Phosphatidylinositol 3-Kinase-dependent Suppression of the Human Inducible Nitric-oxide Synthase Promoter Is Mediated by FKHRL1

The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-κB) to its 5 ′-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-γ). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-γ-stimulated hiNOS promoter activity. In response to LPS/IFN-γ, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-γ- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products. The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-κB) to its 5 ′-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-γ). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-γ-stimulated hiNOS promoter activity. In response to LPS/IFN-γ, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-γ- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products. NO is an important signaling molecule produced by a family of nitric-oxide synthases (NOS) 2The abbreviations used are: NOS, nitric-oxide synthase; iNOS, inducible NOS; hiNOS, human iNOS; FKHRL1, forkhead (Drosophila) homolog (rhabdomyosarcoma)-like 1; LPS, lipopolysaccharide; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase: STAT, signal transducer and activator of transcription; SEAP, soluble excreted alkaline phosphatase; IFN, interferon; HA, hemagglutinin; dn, dominant negative; GST, glutathione S-transferase. 2The abbreviations used are: NOS, nitric-oxide synthase; iNOS, inducible NOS; hiNOS, human iNOS; FKHRL1, forkhead (Drosophila) homolog (rhabdomyosarcoma)-like 1; LPS, lipopolysaccharide; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase: STAT, signal transducer and activator of transcription; SEAP, soluble excreted alkaline phosphatase; IFN, interferon; HA, hemagglutinin; dn, dominant negative; GST, glutathione S-transferase. during the conversion of l-arginine to l-citrulline (1Stuehr D.J. Griffith O.W. Adv. Enzymol. Relat. Areas. Mol. Biol. 1992; 65: 287-346PubMed Google Scholar). Both constitutive and inducible NOS isoforms have been described (2Michel T. Feron O. J. Clin. Invest. 1997; 100: 2146-2152Crossref PubMed Scopus (846) Google Scholar). Biological processes regulated by calcium-dependent constitutive NO synthesis include vascular tone and neurotransmission and are catalyzed by the neuronal and endothelial NOS isoforms, respectively (3Lowenstein C.J. Dinerman J.L. Snyder S.H. Ann. Intern. Med. 1994; 120: 227-237Crossref PubMed Scopus (850) Google Scholar, 4Nathan C. Xie Q.W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2741) Google Scholar, 5Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar). In contrast, induction of the calcium-independent inducible isoform of NO synthase (iNOS, or NOS2) by inflammatory stimuli results in the sustained release of higher levels of NO (1Stuehr D.J. Griffith O.W. Adv. Enzymol. Relat. Areas. Mol. Biol. 1992; 65: 287-346PubMed Google Scholar, 6Nathan C. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (837) Google Scholar). In vitro and in vivo studies demonstrated a role for iNOS-derived NO in tissue injury, likely via oxidative DNA and membrane damage, inhibition of cellular respiration, and direct modification of proteins (7Murphy M.P. Biochim. Biophys. Acta. 1999; 1411: 401-414Crossref PubMed Scopus (361) Google Scholar). Moreover, depending on cell type or stimulus, NO production was directly linked to the initiation of, or protection from, cellular apoptosis (8Brune B. von Knethen A. Sandau K.B. Cell Death. Differ. 1999; 6: 969-975Crossref PubMed Scopus (264) Google Scholar). Activation of human iNOS (hiNOS) transcription is central to the cellular response to inflammatory stimuli. Cytokine-activated signaling proteins are involved in iNOS gene expression and de novo NO production (e.g. mitogen-activated protein kinases (MAPKs), Janus tyrosine kinase/signal transducer and activator of transcription (STAT), protein kinase A, and protein kinase C) (9Diaz-Guerra M.J. Bodelon O.G. Velasco M. Whelan R. Parker P.J. Bosca L. J. Biol. Chem. 1996; 271: 32028-32033Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Xu W. Comhair S.A. Zheng S. Chu S.C. Marks-Konczalik J. Moss J. Haque S.J. Erzurum S.C. Am. J. Physiol. 2003; 285: L137-L148PubMed Google Scholar, 11Kleinert H. Euchenhofer C. Fritz G. Ihrig-Biedert I. Forstermann U. Br. J. Pharmacol. 1998; 123: 1716-1722Crossref PubMed Scopus (35) Google Scholar). Studies of the hiNOS 5′-flanking region identified the involvement of several inducible transcription factors. The hiNOS promoter contains enhancer regions located up to 16 kb upstream of the transcription start site (12de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M.J. