CD13/APN transcription is regulated by the proto-oncogene c-Maf via an atypical response element

Abstract

Angiogenic growth factors induce the transcription of the cell surface peptidase CD13/APN in activated endothelial cells of the tumor vasculature. Inhibition of CD13/APN abrogates endothelial invasion and morphogenesis in vitro and tumor growth in vivo suggesting a critical functional role for CD13 in angiogenesis. Experiments to identify the transcription factors responsible for this regulation demonstrated that exogenous expression of the proto-oncogene c-Maf, but not other bZip family members tested, potently activates transcription from a critical regulatory region of the CD13 proximal promoter between -115 and -70 bp which is highly conserved among mammalian species. Using promoter mutation, EMSA and ChIP analyses we established that both endogenous and recombinant c-Maf directly interact with an atypical Maf response element contained within this active promoter region via its basic DNA/leucine zipper domain. However full activity of c-Maf requires the amino-terminal transactivation domain, and site-directed mutation of putative phosphorylation sites within the transactivation domain (serines 15 and 70) shows that these sites behave in a dramatic cell type-specific manner. Therefore, this atypical response element predicts a broader range of c-Maf target genes than previously appreciated and thus impacts its regulation of multiple myeloma as well as endothelial cell function and angiogenesis.

Figure 1. The region between −153 and −70 of the proximal promoter of CD13/APN is essential for activity in endothelial cells

A. Equal amounts of plasmids containing 5′ deletions of the proximal promoter of CD13 in the pGL2 luciferase gene reporter plasmid were transiently transfected into EOMA cells. Luciferase activity is normalized to total protein and is represented as percent activation of activity of the intact promoter. Error bars refer to the standard derivation within one experiment performed in triplicate. The experiment was performed on at least three different days and a representative experiment is shown. B. The essential region of the CD13 proximal promoter between −153 and −70 is highly conserved across mammalian species. The transcription factor Ets-2 activates the CD13 promoter via the GGAG (bold) motif.

Figure 2. c-Maf transactivates the CD13 promoter in endothelial cells via a response element between −153 and −70

A. EOMA cells were transiently cotransfected with 0.5 μg/well 1kb/luc reporter and increasing amounts of plasmids expressing bZip family members: c-Maf, c-Jun, c-Fos, MafB, and MafK. Maximum fold activation was extrapolated from experimental data using GraphPad Software. Whole cell lysates from cells transfected with the bZip expression plasmids were analyzed for protein expression by Western blot. B. c-Maf, MafB, and c-Fos expression plasmids were cotransfected with the panel of siRNAs and assayed for protein expression of newly transcribed mRNA to confirm the efficacy and specificity of c-Maf siRNA treatment. Membranes were stripped and reprobed with the GAPDH antibody as a loading control. C. The panel of siRNA was cotransfected with the c-Maf expression plasmid and the CD13 proximal promoter reporter plasmid to confirm that CD13 transactivation is c-Maf specific.

Figure 3. c-Maf directly binds the first 153bp of the the proximal promoter

A. Schematic representation of the structure of c-Maf. H and G represent repeats of histidine and glycine, respectively. EHR refers to the extended homology region upstream of the basic DNA binding region. Arrows indicate two putative phosphorylation residues. Arrowheads indicate the location of point mutations within c-Maf associated with dominant negative cataracts in humans and mice. Open arrows indicate mutations in expression plasmids used in B, within the basic DNA binding domain (PLER, R22E) and leucine zipper domain (L2P4P). B. Equal amounts of the c-Maf or the mutant c-Maf expression plasmids were cotransfected with 1kb/luc reporter plasmid into EOMA cells. The C-term truncation has a 5′ deletion of amino terminal transactivation domain; L2P4P, mutation of the leucine zipper; PLER, insertion of 4 amino acids in basic DNA binding domain; and R22E, mutation in basic DNA binding domain. C. Equal amounts of the 5′ deletion fragments of the proximal promoter of CD13 (153/luc, 115/luc, and 70/luc reporter plasmids) were cotransfected with c-Maf expression plasmid into EOMA cells. Luciferase activity is normalized to μg protein and is represented as percent transactivation of values obtained with full length c-Maf. Error bars refer to the standard derivation within one experiment performed in triplicate. Each experiment was performed at least three times and a representative experiment is shown. D. EMSA with the recombinant GST fusion protein containing the bZip domain of c-Maf. Competitions were performed with excess oligonucleotide with either the MARE sequence (lane 3) or the sequence between −153 and −115 (lane 4).

