various 5′-flanking regions (between −2000 and +9 relative to the transcription start site) of the human SPR1 gene were amplified from human genomic clone (11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar) by polymerase chain reaction (PCR) with two restriction sites attached to the two specific primers: SalI to the 5′-primer and XbaI to the 3′-primer. Positive clones containing the appropriate inserts were determined by PCR screening and confirmed by restriction mapping and DNA sequencing. The generation of various chimeric constructs is described in Fig. 1. The 2000-CAT3, 622-CAT3, 557-CAT3, 162-CAT3, 113-CAT3, 98-CAT3, and 67-CAT3 constructs consist of DNA fragments between −2000 and +9, −622 and +9, −557 and +9, −162 and +9, −113 and +9, −98 and +9, and −67 and +9, respectively, of the SPR1 promoter in the pBL-CAT3 vector under XbaI and SalI cloning sites. The chimeric constructs 622/81-tk-CAT2, 622/540-tk-CAT2, 193/81-tk-CAT2, and 162/96-tk-CAT2 contain various 5′-flanking sequences of SPR1 gene between −622 and −81, −622 and −540, −193 and −81, and −162 and −96 relative to the transcription start site, respectively, in the pBL-CAT2 vector under XbaI and SalI cloning sites. The PCR reactions were carried out in a total volume of 100 μl containing 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.2 mM MgCl2, 0.01% gelatin, 250 μM each of dATP, dGTP, dCTP, and dTTP (Pharmacia). Initial denaturation was at 95°C for 5 min followed by 30 cycles of 94°C denaturation (1 min), 55°C annealing (1 min), and 72°C extension (4 min), with a final 72°C extension for 7 min in an automated thermal cycler (Perkin-Elmer Corp.). The 451-CAT3 and 135-CAT3 constructs containing regions between −451 to +9 and −135 to +9 of the SPR1 promoter, respectively, were constructed by ligation of SspI-XbaI and HincII-XbaI DNA fragments, digested from 622-CAT3 construct into pBL-CAT3 vector. DNA transfection was performed using a liposome technique (Lipofectin™) according to the procedure suggested by the manufacturer (Life Technologies, Inc.). Each culture dish at 70-80% confluence was washed with serum-free culture medium and then transfected with 3 μg of chimeric construct DNA, 1 μg of pCH110 plasmid DNA, and 1 μg of expression plasmid DNA when the cotransfection experiments were carried out. The pUC18 plasmid DNA was added to ensure that all the individual experiments contained the same total amount of DNA. TPA was added to transfected cultures at 10 ng/ml for 48 h before the harvesting. Cell extracts were prepared by freeze-thaw in 0.25 M Tris-HCl (pH 8.0). The protein concentration of cell extracts was determined by a modified Bradford technique (Bio-Rad). Equal amounts of protein were assayed for CAT activity by either a liquid scintillation method (11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar) or an enzyme-linked immunosorbent assay kit (Boehringer Mannheim) following the manufacturer's suggested protocol. The β-galactosidase activity, used as the internal control that normalized transfection efficiency, was determined by a β-galactosidase enzyme assay system (Promega). Nuclear extracts were prepared according to the method of Dignam et al.(17Dignam J. Lebowitz R. Roeder R. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar) from cultured cells, except that during extraction 600 mM NaCl was used instead of 400 mM NaCl. The binding was performed in a 20-μl reaction volume containing 25 mM HEPES, pH 7.9, 10% (v/v) glycerol, 30 mM NaCl, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 5 mM MgCl2, 0.5 mM EDTA, 0.5 μg of salmon sperm DNA, and 50-100 ng of poly(dI-dC). After incubation on ice for 10 min with 3-5 μg of nuclear extract, 0.1-0.5 ng of end-labeled probe (~20,000 cpm) was added and incubated at room temperature for 15-20 min. The DNA-protein complexes were resolved in native 4% acrylamide gels (30:1 ratio of acrylamide to bisacrylamide) in 0.5 × Tris borate-EDTA buffer. In supershift analysis, nuclear extracts were mixed with 1-2 μg of anti-c-JUN antibody (Santa Cruz Biotechnology, CA) and incubated on ice prior to adding the labeled DNA probe. For competition experiments, cold consensus AP1 oligonucleotide (Promega) was added to the reaction mixture prior to adding labeled probe. The method of in vivo DMS footprinting was described by Mueller and Wold(18Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (787) Google Scholar). The primary TBE cells were cultured to 70-80% confluence followed by incubation with or without TPA (10 ng/ml, Sigma) for 12 h. For in vivo DMS treatments, cells were treated with 0.1% DMS (Aldrich) for 2 min at room temperature. The DMS-treated cells were lysed in SDS buffer (0.5% SDS, 20 mM Tris-Cl, pH 8.0, 200 mM NaCl, and 20 mM EDTA) and then incubated with proteinase K (250 μg/ml) (Sigma) at 37°C for 12 h. Genomic DNA was then isolated by phenol/chloroform extraction. As an in vitro control, protein-free genomic DNA was prepared and treated with DMS for 20-30 s at room temperature. It was then cleaved by piperidine (Aldrich) and mapped by the ligation-mediated PCR method of genomic DNA sequencing(19Reddy P.M. Stamatoyannopoulos G. Papayannopoulou T. Shen C.K. J. Biol. Chem. 1994; 269: 8287-8295Abstract Full Text PDF PubMed Google Scholar). DNA fragments were analyzed on 6% denaturing urea-polyacrylamide gels. The bands on the autoradiogram were detected after 24-48 