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (356) Google Scholar). Two AP-1 and two NF-κB binding sites were necessary for cytokine stimulation of hiNOS promoter activation (13Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Specific AP-1 heterodimers (JunD/Fra-2) bound the hiNOS promoter in extracellular signal-regulated kinase- and p38 MAPK-dependent fashion, providing one molecular mechanism for hiNOS transcriptional activation (14Kristof A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2001; 276: 8445-8452Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In separate studies, NF-κB or c-Fos interacted with STAT-1 to regulate hiNOS promoter activity in response to cytokines (10Xu W. Comhair S.A. Zheng S. Chu S.C. Marks-Konczalik J. Moss J. Haque S.J. Erzurum S.C. Am. J. Physiol. 2003; 285: L137-L148PubMed Google Scholar, 15Ganster R.W. Taylor B.S. Shao L. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8638-8643Crossref PubMed Scopus (278) Google Scholar). Because overproduction of NO by iNOS might cause abnormal inflammation, cell proliferation, or apoptosis, signaling mechanisms that suppress basal iNOS promoter activity have attracted growing interest. In contrast to MAPKs and Janus tyrosine kinases, phosphatidylinositol 3-kinase (PI3K) activity limited hiNOS gene induction (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar). PI3K is a key signaling in cellular responses to factors and cytokines transcriptional initiation of protein cell proliferation, J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.P. S. Cell. 1998; Scopus Google Scholar). of the PI3K to to of the to membrane D.A. 1998; PubMed Scopus Google Scholar). The of phosphatidylinositol of proteins to and activation of kinases (e.g. kinase and protein kinase B PubMed Scopus Google Scholar). PI3K the activity of transcription factors and NF-κB J. H. S. M. C. J. Cell Biol. PubMed Scopus Google Scholar, H. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. 2001; PubMed Scopus Google Scholar). However, PI3K suppressed hiNOS transcription (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar, I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google and inhibitory mechanisms are likely to a The forkhead transcription factors and FKHRL1 are phosphorylated by J. Mol. Med. 1999; PubMed Scopus Google Scholar). FKHRL1 the of involved in or apoptosis (e.g. and L. P.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. S. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, C. L. L. P.J. Mol. Cell. Biol. PubMed Scopus Google Scholar, S. C. R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). of FKHRL1 or to a in FKHRL1, in the of apoptosis A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). consensus binding sites for FKHRL1 are in the hiNOS 5′-flanking region. We hypothesized in the of inflammatory the PI3K pathway hiNOS promoter induction via and of that PI3K hiNOS promoter activity in a fashion. Moreover, FKHRL1 and FKHRL1 the hiNOS promoter via a enhancer a role for FKHRL1 in the of hiNOS gene Cell and cells a human lung type epithelial adenocarcinoma cell were described (14Kristof A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2001; 276: 8445-8452Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). cells were with and with or for with LPS, or with in was directly to the cells of hiNOS FKHRL1-binding sites or and or were identified in the 8.3-kb hiNOS promoter The 8.3-kb hiNOS promoter linked to the luciferase was described S.C. Marks-Konczalik J. Moss J. 1998; PubMed Scopus Google Scholar). in FKHRL1-binding were by The of the corresponding to the regions of the hiNOS promoter were are and used in the is in to to to to and were by and of cells were transfected with kinase promoter linked to luciferase and or S.C. Marks-Konczalik J. Moss J. 1998; PubMed Scopus Google or with expression the dominant-negative and in separate with or constitutively active FKHRL1 M. and A. A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). expression for dominant-negative FKHRL1 the FKHRL1, or expression siRNA or were used to endogenous FKHRL1 protein levels or activity. were reporter or transfected with of and activity was to cell protein cells were with and or with the of for and for of luciferase activity luciferase or to LPS/IFN-γ-stimulated promoter activity of hiNOS mRNA cells were in and of the was and of was with of and probe for or hiNOS or in for a of was the for and for by of for and for expression to the was the 2001; PubMed Scopus Google Scholar). for hiNOS mRNA or were and were by hiNOS were by in mRNA levels to of FKHRL1 and cells were or with for and or with in 5 and were for 5 of the and