3.1. Human CAIII promoter is transcriptional active in Saos-2 and HT-29 cells
To identify regulatory elements in human CAIIII gene, we cloned a 1027 bp of promoter sequence and sequenced (accession number: MF374499.1). Transcriptional start Site (TSS) and Translation Start codon (TSC) were indicated in the 1027 bp of the CAIII promoter sequence (Fig. 1a). 5’UTR region is about 86 bp. Several potential TF recognition sites, such as Kruppel-like transcription factors, Myo-D, activator protein 2, and activator protein 4 (Data not shown) were identified using MathInspector program. The most interesting feature of the promoter is to possess many consensus sequences of GC-box factors SP1/GC (Fig. 1a and 1b). There are 9 putative SP1 binding elements. To determine the conserved regions of CAIII promoter, 429 bp of human, rat and mouse CAIII promoters were aligned using BioEdit program. Aligned region of CAIII promoter site showed the high similarity in all species (Fig. 1b). CAIII promoters form three species were compared and the results showed 73% identity between rat and human sequences, 85% identity between rat and mouse sequences, and 75% identity between mouse and human sequences. (Fig. 1a). The human CAIII gene promoter does not possess classical TATA box but includes many putative transcription factor binding elements as well as classical GC box (Although the TATA box and GC boxes are conserved between mouse and rat CAIII promoter, Fig. 1a, b).
To find out the CAIII mRNA expression pattern in several cancer cells, prostate cancer cells (PC3), colorectal cancer cells (HT-29) and osteosarcoma cells (Saos-2), we used qRT-PCR strategy (Fig. 1c). qRT-PCR analysis showed that CAIII and had the highest expression in the osteosarcoma cells, Saos-2 and HT-29 cells and with moderate expression in PC3 cells. Immunofluorescence analysis was used for determination of the location of the human CAIII. CAIII was in both the nucleus and the cytoplasm in PC3 and Saos-2 cells (Fig. 1d).
To determine the minimal promoter region for human CAIII gene promoter, four truncated deletion plasmids were constructed in pMetluc vector with progressively larger deletions from the 5’ end of the promoter containing – 939/+86, -699/+86, -236/+86, and − 108/+86 (Fig. 1e). All promoter constructs are active in human colon carcinoma cells, HT-29 cells and human osteosarcoma, Saos-2. 194 bp fragment P4 (-108/+86) displayed the most active promoter activity in both cells (Fig. 1e). In addition, a central functional promoter of the human CAIII gene is located within the − 108/+86 region. However, other promoter fragments are more active than the activities of fragments in HT-29 cells (Fig. 1e). The difference of the promoter activity in Saos-2 and HT-29 cells might be resulted from the presence of cell type-specific regulatory elements.
3.2. Overexpression of SP1 transcription factor upregulates CAIII gene expression
Transcription factor binding search revealed multiple SP1 binding elements in the promoter. Bioinformatics analysis showed that the several putative TFs SP1 Motifs bind at-939/+86 (Fig. 1a). Thus, we hypothesized that the transcriptional activity of the human CAIII gene may be significantly regulated by SP1. (Fig. 2a). In order to show if SP1 transcription factor regulates CAIII transcription, four different CAIII promoters were co-transfected into Saos-2 with or without SP1 expression vector. Three of promoter constructs (699/+86, -236/+86, and − 108/+86) could be highly upregulated by SP1 overexpression based on the basal activities of them (p ≤ 0.05) (Fig. 2a). Therefore, these sites may involve in the activation of gene transcription.
In order to demonstrate if this activation would be carried on at transcriptional and translational level, CAIII mRNA levels were determined upon SP1 overexpression. Firstly, SP1 overexpression was confirmed by qRT-PCR in osteosarcoma model, Saos-2 cells (Fig. 2b). As indicated in Fig. 2b, SP1 was successfully overexpressed in Saos-2 cells. Only vector -transfected cells were used as control groups for overexpression studies (Fig. 2c). In Fig. 2c, CAIII expression was increased by SP1 nearly about 6- fold, especially at 72 h. SP1 overexpression led to a time-dependent increase of CAIII mRNA level. Consistent with mRNA level, SP1 increased CAIII protein level to 2-fold in Saos-2 cells (Fig. 2d).
3.3. SP1 activates CAIII not only activate in osteosarcoma but also in prostate cancer cells.
In order to reveal the SP1-mediated activation in tissue specific manner, the SP1 overexpression studies were conducted in PC3 cells. As seen in Fig. 3a, SP1 overexpression was successfully performed in PC3 cells. This overexpression continues steadily for 72 hours. Like, Saos-2 cell, there is also time dependent SP1 mediated CAIII activation in PC3 cells (Fig. 3b).
Upon, SP1 overexpression, transcriptional activities of three promoter fragments, 699/+86, -236/+86, and − 108/+86 were upregulated in statistically significant manner (Fig. 2A). For analyses of Sp1 binding sites, P3 − 236/+86 region was subdivided into three parts (-181/-153, -158/-134 and + 28/+62) for EMSA analysis. These three probes were synthesized and labelled with biotin for EMSA. Firstly, to elucidate Sp1-mediated regulation of CAIII promoter, we carried-out DNA-protein interaction assay with a probe − 158/-134 with nuclear extract derived from different cells. Different binding patterns was obtained from different nuclear extract, osteoma sarcoma cells (MG-63 and Saos-2) and prostate cancer cells. Interestingly, no DNA-protein complex formed except for PC3 in the EMSA analysis of probe − 158/-134. There is one and very strong DNA-protein complex in PC3 nuclear extract (Fig. 3). Differential TF binding on a promoter is common transcriptional regulation mechanism as a result of tissue specific regulation of the genes.
3.4. SP1 directly binds on multiple putative SP1 sites of CAIII promoter
Three different biotin-labelled oligonucleotide probes, (-181/-153), (-158/-134) and (+ 28/+62) and nuclear extracts from PC3 was used in EMSA analysis (Fig. 4A). EMSA analysis revealed that probes (-181/-153), (-158/-134) and (+ 28/+62) had generated one DNA-protein complex. With the use of unlabelled probe competition, the specificity of the complexes was assessed (Fig. 4B, C and D). Additionally, the inclusion of the unlabeled SP1 probe competitively reduced the binding of biotin-labelled probes to nuclear extract, indicating that SP1 binds to the related regions based on three independent experiments. Probe 2 (-158/-134) contains three consensus SP1/GC-rich binding elements. These potential SP1 sites were deleted and the mutated EMSA probe was performed in EMSA analysis. EMSA with the SP1-mutated primers diminished the DNA-protein complex (Fig. 4E). This result reveals that SP1 transcription factor directly binds the putative SP1 binding sites residing between − 153 and − 158 bp in CAIII promoter sequence