h of exposure. The reproducibility of the in vivo footprinting data was checked by analyzing genomic DNA samples prepared from three or more separate batches of DMS-treated cells. The StatView™ statistical program on the Macintosh computer was used to perform analysis of variance between different CAT reporter constructs. The Fisher PLSD (Protected Least Significant Difference) test was used to determine whether existing differences were significant at the 95% confidence level. Previous research based on the nuclear run-on assay and transient transfection study suggests that the treatment of primary airway epithelial cells with TPA stimulates the expression of the SPR1 gene at the transcriptional level (11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar). DNA sequence analysis of genomic clones revealed the following motifs: “TATA” box at −28; three TRE-like sites at −49, −111, and −472; ETS at −55 and ETS-like at −123; GT motif (GGTGG) at −131; TRE site at −141; and CRE-like at −588 relative to the transcription start site in the 5′-flanking region of the human SPR1 gene(11An G. Tesfaigzi J. Chuu Y.J. Wu R. J. Biol. Chem. 1993; 268: 10977-10982Abstract Full Text PDF PubMed Google Scholar). The −49 TRE-like site and −55 ETS site are merged together. To elucidate the functional roles of these putative cis-acting elements, a number of chimeric constructs containing various lengths of the 5′-flanking region and the CAT reporter gene were prepared (Fig. 1). The functional roles for the motifs of −141 TRE, −131 TRE-like, −55 ETS, and −49 TRE-like were further analyzed by site-directed mutagenesis. These constructs were used in transient transfection studies. Initially, we observed no significant difference in either the basal or the TPA-induced CAT activities between 622-CAT3 and 2000-CAT3 construct transfected cells of primary TBE cultures (data not presented). Therefore, we focused the deletional analysis on the region from −622 to +9. As presented in Fig. 2, the relative CAT activity in 622-CAT3 transient transfected cells without TPA treatment is 24-fold higher than the control cells transfected with the promoterless pBL-CAT3 DNA or 2-fold higher than those cells with pBL-CAT2 containing the tk promoter. The relative CAT activity in the absence of TPA treatment was the same among different chimeric constructs, except for the 162-CAT3, which was 4-fold higher (Fig. 2). This implies that the DNA sequences between −98 and +9 of the 5′-flanking region contain sufficient information for the basal promoter activity. This elevation in the basal promoter activity observed in 162-CAT3, but not in 135-CAT3, implies that the DNA fragment between −162 and −135 contains a sequence responsible for the enhanced expression. In contrast, cells transfected with 451-CAT3 construct, which contains the 162-CAT3 DNA fragment plus the flanking region between −451 and −162, did not exhibit enhanced basal promoter activity. This implies that the region between −451 and −162 may contain a sequence that down-regulates this enhanced activity. To further elucidate the region involved in the basal promoter activity, a 67-CAT3 chimeric construct that includes −55 ETS, −49 TRE-like, and −28 TATA was studied. As presented in Fig. 3, the relative CAT activity in cells transfected with this construct is very low but significantly higher (2-fold) than the promoterless pBL-CAT3 transfected cells; however, this activity is less than 10% of the 98-CAT3 transfection. To clarify this residual activity, site-directed mutations in both −55 ETS and −49 TRE-like sites were prepared. Single or double mutations in these sites have no effect on this residual CAT activity, which suggests that neither site is involved in this residual CAT activity. Both immunohistochemistry (3Kartasova T. van Muijen G.N.P. van Pelt-Heerschap H. van de Putte P. Mol. Cell. Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (68) Google Scholar) and mRNA analyses (4An G. Huang T.H.M. Tesfaigzi J. Garcia-Heras J. Ledbetter D.H. Carlson D.M. Wu R. Am. J. Respir. Cell and Mol. Biol. 1992; 7: 104-110Crossref PubMed Scopus (37) Google Scholar) have demonstrated that the expression of the SPR1 gene is closely associated with squamous epithelial cell types. This specificity is preserved in the basal promoter analysis. As illustrated in Fig. 4, 162-CAT3 transfected primary human TBE cells exhibited the highest CAT activity as compared with the immortalized human normal TBE cell line, S clone, and other ATCC cell lines. The non-epithelial cell line A172 exhibited no CAT activity. In the case of transfection with the 98-CAT3 construct, which contains no enhanced sequence, all TBE cells, regardless of their origin, exhibited significantly lower levels of basal promoter activity. Transfected non-TBE cells exhibited no activity. The relative CAT activity in 622-CAT3 transfected cells was further enhanced 4-fold by TPA (Fig. 2). This enhancement of promoter activity by TPA is maintained in various deletion-construct transfected TBE cells, until the TRE-like motif at −111 is deleted in such constructs as 98-CAT3 and 67-CAT3. These results imply that the CRE-like motif at −588 and the two TRE-like motifs at −472 and −49 are not involved in mediating the TPA response. We observed that TPA transiently stimulates the expression of the JUN family gene products (data not shown) in TBE cells prior