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• W2021330348 abstract "Several of the complications seen in patients with both type I and type II diabetes mellitus are associated with alterations in the expression of matrix metalloproteinases. To identify the cis-acting elements that mediate the stimulatory effect of insulin on collagenase-1 (matrix metalloproteinase-1) gene transcription a series of collagenase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into HeLa cells. Multiple promoter elements, including an Ets and activator protein-1 (AP-1) motif, were required for the effect of insulin. The AP-1 motif appears to be a target for insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene, whereas the data suggest that the Ets motif acts to enhance the effect of insulin mediated through the AP-1 motif. Multiple promoter elements were also required for the stimulatory effect of phorbol esters on collagenase-CAT gene transcription, and the AP-1 motif was also a target for phorbol ester signaling. However, thecis-acting elements required for the effects of insulin and phorbol esters were not identical. Moreover, phorbol esters were a much more potent inducer of collagenase-CAT gene transcription than insulin, a difference that may be explained by selective effects of insulin and phorbol esters on AP-1 expression. Several of the complications seen in patients with both type I and type II diabetes mellitus are associated with alterations in the expression of matrix metalloproteinases. To identify the cis-acting elements that mediate the stimulatory effect of insulin on collagenase-1 (matrix metalloproteinase-1) gene transcription a series of collagenase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into HeLa cells. Multiple promoter elements, including an Ets and activator protein-1 (AP-1) motif, were required for the effect of insulin. The AP-1 motif appears to be a target for insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene, whereas the data suggest that the Ets motif acts to enhance the effect of insulin mediated through the AP-1 motif. Multiple promoter elements were also required for the stimulatory effect of phorbol esters on collagenase-CAT gene transcription, and the AP-1 motif was also a target for phorbol ester signaling. However, thecis-acting elements required for the effects of insulin and phorbol esters were not identical. Moreover, phorbol esters were a much more potent inducer of collagenase-CAT gene transcription than insulin, a difference that may be explained by selective effects of insulin and phorbol esters on AP-1 expression. The maintenance of the extracellular matrix is accomplished by a balance of synthesis and degradation, the latter being determined by the relative activities of a family of extracellular matrix proteinases, the matrix metalloproteinases (MMPs), 1The abbreviations used are: MMP, matrix metalloproteinase; PEPCK, phosphoenolpyruvate carboxykinase; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; ME, malic enzyme; IRS, insulin response sequence.1The abbreviations used are: MMP, matrix metalloproteinase; PEPCK, phosphoenolpyruvate carboxykinase; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; ME, malic enzyme; IRS, insulin response sequence. and the tissue inhibitors of MMPs (1Vincenti M.P. White L.A. Schroen D.J. Benbow U. Brinckerhoff C.E. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 391-411Crossref PubMed Scopus (242) Google Scholar, 2Borden P. Heller R.A. Crit. Rev. Eukaryotic Gene Expression. 1997; 7: 159-178Crossref PubMed Scopus (290) Google Scholar, 3Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar). Tissue inhibitors of MMPs block MMP action by binding covalently to MMPs and preventing both activation of the MMP by enzymatic modification and the ability of MMPs to bind substrate (1Vincenti M.P. White L.A. Schroen D.J. Benbow U. Brinckerhoff C.E. Crit. Rev. Eukaryotic Gene Expression. 1996; 6: 391-411Crossref PubMed Scopus (242) Google Scholar, 2Borden P. Heller R.A. Crit. Rev. Eukaryotic Gene Expression. 1997; 7: 159-178Crossref PubMed Scopus (290) Google Scholar, 3Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar). The regulation of MMP gene expression has received considerable attention primarily because of the involvement of increased MMP activity in tumor progression, specifically the development of malignant carcinomas (4Crawford H.C. Matrisian L.M. Enzyme Protein. 1996; 49: 20-37Crossref PubMed Scopus (184) Google Scholar). However, several of the complications associated with both type I, insulin-dependent diabetes mellitus and type II, non-insulin-dependent diabetes mellitus, including glomerulosclerosis of the kidney (5Schleicher E.D. Olgemoller B. Eur. J. Chem. Clin. Biochem. 