for of protein were to and to a of FKHRL1 or FKHRL1 phosphorylated was by or in were by The of the corresponding to the regions of the hiNOS or promoter were are to to to to to and A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in were and by to to for 5 oligonucleotide were with a kinase and on of and cells to were for or with stimulation with for and of proteins described (13Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, A. W. Mol. Cell 2001; PubMed Scopus Google Scholar). of proteins were with a probe hiNOS promoter or FKHRL1 binding or one the in the promoter A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google for of the binding were in in which a or of or oligonucleotide was to cells the were by in a with was to and an PI3K and of hiNOS in and studies the PI3K pathway a of iNOS and NO synthesis (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar, I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, B. R. A. J.S. S. J. Immunol. 1998; Google Scholar, Biophys. 1997; PubMed Scopus Google Scholar). In the PI3K potentiated activation of the iNOS promoter (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar). in human lung epithelial adenocarcinoma (A549) cells demonstrated an inhibitory role for PI3K in human iNOS promoter induction A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). PI3K effects on gene transcription A. M.E. 1999; PubMed Scopus Google Scholar). of dominant-negative LPS/IFN-γ-stimulated hiNOS promoter activity Co-expression of dominant-negative increased basal promoter activity. The induction of hiNOS promoter activity by by basal hiNOS promoter activity was in cells and in dominant-negative results that a PI3K- and transcription factor might regulate basal hiNOS transcription and a effect during to innate immune is necessary for hiNOS transcriptional in cells to an effect of on activation of A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Two NF-κB and two AP-1 binding sites in the hiNOS 5′-flanking region are necessary for hiNOS transcriptional induction by cytokines and (13Marks-Konczalik J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2001; 276: 8445-8452Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). We in NF-κB or AP-1 binding or activity might hiNOS promoter activity. By gel-shift analysis, LPS/IFN-γ-stimulated binding to the site effect on AP-1 activity that the of hiNOS promoter activity by by effects on Moreover, LPS/IFN-γ-stimulated NF-κB binding or reporter activity and and transcriptional of hiNOS by the PI3K the of FKHRL1 in a a activator of gene phosphorylated and by In a a of and to maximal hiNOS promoter effect (14Kristof A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2001; 276: 8445-8452Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). the of signaling the of FKHRL1 in cell cells with In the of LPS/IFN-γ, of FKHRL1 increased and for FKHRL1 levels In separate cells were or with for with PI3K of FKHRL1, basal and LPS/IFN-γ-stimulated of FKHRL1 with effect on FKHRL1 levels We the role of or in the of of to of FKHRL1 effect of FKHRL1 in response to of FKHRL1 levels. The maximal effect and FKHRL1 is a of signal in response to FKHRL1 a of the hiNOS on its consensus several FKHRL1-binding sites were in the hiNOS 5′-flanking region sites were and upstream of the transcription start site to consensus binding sites were and on the and on the FKHRL1 regulate the hiNOS and were with In cells transfected with and with induction of hiNOS promoter activity by LPS/IFN-γ, with in of FKHRL1 basal and LPS/IFN-γ-stimulated hiNOS promoter activity. In cells FKHRL1, induction of hiNOS promoter activity by was by induction in cells and with that FKHRL1 is to hiNOS promoter activity. of FKHRL1, the of of FKHRL1 (e.g. W. S. H. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. H. M.E. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). the effect of a of FKHRL1 that phosphorylated by FKHRL1 contains sites and or to sites a constitutively active FKHRL1 A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of constitutively active FKHRL1 to a in basal and hiNOS promoter activity that was by induction of promoter activity by in cells and with By of constitutively active FKHRL1 increased basal and hiNOS mRNA levels by and respectively The induction of hiNOS mRNA by was in cells and in constitutively active in contrast to promoter activity hiNOS mRNA levels inducible in cells constitutively active FKHRL1 by mechanisms (e.g. mRNA the results that FKHRL1 endogenous hiNOS transcription and that of hiNOS promoter is inhibitory by FKHRL1 for of hiNOS by a role for FKHRL1 in the of hiNOS transcriptional its dominant-negative or a