Figure 4. c-Maf acts through an atypical response element within the proximal promoter of CD13

A. EMSA with recombinant c-Maf and 153bp probe. Competitions were performed with a series of oligonucleotides spanning the length of the 153bp fragment, as indicated above each lane. B. Unlike other bZip transcription factors which interact with the core TGACGTCA palindromic response element, Maf transcription factors interact with the flanking TGC motifs of the canonical MARE, as described (Dlakic et al., 2001). C. The sequence between −120 and −87 contains two TGC motifs (−113 and −104) and one TGT motif (−97). Site-directed mutations were introduced into the three separate motifs within the −120 to −87 region in the context of the 153bp/luc plasmid. D. Equal amounts of the reporter plasmids containing the mutations were transiently transfected into EOMA cells and harvested after 24 hrs. Luciferase activity is normalized to μg protein and is represented as percent activation of the intact −153 bp promoter. Error bars refer to the standard deviation within one experiment performed in triplicate. The experiment was performed on at least three times and a representative experiment is shown.

Figure 5. c-Maf activity is dependent on two highly conserved c-Maf serine residues that are regulated differently in endothelial and liver cells

A. Two serine residues (S15 and S70 in c-Maf) within the amino-terminal transactivation domain are conserved across the large Maf family. B. Whole cell lysates from cells transfected with RcRSV, c-Maf or the c-Maf phosphorylation site mutants (S15A, S70A, S15A/S70A) were probed with the M153 c-Maf antibody and were stripped and probed with the GAPDH antibody, as a loading control. Arrows indicate mutation of the S70A within c-Maf alters the electrophoretic mobility in SDS-PAGE to a greater degree than the S15A mutation. C. The c-Maf and the c-Maf point mutant expression plasmids (S15A, S70A, S15A/S70A) were co-transfected with the 153/luc CD13 reporter plasmid into endothelial (EOMA) or liver (HepG2) cell lines. In EOMA cells, the c-Maf ^S15A had significantly less activity on the CD13 promoter than c-Maf, while in HepG2 cells, c-Maf ^S70A and c-Maf ^S15A/S70A has significantly less activity than wild type c-Maf. D. The c-Maf and the c-Maf phosphorylation site mutant plasmids were co-transfected with the IL-4/luc reporter plasmid into EOMA or HepG2 cells. In HepG2 cells, the c-Maf ^S70A and the c-Maf ^S15A/S70A had significantly greater activity than c-Maf, while the difference seen between c-Maf^S70A and c-Maf^S15A/S70A was not significant (ns., p=0.376). Luciferase activity was normalized to μg protein and is represented as fold transactivation over transient transfection with the empty expression vector RcRSV. Error bars refer to the standard derivation within one experiment performed in triplicate. The experiment was performed at least three times and a representative experiment is shown. An asterisk represents statistical significance versus the appropriate control in each group (*p<0.05, ns. not significant).

Figure 6. c-Maf is expressed in endothelial cells and directly binds the first 153bp of the endogenous proximal promoter of the CD13 gene

A. Southern blot analysis of c-Maf after RT-PCR of total RNA from KS1767, EOMA, HUVEC or OPM2 multiple myeloma positive control cells. B. EMSA with nuclear lysates from KS1767 cells with the 153bp probe. Competitions were performed with excess oligonucleotide with either the canonical Maf response element (MARE) sequence (lanes 2, 4, 7) or the sequence between −153 and −115 (lane 8), as indicated above each lane. Arrows indicate the band specifically competed by the MARE oligonucleotide. C. Western blot analysis of immunoprecipitated c-Maf from KS1767 nuclear lysates. D. Chromatin immunoprecipitation from formaldehyde fixed KS1767 cells. Genomic DNA was PCR amplified with primers for the CD13 proximal promoter (27 cycles), the CD13 distal promoter (negative control, 28 cycles), and the IL-4 promoter (positive control, 26 cycles). Relative intensities of c-Maf immunoprecipitated PCR products were compared to rabbit IgG chromatin immunoprecipitated PCR products. Each experiment was performed at least three times and a representative experiment is shown.