1992; 30: 635-640PubMed Google Scholar, 6Adler S. J. Am. Soc. Nephrol. 1994; 5: 1165-1172PubMed Google Scholar), retinopathy (7Jiang Z.Y. Towler H.M.A. Luthert P. Lightman S. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus. A Fundamental and Clinical Text. Lippincott-Raven, Philadelphia1996: 719-727Google Scholar) periodontal disease (8Birkedal-Hansen H. J. Periodontol. 1993; 64 Suppl. 5: 474-484Google Scholar), and some forms of cardiac disease (9Shehadeh A. Regan T.J. Clin. Cardiol. 1995; 18: 301-305Crossref PubMed Scopus (150) Google Scholar), are also characterized, in part, by alterations in the amount and composition of extracellular matrix protein. In the streptozotocin rat model of type I diabetes, collagenase-1 (MMP-1; referred to henceforth as collagenase) gene expression in the glomerulus is decreased (10Nakamura T. Fukui M. Ebihara I. Osada S. Tomino Y. Kiode H. Renal Physiol. Biochem. 1994; 17: 316-325PubMed Google Scholar), and this would be predicted to contribute to an increase in the mesangial matrix and/or the glomerula basement membrane, one of the characteristics of glomerulosclerosis (5Schleicher E.D. Olgemoller B. Eur. J. Chem. Clin. Biochem. 1992; 30: 635-640PubMed Google Scholar, 6Adler S. J. Am. Soc. Nephrol. 1994; 5: 1165-1172PubMed Google Scholar). This decrease in collagenase gene expression is reversed by insulin treatment (10Nakamura T. Fukui M. Ebihara I. Osada S. Tomino Y. Kiode H. Renal Physiol. Biochem. 1994; 17: 316-325PubMed Google Scholar), but it is unclear whether this represents a direct effect of insulin as opposed to an indirect effect mediated through changes in glucose concentration. The effect of insulin on collagenase expression in glomerula-derived cell lines has not been studied; however, in NIH 3T3 (11Medema R.H. Wubbolts R. Bos J.L. Mol. Cell. Biol. 1991; 11: 5963-5967Crossref PubMed Scopus (110) Google Scholar), Chinese hamster ovary (12Rutter G.A. White M.R.H. Tavaré J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and HeLa cells (13Streeper R.S. Chapman S.C. Ayala J.E. Svitek C.A. Goldman J.K. Cave A. O'Brien R.M. Mol. Endocrinol. 1998; 12: 1778-1791Crossref PubMed Scopus (22) Google Scholar) insulin stimulates the expression of reporter genes when ligated to the collagenase promoter. Interestingly, if insulin does directly regulate collagenase gene expression in the glomerulus, then collagenase expression would actually be predicted to be increased in hyperinsulinemic individuals because the insulin resistance associated with muscle, adipose tissue, and liver may not be manifest in the kidney (14Reaven G.M. Am. J. Kidney Dis. 1997; 30: 928-931Abstract Full Text PDF PubMed Scopus (128) Google Scholar). cis-Acting elements that mediate the action of insulin on gene transcription are referred to as insulin response sequences (IRSs) or elements (15O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (436) Google Scholar). Several IRSs have been identified, but it is apparent that a single consensus IRS does not exist (15O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (436) Google Scholar). Instead, it is predicted that multiple classes of consensus IRSs will be found, only three of which have currently been identified (15O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (436) Google Scholar). One of these has the consensus sequence T(G/A)TTT(T/G)(T/G) and mediates the insulin-dependent transcriptional inhibition of several hepatic genes such as those encoding phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor-binding protein-1, tyrosine aminotransferase, apolipoprotein CIII, and glucose-6-phosphatase (16O'Brien R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar, 17Suwanickul A. Morris S.L. Powell D.R. J. Biol. Chem. 1993; 268: 17063-17068Abstract Full Text PDF PubMed Google Scholar, 18Ganss R. Weih F. Schütz G. Mol. Endocrinol. 1994; 8: 895-903Crossref PubMed Scopus (56) Google Scholar, 19Li W.W. Dammerman M.M. Smith J.D. Metzger S. Breslow J.L. Leff T. J. Clin. Invest. 