siRNA its of dominant-negative FKHRL1 and endogenous FKHRL1 were by the of the effect of FKHRL1 used a of one that was to hiNOS promoter induction A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In cells a FKHRL1 its potentiated hiNOS mRNA induction In the of hiNOS induction was that FKHRL1 represents a for the of hiNOS transcription by FKHRL1 was for hiNOS gene induction. the of sites to hiNOS induction in the of used siRNA FKHRL1 during of a FKHRL1 of FKHRL1 induction of hiNOS mRNA the of to hiNOS gene induction of a siRNA effect on the of hiNOS induction by results that inhibition of FKHRL1 transcriptional activity by is necessary for of hiNOS gene induction. The hiNOS a FKHRL1-binding for in the hiNOS 5′-flanking region that endogenous effects on promoter were of FKHRL1-binding sites and the of to LPS/IFN-γ-stimulated promoter mutation of the or and The induction of promoter activity in cells with were and for the and respectively and In contrast, mutation of the the of to hiNOS promoter activity in the of LPS/IFN-γ, a of inhibition and of promoter by was upon mutation of the site and and with the mutation to a in basal and LPS/IFN-γ-stimulated promoter activity in the of that of its effects to FKHRL1 its consensus in the hiNOS promoter gel-shift were was by and and of a probe the site was by with proteins were cells or with and or with Regulation of FKHRL1 binding was with with the a was with FKHRL1 the the same that corresponding to to increased molecular or the a that endogenous However, the that bound the the same that bound a FKHRL1-binding the promoter S. A. S. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). with the involvement of FKHRL1, the of in LPS/IFN-γ-stimulated to and of FKHRL1 the A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). increased binding in basal and LPS/IFN-γ-stimulated inhibitory by of endogenous FKHRL1 to the probe the site was by the of corresponding probe the of the of was that of endogenous to the of binding or or by In the consensus FKHRL1-binding the in the hiNOS promoter of hiNOS transcription and FKHRL1 in activation of bacterial and inflammatory responses that are of innate G. Immunol. 2003; PubMed Scopus Google Scholar). to the and a signal in to the transcription of B. J. Biol. 2003; PubMed Scopus Google Scholar). of iNOS mRNA is a in the production of NO in response to and C. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (837) Google Scholar). NO results in DNA damage, killing of and of immune cell C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In NO regulate cellular apoptosis or proliferation, responses that important in tissue and the of (8Brune B. von Knethen A. Sandau K.B. Cell Death. Differ. 1999; 6: 969-975Crossref PubMed Scopus (264) Google Scholar, S. J. M. J. Med. 1997; PubMed Scopus Google Scholar). Human iNOS transcription is via binding of and transcription factors (e.g. and NF-κB) to the iNOS gene 5′-flanking region (10Xu W. Comhair S.A. Zheng S. Chu S.C. Marks-Konczalik J. Moss J. Haque S.J. Erzurum S.C. Am. J. Physiol. 2003; 285: L137-L148PubMed Google Scholar, J. Chu S.C. Moss J. J. Biol. Chem. 1998; 273: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 15Ganster R.W. Taylor B.S. Shao L. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8638-8643Crossref PubMed Scopus (278) Google Scholar, B.S. de Vera M.E. R.W. Shapiro R.A. Morris Billiar T.R. Geller D.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In to in gene the of signaling mechanisms that transcriptional responses been an in was used to a that basal activity Biol. PubMed Scopus Google Scholar). The that PI3K activity the induction of iNOS in cells A.S. Marks-Konczalik J. Moss J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google that protein kinases might a inhibitory role in the transcriptional response to of innate In the the transcription factor FKHRL1 regulated hiNOS promoter activity in PI3K- and via binding to a in the hiNOS promoter. with inhibition of iNOS induction (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar, I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, B. R. A. J.S. S. J. Immunol. 1998; Google Scholar, Biophys. 1997; PubMed Scopus Google to the of FKHRL1, a modification to FKHRL1 transcriptional and the induction of By PI3K or PKB, a to in hiNOS promoter activity or mRNA levels in lung epithelial cells to and FKHRL1, its and its sites were for In contrast to or FKHRL1 was a of basal hiNOS promoter in to induction by B and with FKHRL1 was constitutively to the by or A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of FKHRL1 its in induction of A. A. M.J. P. M.J. