Identification of a Prostaglandin-responsive Element in the Na,K-ATPase β1 Promoter That Is Regulated by cAMP and Ca2+

Abstract

The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E1 (PGE1) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1–14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337–346). In this work, we present evidence that transcription of the Na,K-ATPase β1 subunit is stimulated by PGE1, an effect that may be mediated through the cAMP and Ca2+ pathways. Transient transfection studies using 5′-deletion mutants of the human β1 subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE1, 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the β1 subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE1 was further confirmed by the increased PGE1 stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-β72–167, which contains region -167 to -72 in the human β1 subunit promoter. The PGE1 stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-β72–167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used. The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E1 (PGE1) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1–14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337–346). In this work, we present evidence that transcription of the Na,K-ATPase β1 subunit is stimulated by PGE1, an effect that may be mediated through the cAMP and Ca2+ pathways. Transient transfection studies using 5′-deletion mutants of the human β1 subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE1, 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the β1 subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE1 was further confirmed by the increased PGE1 stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-β72–167, which contains region -167 to -72 in the human β1 subunit promoter. The PGE1 stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-β72–167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used. The Na,K-ATPase is a ubiquitous transmembrane protein that establishes and maintains an electrochemical gradient across the plasma membrane in virtually all mammalian cells (3Lingrel J.B. Orlowski J. Shull M.M. Price E.M. Prog. Nucleic Acid Res. Mol. Biol. 1990; 38: 37-89Crossref PubMed Scopus (360) Google Scholar). In addition, the Na,K-ATPase plays specialized roles in specific cell types. For example, in nerve and muscle cells, the Na,K-ATPase is involved in the propagation of an action potential. In the kidney, where the Na,K-ATPase is present at higher levels than in most tissues, the Na,K-ATPase plays an important role in Na+ a process that in In kidney cells, the Na,K-ATPase is present in the M. J. Cell Biol. PubMed Scopus Google Scholar). Na+ in the of the is into the cells through to the the Na+ is of the membrane through the Na,K-ATPase in for a process that The subunit of the Na,K-ATPase (a the the subunit to the and of the subunit from the to the plasma The Na,K-ATPase is subject to regulation by a of as protein PGE1, prostaglandin Madin-Darby canine prostaglandin phorbol 12-myristate CREB, cAMP regulatory element-binding CREB-binding protein transcription and as may effects the activity of M.M. PubMed Scopus Google as well as effects from in the of present J. Cell. Physiol. PubMed Scopus Google Scholar). effects may be the of transcriptional post-transcriptional the effects of of a of regulatory effects the Na,K-ATPase are to as by the of In expression at the post-transcriptional I. J. Physiol. PubMed Google Scholar). in kidney Na,K-ATPase and β1 subunit expression at the transcriptional J. Biol. PubMed Google Scholar). The by which regulatory Na,K-ATPase activity are well are among the regulatory that the activity of the Na,K-ATPase M.M. PubMed Scopus Google PubMed Scopus Google PubMed Scopus Google J. Physiol. Google Scholar). regulatory effects as the Na,K-ATPase through interaction with as the cAMP and protein may be the that is is with the transcriptional regulation of the Na,K-ATPase by PGE1, using a well in of the the Madin-Darby canine kidney cell M. Google Scholar). Previously, we observed that PGE1 the activity of the Na,K-ATPase in cells, an effect that be by increased expression of the for the and of the Na,K-ATPase M. M. J. C. Exp. Cell Res. PubMed Scopus Google J. Cell. Physiol. PubMed Scopus Google Scholar). obtained evidence of a stimulatory effect of PGE1 β1 subunit transcription using a human Na,K-ATPase β1 promoter/luciferase construct J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google In this work, we a prostaglandin-responsive element that is present in the human Na,K-ATPase β1 subunit promoter. The results of our transfection studies with 5′-deletion mutants of the β1 subunit promoter the of a PGRE with the sequence AGTCCCTGC at a within region to The observed the phorbol 12-myristate and the okadaic the PGE1 were all the of the element in the β1 subunit suggesting that this element is a element Electrophoretic mobility shift assays indicated the of a sequence between