1995; 96: 2601-2605Crossref PubMed Scopus (242) Google Scholar, 20Streeper R.S. Svitek C.A. Chapman S. Greenbaum L.E. Taub R. O'Brien R.M. J. Biol. Chem. 1997; 272: 11698-11701Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The other consensus IRSs are the Ets motif (21Jacob K.K. Ouyang L. Stanley F.M. J. Biol. Chem. 1995; 270: 27773-27779Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and the serum response element (15O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (436) Google Scholar, 22Thompson M.J. Roe M.W. Malik R.K. Blackshear P.J. J. Biol. Chem. 1994; 269: 21127-21135Abstract Full Text PDF PubMed Google Scholar), which mediate stimulatory effects of insulin on several genes. However, unlike the PEPCK-type IRS that confers a selective effect of insulin (15O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (436) Google Scholar), the Ets motif and serum response element can mediate the effects of multiple other hormones on gene transcription (23Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (346) Google Scholar, 24Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar). The activator protein-1 (AP-1) motif, which binds members of the Fos and Jun transcription factor families (25Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3256) Google Scholar) and, like the Ets motif and serum response element, mediates transcriptional changes in response to multiple ligands (26Karin M. Liu Z.-g. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2289) Google Scholar), may represent a fourth class of consensus IRS. Thus, Rutter et al. (12Rutter G.A. White M.R.H. Tavaré J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) have shown that in Chinese hamster ovary cells, mutation of the AP-1 motif in the collagenase promoter abolishes the stimulatory effect of insulin on the expression of a collagenase-luciferase fusion gene. Similarly, in rat H4IIE hepatoma cells, an AP-1 motif is required for the stimulatory effect of insulin on the expression of a malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) fusion gene (13Streeper R.S. Chapman S.C. Ayala J.E. Svitek C.A. Goldman J.K. Cave A. O'Brien R.M. Mol. Endocrinol. 1998; 12: 1778-1791Crossref PubMed Scopus (22) Google Scholar). However, paradoxically, in HeLa cells insulin has almost no effect on ME gene transcription, whereas it markedly stimulates collagenase gene transcription (13Streeper R.S. Chapman S.C. Ayala J.E. Svitek C.A. Goldman J.K. Cave A. O'Brien R.M. Mol. Endocrinol. 1998; 12: 1778-1791Crossref PubMed Scopus (22) Google Scholar). This observation raises the question as to why the AP-1 motif appears to only mediate an insulin-dependent activation of gene transcription in some cell contexts. To indirectly address this question, we have analyzed the promoter elements required for the stimulatory effect of insulin on collagenase gene transcription in HeLa cells. The results demonstrate that multiple promoter elements are required for the effect of insulin on collagenase gene transcription in addition to the AP-1 motif. However, the collagenase AP-1 motif appears to be a target of insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene. The additional collagenase promoter elements that are required for the full stimulatory effect of insulin may bind accessory factors that act to enhance the effect of insulin mediated through the AP-1 motif. The data also suggest that the mechanism of insulin and phorbol ester signaling through the collagenase-1 AP-1 motif are distinct. [α-32P]dATP (>3000 Ci·mmol−1) and [3H] acetic acid, sodium salt (>10 Ci·mmol−1) were purchased from Amersham Pharmacia Biotech and ICN, respectively. Insulin was obtained from Collaborative Bioproducts, and phorbol 12-myristate 13-acetate (PMA) and α-chymotrypsin were from Sigma. Specific antisera to c-Fos (sc-52), Fra-1 (sc-183), Fra-2 (sc-604), c-Jun (sc-45), Jun B (sc-46), and Jun D (sc-74) were all obtained from Santa Cruz Biotechnology, Inc. DNA manipulations were accomplished by standard techniques (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), and DNA sequencing was performed using the USB Sequenase kit. All plasmid constructs were purified by centrifugation twice through cesium chloride gradients (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A BglII-HindIII fragment of the human collagenase-1 promoter, spanning the sequence from −518 to +64, was isolated from the plasmid pCol·Luc (12Rutter G.A. White M.R.H. Tavaré J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and ligated, in the same orientation as that of the endogenous gene, into theBglII-digested polylinker of the pCAT(An) expression vector, a generous gift from Dr. Howard Towle (28Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar). The noncompatible 3′HindIII-BglII junction was filled in using the Klenow fragment of Escherichia coli DNA polymerase I prior to blunt-end ligation. The pCAT(An) vector has polyadenylation signals located 5′ of the polylinker to prevent read-through transcription (28Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar). Control experiments demonstrated that there was no basal CAT expression and no effect of insulin or PMA when the pCAT(An) vector, minus the collagenase promoter, was transiently transfected into HeLa cells (data not