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of FKHRL1 to the hiNOS promoter was necessary for hiNOS induction 5 and of FKHRL1, and DNA binding in response to and activation of transcription by the in In the of and PI3K activity basal hiNOS transcription and binding to the site in hiNOS promoter activity the of stimulatory transcription or AP-1 of activation of the hiNOS promoter by transcription to of FKHRL1, maximal transcription Full induction upon inhibition of PI3K and of FKHRL1 to the hiNOS promoter. FKHRL1 a molecular mechanism for the induction of basal or in the of The of inhibitory transcriptional mechanisms on the synthesis of hiNOS mRNA been in factor suppressed hiNOS promoter activity via a binding site upstream of the transcription start site M. H. R. Shao L. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a transcriptional by iNOS promoter induction in in a C.J. J. Immunol. 2003; PubMed Scopus Google Scholar). The a mechanism by which hiNOS mRNA levels limited in response to inflammatory stimuli. observed a of hiNOS promoter activity and mRNA levels. potentiated hiNOS mRNA promoter in cells constitutively active FKHRL1 B and is that transcriptional response in the endogenous 5′-flanking region 16 kb (12de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M.J. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (356) Google that are in the transfected 8.3-kb hiNOS construct. of constitutively active FKHRL1 might regulate hiNOS mRNA levels of transcriptional for by levels of proteins involved in mRNA direct effects on FKHRL1, signaling mechanisms the mRNA levels of inflammatory inhibition of p38 to mRNA levels to a in mRNA J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In human inhibition of PI3K to increased NF-κB and AP-1 via with mitogen-activated protein kinase and by the inhibition of synthase M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PI3K NF-κB to the to the of iNOS promoter activity (16Diaz-Guerra M.J. Castrillo A. Martin-Sanz P. Bosca L. J. Immunol. 1999; 162: 6184-6190PubMed Google Scholar, I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in LPS/IFN-γ-stimulated cells to an effect of PI3K inhibition on NF-κB binding or activity and by FKHRL1 and the PI3K pathway hiNOS to or that regulated during In sites in FKHRL1 were necessary for of hiNOS promoter activity In a FKHRL1-binding site in the hiNOS promoter was identified and In studies transcriptional of apoptosis and cell proliferation, of FKHRL1 increased and promoter activity C. L. L. P.J. Mol. Cell. Biol. PubMed Scopus Google Scholar, S. C. R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar, W. S. H. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). FKHRL1 interacted with to activation of the promoter J. L. S.A. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). FKHRL1 represents a molecular for the of PI3K and transcriptional that regulate cell The of FKHRL1 by described represents one mechanism that might DNA and apoptosis during the innate immune In the protein iNOS induction by cytokines cells were to NO S. Geller D.A. Billiar T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). activity was to and and of S. C. P. I. D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar). FKHRL1 was in the cellular response to DNA damage. FKHRL1 and DNA by the transcription of the and DNA gene H. A. M.E. PubMed Scopus Google Scholar). with of involved in FKHRL1 of transcription and cell S. T. PubMed Scopus Google Scholar). of the gene was regulated by FKHRL1 H. A. M.E. PubMed Scopus Google Scholar). studies FKHRL1 a molecular and the of cell or of the hiNOS FKHRL1 binding site of dominant FKHRL1, or of siRNA of hiNOS promoter activity by a of binding of endogenous FKHRL1, mechanisms of FKHRL1 phosphorylated FKHRL1 FKHRL1 and W. S. H. R. Cell. Full Text Full Text PDF PubMed Scopus Google sites in In and kinase phosphorylated the and A. J. H. M.E. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). studies demonstrated a role for of FKHRL1 and of its transcriptional activity M. C. W. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). an important role for of FKHRL1 in the of hiNOS transcription upon to bacterial The FKHRL1-binding consensus binding of hiNOS promoter activity by PI3K and to its with and of promoter FKHRL1 might or depending on promoter and binding J. L. S.A. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). studies the of FKHRL1-binding sites in the hiNOS the of protein that the The a role for FKHRL1 in the of hiNOS transcription by activation of We and for of the G. and S. and R. of for We M. and A. for providing FKHRL1 and bacterial expression with

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