to the stimulation of SPR1 gene expression. We then examined whether overexpression of JUN family proteins can enhance the CAT activity in the absence of TPA treatment. Cotransfection with one of the JUN family genes in 622-CAT3 transfected cells stimulated CAT activity 2-10-fold in the absence of TPA treatment (Fig. 5). Among the JUN family genes, c-JUN cotransfection was the most active. TPA treatment of these c-JUN cotransfected cells resulted in a 40-fold increase of CAT activity; however, TPA has no effect on the expression of JUN family proteins in cotransfected cells (data not shown). These results suggest that the SPR1 promoter is synergistically stimulated by TPA and c-JUN. To further elucidate whether the regulatory sequences at the 5′-flanking region can enhance the promoter activity in heterologous constructs, several selective DNA fragments of SPR1 promoter were cloned to the pBL-CAT2 vector carrying the heterologous HSV-tk promoter (Fig. 1C). As shown in Fig. 6, in the absence of TPA and c-JUN cotransfection, the relative CAT activities in both 622/81-tk-CAT2 and 193/81-tk-CAT2 transfected cells were 13- and 11-fold higher than the pBL-CAT2 control. However, this enhanced activity cannot be seen in 622/540-tk-CAT2 transfected cells. This suggests that the enhanced activity located at the −193 and −81 5′-flanking region is capable of stimulating a heterologous promoter. The stimulations by TPA and c-JUN cotransfection are not as significant as in the homologous promoter system, only a 20-40% stimulation. However, this stimulation was not seen in 622/540-tk-CAT2 transfected cells. These results suggest that the DNA sequence located between −193 and −81 contains the cis-element that is also capable of activating the heterologous tk promoter in response to TPA/c-JUN treatment. To elucidate the site(s) responsible for this stimulation, we carried out site-direct mutations in −141 TRE and −111 TRE-like motifs. As illustrated in Fig. 7, double mutations at these two sites (−141M, −111M) significantly reduced the enhancement on tk promoter activity; however, a single mutation at −141 TRE motif (−141M) has no effect. Yet, a single mutation at the −111 TRE-like site (−111M) reduced the enhanced activity by 50%. In contrast to the enhanced tk promoter activity, a single mutation in either of these two sites has no effect on the stimulating activity exerted by the c-JUN cotransfection. These results suggest that any one of these two TRE motifs can participate in c-JUN mediated stimulation. To further understand the nature of the regulation of SPR1 gene expression in vivo, the interactions between the promoter DNA sequence and transcriptional proteins were studied by DMS footprinting. Using appropriate primers as described in Fig. 8A, the DNA-protein interaction sites of the SPR1 promoter region were mapped. Genomic footprinting data of the DNA fragment between −164 and −94 are displayed in Fig. 8B. The G residue at −140 of the coding strand of the TRE motif was partially protected, while the flanking G residue at −143 was hyperreactive in primary TBE cells. This protection at −140 G residue was slightly enhanced by TPA. In addition, a displayed protection pattern at −138 G residue was observed only in TPA-treated cells. Other protection residues in the coding strand included the G residues at −130, −127, −126, −121, −120, which are parts of the GT (−131 to −127) and ETS-like (−123 to −116) motifs. At the −111 TRE-like motif site, the G residue at −110 was partially protected, and this protection was slightly enhanced in cells after the TPA treatment. The G residues at −113, −112, and −99 in other flanking regions were partially protected, whereas the G residue at −94 was hyperreactive to piperidine cleavage in both TPA-treated or untreated cells. On the noncoding strand, only G residues at −104 and −106 displayed protections (Fig. 8B). Other DMS footprints were detected around the CRE-like motif. On the coding strand, similar protections occurring at −587 and −606 G residues were observed in both TPA-treated and untreated cells (Fig. 9B). On the noncoding strand, G residue at −582 was partially protected, whereas G residues at −589, −593, and −598 were hyperreactive. Again, TPA had no effect on the footprinting pattern in this region. A summary of these footprinting studies is presented in Fig. 10. There are multiple protections in the region between the −141 TRE and the −111 TRE-like motifs. These protections, presumably due to the interactions between the trans-activation proteins and the DNA sequence, are further enhanced on both −141 TRE and −111 TRE-like motifs by TPA. In contrast, the protections in the CRE-like motif at −588 are not affected. The DNA-protein interactions were further studied" @default.
• W2085280167 created "2016-06-24" @default.
• W2085280167 creator A5019299639 @default.
• W2085280167 creator A5021530808 @default.
• W2085280167 creator A5023146592 @default.
• W2085280167 creator A5032733372 @default.
• W2085280167 creator A5034002289 @default.
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• W2085280167 date "1995-11-01" @default.
• W2085280167 modified "2023-09-27" @default.
• W2085280167 title "Expression of Human Squamous Cell Differentiation Marker, SPR1, in Tracheobronchial Epithelium Depends on JUN and TRE Motifs" @default.
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