shown). A series of truncated collagenase-CAT fusion genes was then generated, with the 5′ end points shown in Fig. 4, using the −518/+64 pCAT(An) construct as a template, by either restriction enzyme digestion or polymerase chain reaction, using standard techniques (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The 3′ polymerase chain reaction primer was designed to conserve the junction between the collagenase promoter and CAT reporter gene to be the same as that in all other collagenase-CAT fusion gene constructs. All promoter fragments generated by polymerase chain reaction were completely sequenced to ensure the absence of polymerase errors, whereas promoter fragments generated by restriction enzyme digestion were only sequenced to confirm the 5′ end points. Site-directed mutants of the collagenase Ets and AP-1 motifs in the context of the −97 to +64 promoter fragment, as shown in Fig. 5, were generated by polymerase chain reaction in conjunction with 5′ primers that incorporated the mutated sequence (Table I).Figure 5Site-directed mutation of the Ets and AP-1 motifs in the collagenase promoter reduces both basal collagenase-CAT fusion gene transcription and the stimulatory effects of insulin and PMA . HeLa cells were transiently co-transfected, as described under “Experimental Procedures,” with a series of collagenase-CAT fusion genes, with 5′ deletion end points and mutations as shown on theabscissa, and expression vectors encoding β-galactosidase and the insulin receptor. Following transfection, cells were incubated for 24 h in serum-free medium in the presence or absence of 10 nm insulin or 100 nm PMA. The cells were then harvested, and both CAT and β-galactosidase activity were assayed as described previously (20Streeper R.S. Svitek C.A. Chapman S. Greenbaum L.E. Taub R. O'Brien R.M. J. Biol. Chem. 1997; 272: 11698-11701Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 30O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.-C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar). Results are presented as the ratio of CAT activity, corrected for protein concentration in the cell lysate, in insulin-treated (A) or PMA-treated (B)versus control cells and are expressed as fold induction. InC, results are presented as the ratio of CAT to β-galactosidase activity in control cells and are expressed as arbitrary units. Results represent the mean of ± S.E. of 4–13 (insulin) or 3 (PMA) experiments, in which each construct was assayed in duplicate.View Large Image Figure ViewerDownload (PPT)Table ISequence of oligonucleotides used in these studiesView Large Image Figure ViewerDownload (PPT)All nucleotide positions are relative to the collagenase gene transcription start site at +1 (33Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (585) Google Scholar). The collagenase AP-1 and core Ets motifs are boxed. Wild-type (WT) and mutated (MUT) sequences are shown in uppercase and lowercase letters, respectively. The oligonucleotide representing the collagenase sequence between −97 and −64 was synthesized with either BamHI (GATC)- or HindIII (AGCT)-compatible ends. Both the BamHI- andHindIII-compatible ends contribute to the collagenase sequence such that the oligonucleotide with BamHI ends actually represents the collagenase sequence between −98 and −64, whereas the oligonucleotide with HindIII ends actually represents the collagenase sequence between −97 and −61. Open table in a new tab All nucleotide positions are relative to the collagenase gene transcription start site at +1 (33Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (585) Google Scholar). The collagenase AP-1 and core Ets motifs are boxed. Wild-type (WT) and mutated (MUT) sequences are shown in uppercase and lowercase letters, respectively. The oligonucleotide representing the collagenase sequence between −97 and −64 was synthesized with either BamHI (GATC)- or HindIII (AGCT)-compatible ends. Both the BamHI- andHindIII-compatible ends contribute to the collagenase sequence such that the oligonucleotide with BamHI ends actually represents the collagenase sequence between −98 and −64, whereas the oligonucleotide with HindIII ends actually represents the collagenase sequence between −97 and −61. A plasmid designated X(An) was generated by ligating a minimalXenopus 68-kDa albumin promoter (29Schorpp M. Kugler W. Wagner U. Ryffel G.U. J. Mol. Biol. 1988; 202: 307-320Crossref PubMed Scopus (59) Google Scholar) with the following sequence (5′-AAGCTTGATCTCTCTGAGCAATAGTATAAAACTCGAG-3′;HindIII and XhoI restriction enzyme sites underlined; TATA box in italics) intoHindIII-XhoI cleaved pCAT(An). The plasmid XMB was then generated by removing a BamHI site