and that to the cAMP regulatory element-binding protein (CREB) and Sp1 transcription further the of the involvement of CREB and Sp1 in PGE1 the PGRE was into a heterologous and studies were conducted with containing a or a region of the β1 subunit promoter. In addition, evidence for an interaction between CREB and Sp1 was obtained from immunoprecipitation and The results of studies that a interaction between transcription is involved in regulation by through both and pathways. human PGE1, and were from the and were from and was from The was obtained from The and the Sp1 and were from The was obtained from against a the of human a at the of human Sp1 and a protein to the of human CREB-binding protein were obtained from and the were from and were from The expression was obtained from of M. S. Mol. Cell. Biol. PubMed Scopus Google Scholar). The and were obtained from of Mol. Google Scholar). and were from J.B. M. Mol. Cell. PubMed Scopus Google I. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). The expression contains a promoter with a the the and the were all obtained from The contains a promoter with of the that expression of the The a protein that contains the of CREB and the the transcriptional CREB is and the protein to the sites the and transcription of the Cell and of a of and with and as M. S. PubMed Scopus Google Scholar). The for of cells was further with human PGE1, and were using and in by of action with as M. S. PubMed Scopus Google Scholar). cells to be for transfection studies were at into containing with and The the were with the as well as with the as to for transfection For were in in with and The the was to with and and the were to a and were to the and the were to for indicated the of the the were in the cell was for in a the were immediately for assays of and or at used. an of the cell was in of containing and The were using a with the the activity of the transfected cells, of cell were at in containing the in the and the was as activity is as a of the activity in transfected of activity was with to the activity of the The obtained in was the of The was with the indicated from the The of observed stimulatory effect was by of and the in were as when the human Na,K-ATPase β1 subunit promoter was into the the construct J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google Scholar). of mutants within the human Na,K-ATPase β1 subunit promoter and was as by J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google Scholar). we mutants and by of following of at the and immediately to the β1 subunit promoter with a sequence to -167 to -72 of the human Na,K-ATPase β1 the GC boxes and the PGRE and was into the sites the of the upstream from the the expression In addition, a in which the two GC boxes were translocated as was into the sites of the expression with a sequence to to of the human fibronectin promoter M. S. Mol. Cell. Biol. PubMed Scopus Google four GC boxes was into the sites of the expression The with the sequence from to with the PGRE from was into the sites of of were from cells by a Nucleic Res. PubMed Scopus Google of the of Nucleic Res. PubMed Scopus Google Scholar). cells were to in in with and human The were with from the using a into and at for at the the cell was in and at in a that was than the cell for the cells were for and at for at The was in and for the was in a at for at The was into and in The protein of the was by the of M.M. PubMed Scopus Google Scholar). Electrophoretic were by the using The the and the Sp1 were when as were as Included among the were the PGRE to the PGRE to the a and the Sp1 the were at for in containing and in a of of was in The was to the and the was for at In studies using the antibody was the with the or this as The was by the of a of and and from the were immediately and The were and to the of the the were with a and using the and cell for and assays were in in with and The were transiently transfected with of the and of the the were for with the were in containing and from the with a and in a at for were conducted by a of the by M. M. J. C. Exp. Cell Res. PubMed Scopus Google Scholar). of cell with to were with to protein in and by with or antibody to protein in for at The were by in a at using and in at for by in a at for and were in M. PubMed Scopus Google Scholar). The assays were by cell as in using of or by for at were by in and by in at for The obtained from and assays were by using (CREB) or were to The were with or and were by of PGE1 and the regulation of the human Na,K-ATPase β1 promoter activity by PGE1 and cells were transiently transfected with a human Na,K-ATPase β1 promoter/luciferase construct J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google Scholar). the were for in the of PGE1, or further that PGE1 a in activity to the a Previously, both a element to J.B. J. Biol. PubMed Scopus Google and a element to J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google were the human Na,K-ATPase β1 subunit promoter of is required for PGE1 