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Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting electrophoresis mobility shift assay (WEMSA).

Moeenrezakhanlou A, Nandan D, Reiner NE - Biol Proced Online (2008)

Bottom Line: Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1.WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA.In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada.

ABSTRACT
Calcitriol (1alpha, 25-dihydroxyvitamin D(3)) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.

No MeSH data available.


Related in: MedlinePlus

Fig. 4                     EMSA analysis confirms results obtained through WEMSA.EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter. Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.
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f4: Fig. 4 EMSA analysis confirms results obtained through WEMSA.EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter. Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.

Mentions: The analysis of calcitriol-regulated Sp-1 binding to this site in the CD14 promoter using WEMSA suggested that calcitriol negatively regulated Sp-1 binding to a specific DNA sequence (Figs. 2 and 3). In order to validate the results of WEMSA, we used the same candidate Sp-1-like oligo (5'-AGAGGTGGGGAGG-3') in a classical EMSA. THP-1 cells were treated with or without calcitriol and nuclear extracts were prepared and incubated with this Sp-1-like sequence. DNA-protein complexes were then separated by polyacrylamide gel electropheresis for EMSA. The results shown in Fig. 4 clearly demonstrate clacitriol-mediated down-regulation of Sp-1 binding to the candidate Sp-1 binding sequence in the CD14 promoter, thereby corroborating the results obtained by WEMSA. Taken together, these results confirm that the candidate Sp-1-like binding site located at position -91 to -79 is a bona fide site for Sp-1 binding and that calcitriol reduces Sp-1 binding to this position in the CD14 promoter.


Identification of a calcitriol-regulated Sp-1 site in the promoter of human CD14 using a combined western blotting electrophoresis mobility shift assay (WEMSA).

Moeenrezakhanlou A, Nandan D, Reiner NE - Biol Proced Online (2008)

Fig. 4                     EMSA analysis confirms results obtained through WEMSA.EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter. Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.
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Related In: Results  -  Collection

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f4: Fig. 4 EMSA analysis confirms results obtained through WEMSA.EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter. Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.
Mentions: The analysis of calcitriol-regulated Sp-1 binding to this site in the CD14 promoter using WEMSA suggested that calcitriol negatively regulated Sp-1 binding to a specific DNA sequence (Figs. 2 and 3). In order to validate the results of WEMSA, we used the same candidate Sp-1-like oligo (5'-AGAGGTGGGGAGG-3') in a classical EMSA. THP-1 cells were treated with or without calcitriol and nuclear extracts were prepared and incubated with this Sp-1-like sequence. DNA-protein complexes were then separated by polyacrylamide gel electropheresis for EMSA. The results shown in Fig. 4 clearly demonstrate clacitriol-mediated down-regulation of Sp-1 binding to the candidate Sp-1 binding sequence in the CD14 promoter, thereby corroborating the results obtained by WEMSA. Taken together, these results confirm that the candidate Sp-1-like binding site located at position -91 to -79 is a bona fide site for Sp-1 binding and that calcitriol reduces Sp-1 binding to this position in the CD14 promoter.

Bottom Line: Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1.WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA.In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada.

ABSTRACT
Calcitriol (1alpha, 25-dihydroxyvitamin D(3)) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.

No MeSH data available.


Related in: MedlinePlus