located just 5′ of the HindIII site by the use of mung bean nuclease. Double-stranded complementary oligonucleotides, representing various regions of the collagenase promoter (Table I), were synthesized withHindIII compatible ends and ligated intoHindIII-cleaved XMB in multiple copies. The number of inserts was determined by restriction enzyme analysis and confirmed by DNA sequencing. Human HeLa cervical carcinoma cells were grown to 90% confluence in T150 flasks in DMEM containing 10% (v/v) calf serum and were replated the day before use into 55-cm2 culture dishes (1 flask to 26 dishes). Attached cells were transfected by addition of 0.5 ml of a calcium phosphate-DNA co-precipitate (30O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.-C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar), containing the reporter gene construct (15 μg), an expression vector for β-galactosidase (2.5 μg), and an expression vector encoding the insulin receptor (5 μg), courtesy of Dr. Jonathan Whittaker, to the 10 ml of culture medium. After an overnight incubation the medium was removed and the cells incubated in 10 ml PBS for 10 min at room temperature. The cells were then incubated for a further 8–24 h in 10 ml serum-free DMEM supplemented with or without various concentrations of PMA or insulin, as indicated in the Figure legends, prior to harvesting. Hamster insulinoma tumor cells were grown to 70% confluence in T150 flasks in DMEM containing 2.5% (v/v) fetal bovine serum and 15% (v/v) horse serum and were replated the day before use into 55-cm2 culture dishes (1 flask to 14 dishes). Attached cells were then co-transfected as described previously (31Ebert D.H. Streeper R.S. Chapman S.C. Svitek C.A. Goldman J.K. Mathews C.E. Leiter E.H. Hutton J.C. O'Brien R.M. Diabetes. 1999; 48: 543-551Crossref PubMed Scopus (37) Google Scholar) by addition of 0.5 ml of a calcium phosphate-DNA co-precipitate containing 15 μg of reporter plasmid DNA and expression vectors encoding β-galactosidase (2.5 μg) and the insulin receptor (5 μg) to the 10 ml of culture medium. After incubation for between 4 and 6 h, the cells were treated for 2 min with 20% glycerol in serum-free DMEM (5 ml/dish). The cells were then rinsed for 5 min with serum-free DMEM (5 ml/dish) prior to incubation for 24 h in serum-free DMEM (10 ml/dish) supplemented with or without various concentrations of insulin, as indicated in the Figure legends. Rat hepatoma H4IIE cells were grown in 12-well plates in DMEM containing 10% (v/v) fetal bovine serum. Attached cells were transfected at ∼60% confluence with the plasmid pCol·Luc (2 μg; Ref. 12Rutter G.A. White M.R.H. Tavaré J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) using 4 μl of the Tfx-50 transfection reagent (Promega) in a final volume of 400 μl serum-free DMEM, as described previously (32Dickens M. Svitek C.A. Culbert A.A. O'Brien R.M. Tavaré J.M. J. Biol. Chem. 1998; 273: 20144-20149Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). After incubation for 2 h, the cells were then overlaid with 2 ml of serum-containing DMEM. After an overnight incubation, the cells were washed with serum-free DMEM for 2 h prior to incubation for 24 h in serum-free DMEM supplemented with or without various concentrations of insulin, as indicated in the Figure legends. Under these growth and transfection conditions 100 nm insulin was not toxic to these cells (see Fig. 2), in contrast to previous experiments in which the cells were grown in DMEM containing 2.5% (v/v) fetal calf serum and 2.5% (v/v) newborn calf serum and were transiently transfected in solution using calcium phosphate-DNA co-precipitation (13Streeper R.S. Chapman S.C. Ayala J.E. Svitek C.A. Goldman J.K. Cave A. O'Brien R.M. Mol. Endocrinol. 1998; 12: 1778-1791Crossref PubMed Scopus (22) Google Scholar). Cells were either harvested by trypsin digestion and sonicated in 300 μl of 250 mm Tris (pH 7.8) containing 2 mm phenylmethylsulfonyl fluoride, or for luciferase assays, cells were extracted by scraping into passive lysis buffer (Promega). CAT, β-galactosidase, and luciferase assays were performed exactly as described previously (20Streeper R.S. Svitek C.A. Chapman S. Greenbaum L.E. Taub R. O'Brien R.M. J. Biol. Chem. 1997; 272: 11698-11701Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 30O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.