or transfection studies were conducted with the 5′-deletion in and that the stimulatory effects of PGE1 and were in the at to as by the the at to as by the by PGE1 and was in the in the of activity by PGE1 and was results that a regulatory between and is required to elicit the stimulatory effects of PGE1 and Electrophoretic element (a was that was between and -92 and that was within the region of the β1 promoter required to the stimulatory effects of PGE1 and from to the of this to bind mobility shift assays were conducted with a to to within the β1 subunit a region that contains the PGRE to The was with from cells and to the of that when the PGRE was as a the PGRE sequence to a the effect of the of a was this was in the at a the of four of was reduced and this was was to the observed when the PGRE was as a obtained at the of two following when the was as a the The of the two was reduced and when a of the PGRE was as a In the of the was reduced to by a of the results that the is a of the than the when an was into the and this was as a of the the of the two was reduced and that the is a than the the PGRE as a a CREB be to be a of a was by an antibody against CREB with the PGRE and the protein by in a was observed in the of with the that a was obtained with antibody when a was as a the with the using of a is the that when an antibody against was to the the antibody as a the of by when at The region of the β1 promoter the PGRE is and contains a Sp1 to the that sites be involved in the of the which in this region of the studies were conducted as a a to region to of the β1 promoter The present in this a Sp1 to as a region the PGRE S. S. Nucleic Res. PubMed Scopus Google Scholar). that the was a of the with the PGRE in The a in the of both and as well as a in the with the PGRE and containing the Sp1 sequence as a to the as The of the to with the β1 was that the the of the observed in when the PGRE was as a The was as a as the Sp1 or the a in the Sp1 and the and in the of the studies that Sp1 to the the effect of antibody protein to a was a in the of with the antibody or with results were obtained when a PGRE was used. further the involvement of Sp1 and CREB, studies were conducted using a Sp1 or that an PGRE was an of with the Sp1 a in the of and an in the of when a was the PGRE reduced by and The was as a to the observed when the PGRE was as a of the PGRE and Sp1 in PGE1 results obtained that the Sp1 sites in the human β1 subunit promoter to the PGRE are involved in regulation by further the role of the PGRE and Sp1 sites in regulation by PGE1, transfection studies were conducted with cells using the which contains a promoter with a as well as the which contains region to of the human promoter with four GC boxes immediately upstream from the in a stimulation by PGE1 in cells transfected with as with a stimulation observed in transfected with PGE1 stimulation was in the which contains the PGRE in region to of the promoter from The results that a PGE1 be observed in a heterologous promoter and that both the PGRE and Sp1 sites were involved in PGE1 stimulation mediated by this promoter. further the involvement of the PGRE and Sp1 sites in the β1 subunit the effect of PGE1 was in cells transiently transfected with containing region the -167 to -72 of the human β1 subunit promoter that the PGE1 stimulation was in cells transiently transfected with pLuc-MCS-β72–167 with the expression obtained with in the of -167 to -72 contains two GC boxes immediately upstream from the PGRE that when the two GC boxes in region -167 to -72 were translocated farther upstream from the PGRE in the PGE1 stimulation was reduced to of the observed in cells transfected with in the of that the of the Sp1 sites to the PGRE in the β1 promoter is a in the of the PGE1 between CREB and the results were of an interaction between Sp1 and CREB, the was that CREB and Sp1 be co-immunoprecipitated from this immunoprecipitation studies were conducted with a obtained from cells using antibody against Sp1, or The were by and to the results of a with In the of the containing the from the Sp1 and that with CREB were when the results of an with antibody the containing the from the CREB and were observed to that with the results of a using The protein that with Sp1, the with the of CREB, Sp1, and in PGE1 CREB is involved in PGE1 stimulation observed with the effect of CREB Mol. Google the PGE1 was that in with and PGE1 stimulation was reduced by PGE1 stimulation in cells with with in cells with the a effect in cell in the of the of Sp1, cells were with and which Sp1 S. Mol. Cell. Biol. PubMed Scopus Google Scholar). that the PGE1 stimulation increased in the of with the PGE1 stimulation observed in cells with and the further the involvement of Sp1 in the PGE1 the effect of an that a for J. Biol. PubMed Scopus Google was that reduced the PGE1 stimulation by of PGE1 stimulation by cells were into containing and at The the were transfected with and as was to of the The were for with and the PGE1, the or the PGE1 and the of the the activity was and with to are of The were by of The obtained with in the of PGE1 and in the of PGE1 and from the The obtained with in the of PGE1 and from the obtained in the of PGE1 and in a CREB CREB with which in transcription through activity I. