Identification and characterization of the promoter of fibroblast activation protein

Abstract

Fibroblast activation protein (FAP) is a type II integral membrane glycoprotein belonging to the serine protease family. It is selectively expressed by tumor stromal fibroblasts and transiently in the fibroblasts of healing wounds. FAP has been shown to modulate growth, differentiation, adhesion, and metastasis of tumor cells. Despite the importance of FAP in cancer, the mechanisms that govern its expression have not been defined. In this study, we determined the transcription start site of the FAP gene and identified a 2-kb segment with promoter activity in cells expressing FAP. Truncation of this fragment revealed that the core promoter activity resided in a 245-bp fragment surrounding the transcription start site. Electrophoretic mobility shift assay showed that EGR1 binds to the FAP promoter. Mutation of the EGR1 site within this fragment significantly decreased the promoter activity of FAP and eliminated EGR1 binding. Down-regulation of EGR1 resulted in a significant reduction in endogenous FAP mRNA expression. These findings identify the basal transcriptional requirements of FAP gene expression and show EGR1 is an important regulator of FAP expression.

Figure 1

Promoter sequence analysis of FAP genes. Cluster alignment of the proximal promoter regions of the human and mouse FAP genes. Conserved transcription factor binding sites between human and mouse are boxed or underlined. A 40-bp region with 100% homology between human and mouse is highlighted in bold letters. Arrow indicates the position of the transcription start site.

Figure 2

Transcription start site of FAP genes. 5′- RACE assay was performed with RNAs from mouse or human FAP positive cells. Photograph of ethidium bromide-stained agarose gels showing one dominant 5′- RACE product.

Figure 3

Putative FAP promoter activity is specific. (A) The mRNA level of FAP was detected in various cancer cell lines by qRT-PCR. The percent of FAP mRNA was compared with that of V20 after normalized with actin. (B) The luciferase construct that contains ~2kb 5′ flank region of FAP gene and the renilla control vector were transfected into cell lines with different levels of FAP. 48 hrs later, the luminescence was measured and the ratio of luciferase activity versus renilla control is shown in the graph. Values are mean +/− E representing at least three independent experiments.

Figure 4

Identification of promoter sequences in FAP gene. Control pGL3 luciferase constructs or pGL3 contains various regions of FAP 5′ flank regions indicated in the graph were transiently co-transfected with Renilla control into HOS cells and luciferase activity was measured 48 hrs after transfection. The ratio of luciferase activity versus renilla control is shown in the graph. Values are mean +/− SE representing at least three independent experiments. The constructs are numbered on the left relative to the initiate ATG.

Figure 5

Deletion analysis of the minimal FAP promoter. A series of pGL3-245 internal deletion luciferase constructs were transiently co-transfected with Renilla control into HOS cells and luciferase activity was measured 48 hrs after transfection. The ratio of luciferase activity versus renilla control is shown in the graph. Values are mean +/− SE representing at least three independent experiments.

Figure 6

EGR1 is a potential transcription factor that regulates FAP transcription. Luciferase constructs pGL3-245 and its mutant as indicated were transiently co-transfected with Renilla control into HOS cells and luciferase activity were measured 48 hrs after transfection. The ratio of luciferase activity versus renilla control is shown in the graph. Values are mean +/− SE representing at least three independent experiments.

Figure 7

EGR1 binds to the putative EGR1 binding site in the FAP promoter. [γ-³²P] ATP-labeled oligonucleotides covering −225 to −185 region of FAP promoter fragment was incubated with recombinant EGR1 protein (“Control”). Competition experiment was performed by pre-incubating EGR1 protein with a 100-fold excess of unlabeled consensus (“cold WT probe”) or EGR1 mutant oligos (“cold mutant probe”) prior to the addition of the ³²P-labeled oligo. EGR1 antibody was used for supershift analysis. Arrows point at the DNA/protein complex of EGR1 and supershift band identified by the EGR1 antibody.

Figure 8

The expression of FAP mRNA is partially decreased by knockdown of EGR1 in HOS. The siRNAs of E2F1, HOXA4 and EGR1 or non-target control siRNAs were transfected into HOS cells at 100 nM for 24 hrs before the RNA was harvested. The mRNA level of FAP and genes of target were detected by qRT-PCR. Data is shown as relative amount comparing with non-target siRNA controls after normalized with actin. Values are mean +/− SE.