-C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar, 32Dickens M. Svitek C.A. Culbert A.A. O'Brien R.M. Tavaré J.M. J. Biol. Chem. 1998; 273: 20144-20149Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To compare the relative basal CAT expression obtained with the various reporter gene constructs described, CAT activity in samples from control cells was corrected for the β-galactosidase activity in the same sample. Because phorbol esters and insulin affect Rous sarcoma virus-β galactosidase expression in HeLa cells (data not shown), CAT activity in these samples was corrected for the protein concentration in the cell lysate, as measured by the Pierce BCA assay, and each plasmid construct was analyzed in duplicate in multiple transfections, as specified in the Figure legends. Oligonucleotides representing the sense and antisense strands of the collagenase AP-1 and Ets motifs (Table I) were synthesized with BamHI or HindIII compatible ends, respectively, gel purified, annealed, and then labeled with [α-32P]dATP using the Klenow fragment ofEscherichia coli DNA polymerase I to a specific activity of approximately 2.5 μCi/pmol. HeLa nuclear extracts were prepared exactly as described previously (13Streeper R.S. Chapman S.C. Ayala J.E. Svitek C.A. Goldman J.K. Cave A. O'Brien R.M. Mol. Endocrinol. 1998; 12: 1778-1791Crossref PubMed Scopus (22) Google Scholar). Labeled AP-1 oligonucleotide (∼7.5 fmol, ∼30,000 cpm) was incubated with HeLa (3 μg) nuclear extract in a final reaction volume of 20 μl containing 20 mmHEPES, pH 7.8, 100 mm NaCl, 0.38 mm spermidine, 0.08 mm spermine, 0.1 mm EDTA, 1 mmEGTA, 2 mm dithiothreitol, 12.5% glycerol (v/v), and 1 μg of poly(dI-dC)·poly(dI-dC). After incubation for 10 min at room temperature, the reactants were loaded onto a 6% polyacrylamide gel and electrophoresed at room temperature for 90 min at 150 V in a buffer containing 25 mm Tris·HCl at pH 7.8, 190 mmglycine, and 1 mm EDTA. Following electrophoresis, the gels were dried and exposed to Kodak XAR5 film, and binding was analyzed by autoradiography. When the Ets oligonucleotide was used as the labeled probe, the binding conditions were identical to those described for AP-1 except that the NaCl concentration was decreased to 50 mm and poly(dI-dC)·poly(dI-dC) was reduced to 0.5 μg. In addition, visualization of specific Ets binding (see Fig.8 B) required preincubation of HeLa nuclear extract with 10 ng of chymotrypsin for 2 min at room temperature prior to addition of the labeled probe and binding buffer and a further 10 min of incubation at room temperature. Binding was then analyzed by acrylamide gel electrophoresis as described above. For competition experiments (see Fig. 8), the indicated unlabeled double-stranded oligonucleotides (100-fold molar excess) were mixed with the labeled oligomer prior to addition of nuclear extract. Binding was then analyzed by acrylamide gel electrophoresis as described above. Gel supershift assays (see Fig. 9) were carried out by incubating HeLa nuclear extract (3 μg) with the indicated antisera for 10 min at room temperature, prior to the addition of the labeled AP-1 oligonucleotide probe and binding buffer and incubation for an additional 10 min. To begin to study the regulation of collagenase gene transcription by insulin, a collagenase-CAT fusion gene construct, containing collagenase promoter sequence from −518 to +64, relative to the transcription start site at +1 (33Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (585) Google Scholar), was transiently transfected into HeLa cells (Fig. 1). An effect of insulin on reporter gene expression was only detected when the collagenase-CAT fusion gene was co-transfected with an expression vector encoding the insulin receptor (Fig. 1 A). In the absence of insulin, co-transfection with the insulin receptor alone was insufficient to activate collagenase-CAT fusion gene expression, suggesting a low level of signaling through the basal receptor (Fig.1 B). Stanley (34Stanley F.M. J. Biol. Chem. 1992; 267: 16719-16726Abstract Full Text PDF PubMed Google Scholar) has previously shown that co-transfection with an insulin receptor expression vector is also required to observe an effect of insulin on the expression of a transiently transfected prolactin-CAT fusion gene in rat pituitary tumor GH4 cells. Interestingly, insulin stimulates the expression of the endogenous prolactin gene in these cells in the absence of receptor co-transfection (34Stanley F.M. J. Biol. Chem. 1992; 267: 16719-16726Abstract Full Text PDF PubMed Google Scholar). The maximal effect of insulin on collagenase-CAT gene expression was seen at 100 nm (F" @default.
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• W2021330348 date "1999-06-01" @default.
• W2021330348 modified "2023-10-14" @default.
• W2021330348 title "Multiple Promoter Elements Are Required for the Stimulatory Effect of Insulin on Human Collagenase-1 Gene Transcription" @default.
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