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). or is an of the PGE1 the effect of a was that reduced the PGE1 stimulation by to with with PGE1 that a an effect the PGE1 of in the PGE1 observed to an in levels as well as cAMP levels in cells M. Res. 1990; PubMed Scopus Google M. J. Physiol. Google Scholar). PGE1 may through protein as well as protein the the effect of was in in in the β1 subunit promoter. a stimulation by in by was in mutants from to the stimulatory effect of was observed in suggesting as was the with PGE1, stimulation was region to a in this the was and was to effect transcription by of transcription cells were transiently transfected with and as were in with and or in the further with PGE1, or for the were The activity was and to are of four in a and PGE1 may transcription by by the or a CREB is involved in the stimulatory effects of and PGE1, cells were with the and the or (a The a protein containing the CREB to the the CREB is the protein to the sites that and PGE1 stimulated expression by the by and with expression obtained with the in the of that in and PGE1 stimulated transcription by by and a in to to the obtained in with PGE1 the involvement of the effect of two of and Res. PubMed Scopus Google the PGE1 stimulation of was that and reduced the PGE1 stimulation by and In an of was The of to the PGE1 stimulation of the containing in the β1 subunit promoter was that the PGE1 stimulation of of 5′-deletion from to the of the PGE1 stimulation in the of is region to is and by okadaic and protein J. Biol. PubMed Google S. PubMed Scopus Google Scholar). For this okadaic is a of the effect of okadaic transcription was in as well as in containing 5′-deletion that okadaic a stimulation of activity in in 5′-deletion mutants from to are regulators of in the kidney, the activity of the Na,K-ATPase J. Physiol. Google Scholar). the physiologic of is the by which the activity of membrane to the effects of the in the kidney, studies conducted with studies J. Physiol. Google Scholar). be to a by the of kidney cell In this the cell a of kidney cells, to regulation of transcription of the Na,K-ATPase β1 subunit The Na,K-ATPase is a of an as well as a subunit PubMed Scopus Google Scholar). the the subunit to be a in PubMed Scopus Google J. Physiol. Google Scholar). in a of tissues, of the subunit to the subunit in to is with an in Na+ activity J. Biol. 1990; PubMed Google Scholar). in this PGE1 effects the transcription of the Na,K-ATPase β1 subunit in cells a region in the β1 promoter required for regulation by PGE1, transfection studies were conducted using β1 promoter/luciferase containing in the β1 promoter to the this we a region in the promoter to that is required for regulation by PGE1, and mobility shift studies indicated that within this region between -100 and is of to CREB as well as to Previously, and 5′-deletion mutants were to a to present within the human β1 subunit promoter J. Orlowski J. J.B. Nucleic Res. PubMed Scopus Google in to a to J.B. J. Biol. PubMed Scopus Google Scholar). transfection studies indicated that PGE1, and all stimulated containing within the β1 subunit promoter to we a region from the and between and in the β1 subunit that is required to elicit the effects of PGE1, and a sequence within this region to a β1 that contains a as well as an the the was required to elicit the effects of PGE1, and we the of between and prostaglandins. all of the of cells in M. S. PubMed Scopus Google Scholar). PGE1 to both the cAMP and Ca2+ in cAMP and may transcription through the of CREB PubMed Scopus Google Scholar). both of the subunit of protein to CREB at J. Biol. PubMed Google in addition, and to CREB PubMed Scopus Google J. Biol. PubMed Scopus Google Scholar). The of CREB at of CREB to and increases the of CREB to bind to in for as J. Biol. PubMed Google J. Biol. PubMed Google and Cell. PubMed Scopus Google Scholar). In addition, Ca2+ and to transcription through effects the CREB S. PubMed Scopus Google S. PubMed Scopus Google Scholar). the to a regulatory In the protein and as to expression through a a J. Physiol. Google Scholar). In as which transcription through a a phorbol J. Mol. PubMed Scopus Google C. J. PubMed Scopus Google Scholar). The results of our studies are consistent with the of a a which is responsible for transcriptional of the β1 subunit by the cAMP and pathways. a or CREB may be involved in regulation by and mobility shift assays were The results of our studies that four between a containing a β1 was a to with a β1 in addition, a was observed when a antibody against CREB was present the results that the β1 PGRE as a against a the β1 PGRE was as a than an mobility shift studies indicated that a containing a a GC was to with the β1 a of PGRE was observed when a antibody against Sp1 was in the Sp1 is the of the transcription that to the sequence Cell. PubMed Scopus Google present a of Sp1 to transcription Sp1 plays an important role in regulation of transcription as Mol. Cell. Biol. PubMed Scopus Google Scholar). in two cell and in cells, the increased transcription of the β1 subunit in to is mediated through increased Sp1 J. Biol. PubMed Scopus Google Scholar). in the increased β1 subunit transcription is with increased Sp1 C. J. Biol. PubMed Scopus Google Scholar). transcription observed as a of Sp1 action may be the of as well as interaction with J. Biol. PubMed Scopus Google C. J. Biol. PubMed Scopus Google Scholar). results that regulation of transcription of the human β1 subunit by the cAMP and Ca2+ an interaction between Sp1, CREB, and a regulatory region containing Sp1 and PGRE with an interaction between Sp1 and CREB are our immunoprecipitation experiments indicating that Sp1 be co-immunoprecipitated with CREB and In addition, our results that Also consistent with the involvement of CREB and Sp1 in regulation by PGE1 are our that of with an expression (a in reduced stimulation by PGE1, with an expression Sp1 in an in PGE1 and M. Res. PubMed Scopus Google evidence for an transcription to and an GC to in to a sequence to in the subunit promoter. for in the human subunit promoter was by at the or the GC that in a in promoter that both of sites are for transcription through a interaction between transcription M. Res. PubMed Scopus Google Scholar). the studies of and M. Res. PubMed Scopus Google this evidence indicating that regulation by and PGE1 the of a that is of a as well we observed that the which contains four GC boxes in the promoter was to regulation by insertion of the PGRE between the four GC boxes of the human promoter in the in an increased to PGE1 by cells, of a between the Sp1 sites and the PGRE in the promoter. Further evidence was obtained for the involvement of Sp1 in the PGE1 from the transfection studies conducted with the pLuc-MCS-β72–167 which contains region -167 to -72 in the β1 subunit promoter. the PGE1 stimulation was observed in cells transfected with pLuc-MCS-β72–167, was when the two GC boxes immediately to the PGRE were farther upstream from the for the involvement of Sp1 in regulation by the and in the the phorbol element (a was to be a for Sp1 J. Biol. PubMed Scopus Google and increased Sp1 to the was observed following of the in cells by the protein and to be an which is a for Sp1 S. C. J. Biol. PubMed Scopus Google Scholar). In a evidence indicating that the of Sp1 to a Sp1 the promoter is involved in the protein expression of the C. S. M. J. Biol. PubMed Scopus Google Scholar). CREB and Sp1 in the transcriptional of the in human J. Biol. PubMed Scopus Google Scholar). The increased transcription observed in cells was to between Sp1, and of the cAMP in the in increased of Sp1 from the to the to the observed in of CREB was observed to to CREB to a to a which was immediately from the Sp1 In cells, CREB protein levels were to transcription of the to results with the human β1 subunit promoter from the results obtained in the in that we obtained evidence for the involvement of CREB in to Sp1 in the stimulatory effects of the protein and β1 results that an interaction between Sp1 and CREB is involved in regulation of β1 subunit expression through pathways. an interaction between Sp1 and CREB may be or with an interaction are indicating that both Sp1 and CREB with as well as transcription I. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). the involvement of both Sp1 and CREB in regulation by the protein indicated evidence was for the involvement of a protein Sp1 and Included among studies is a the regulation of the CREB by cAMP in cells, the of CREB to two the CREB promoter as well as the of Sp1 to an Sp1 C. Biol. PubMed Scopus Google Scholar). regulation of the in cells by and cAMP to be an that is in to a M. S. J. Biol. PubMed Scopus Google Scholar). in and between CREB and Sp1 may be involved in or the Na,K-ATPase and subunit are in a by and is the results of our 5′-deletion that PGE1, and okadaic all transcription through the our results obtained using a that PGE1, and all In regulation of the and of the Na,K-ATPase in this regulation of the human and subunit is as an in a in the human subunit when that the and subunit are subject to regulation by PGE1, and the of an both that PGE1, and transcription of the and subunit to the this the of regulation of and subunit levels by PGE1 and as regulation may be the of in at the post-transcriptional level. this is a in as for a of from Cell. 38: PubMed Scopus Google to J. Biol. PubMed Scopus Google to protein M. PubMed Scopus Google boxes M. Res. PubMed Scopus Google Scholar). our is in a to regulatory and the cAMP and Ca2+ pathways. Further studies are in to the by which Sp1 and CREB to expression two pathways. and for and and and at the of at for of the for this