Kinin B1 Receptors Are Quickly Induced Trauma Inflammation Biology Essay

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Kinin B1 receptors are quickly induced/upregulated after tissue trauma or inflammation and are thought to be involved in maintaining the inflammatory response. At the transcriptional level, studies of B1R promoters have greatly increased our understanding of mechanism and signalling pathways in receptor activation. However, the regulation of promoter in pulmonary epithelial cells is still unknown. Here, we show using in vitro transient transfections with a luciferase reporter gene indicate that both B1R promoters have cell-specific basal activity that lead to the preferential use of (-1600bp upstream TIS) 5' flanking promoter in 16HBE cells and the alternative promoter (-1020bp upstream TSS) in H2126. Interestingly, when both promoters are in the same construct, the luciferase activity is always reduced. Deletion constructs of alternative promoter (-766 to +1 and -410 to +1) in combination with 5' promoter have identified a PRE in the -766 to -410bp region that is common to both cell lines while the region -1020 to -766bp acts as a PRE in 16HBE and NRE in H2126. Either of the promoter alone, in combination or in deletion constructs did not respond to stimulation by LPS and desArg10KD in 16HBE and H2126 cell lines indicating that other regions may be required. This study has shown that B1R demonstrate cell-specific promoter activity in pulmonary epithelial cells and have identified general and cell-specific regulatory elements in pulmonary bronchial epithelial cells 16HBE and H2126. In addition, it is now known that the alternative promoter either alone or in combination with the 5' flanking core promoter does not respond to stimulation by LPS and desArg10KD in 16HBE and H2126 suggesting that element/s outside both promoters is most likely necessary for induction in pulmonary epithelial cells.

3.1 Introduction

Kinins (bradykinin, des[Arg9]-BK, and des[Arg10]-KD DAKD) are biologically active peptides formed by the enzymatic action of the classical kallikreins (KLK1, KLKB1) on kininogens. Kinins are primarily pro-inflammatory but can affect processes such as cell proliferation and migration (Bhoola et al 1992). These effects are mediated through two G-protein coupled kinin receptors, one of which is the kinin B1 receptor (B1R) (1, 2). Kinin B1 receptor is usually latent under normal physiological conditions but is quickly upregulated during tissue trauma. Recent evidence has suggested an interesting role of B1R in the development of lung carcinoma through inflammation, cell growth and proliferation (REF). The use of B1R antagonist CU201 inhibited growth in a range of mesothelioma, NSCLC and SCLC cell lines in vitro (3) which is further supported by a similar observation in an in vivo NSCLC xenograft model by reducing growth of cancerous cells by 65% (4). A more recent B1R antagonist B10324 has shown anti-growth activity in an in vivo SCLC model by reducing cancer cell growth up to 86% (5). Lung epithelial cells form a protective lining in the airways which are constantly exposed to many irritants and pathogens. Damage to the epithelial cells usually results in inflammation which under multiple repeated cycles may lead to cells undergoing genetic and epigenetic changes that may favour lung tumorigenesis (Loewen et al 2005 - BMC Cancer). Whilst it is known that A549 lung adenocarcinoma and human bronchial epithelial cells expresses B1R (REF), little is known little is known about the regulation of B1R expression in pulmonary epithelial cells.

The expression of B1R is tightly regulated by two promoters located upstream of exon I and exon III (REF). In cell types including human hepatoma HepG2, rat vascular smooth muscle cells and bovine arterial endothelial cells B1R promoters exhibit cell/tissue specific activity. Whether this is similar in pulmonary epithelial cells is not known. Furthermore, the interaction of promoter regulatory regions resulting in overall gene expression of B1R has not been studied in detail in pulmonary epithelial cells. Finally, the question of the transcription start site from the alternative promoter has not been identified. This study aims to characterise the role of both promoters in pulmonary epithelial cells and in particular determines the combinatorial regulation of both B1R promoters.

3.2 General experimental approach and aims

The first main approach was to determine the cell lines that best reflect higher B1R mRNA expression in lung cancer versus normal immortalised lung cells. B1R expression has been observed in a variety of lung cells including fibroblasts (IMR90), immortalised (BEAS-2B, 16HBE, A549) and primary human bronchial epithelial cells and primary human lung microvascular endothelial cells (6). Incubation of these cells with the B1R agonist desArg10KD upregulated B1R expression suggesting that immortalised lung cells have the capacity to process and express B1R.

The next step was to construct recombinant luciferase plasmids that contain the 5' core promoter, the alternative promoter region or a combination of both regions upstream of the luciferase gene. The method of luciferase gene reporter system to measure promoter activity is well established. The firefly luciferase enzyme is sensitive and does not require post-translation processing for enzyme activity which makes it suitable for a transient transfection assay since luciferase can be measured immediately after translation (Wood et al 1984). In this system, luciferase catalyses a reaction using D-luciferin and ATP in the presence of oxygen and Mg2+ resulting in light emission. The intensity of light emitted correlates to promoter activity (recheck Wood 1991).

The next step was to transfect cells selected from step a) with plasmid constructs from step b). This will lead to an understanding of intrinsic expression of both promoters separately and also the possible change in promoter activity when both are expressed in the cell.

Following on, the activity of both promoters whether alone or in combination was stimulated with a broad-spectrum stimulus and a specific stimulus to determine the activity of promoters under different conditions.

Following that, deletion constructs of the alternative promoter either alone or in combination with the core promoter was used for transfection into selected cells to determine the overall activity and interplay between alternative promoter regions.

Finally, transcription start sites of B1R from the activity of alternative promoter will be identified using 5' RACE PCR.

3.3 METHODS

3.3.1 Culture of 16HBE human bronchial epithelial cells and H2126 human lung adenocarcinoma

16HBE, A549, NHLF, HFLF, H520 and H2126 cells were obtained from the American Type Culture Collection (Rockville, MD). 16HBE, NHLF and HFLF were cultured in complete growth media comprised of Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) containing 10% heat inactivated fetal bovine serum (Sigma, St. Louis, MO), 100 IU/ml penicillin, 100 μg/ml streptomycin and 4 mM l-glutamine (Invitrogen). A549, H520 and H2126 cells were cultured in RPMI 1640 media (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (GIBCO, Invitrogen), 100 IU/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a humidified atmosphere in 5% CO2 at 37°C and were subcultured by incubating with 0.05% trypsin-0.5 mM ethylenediaminetetraacetate (Invitrogen) at a ratio of 1:3 - 1:4, weekly. For cell stimulation purposes, cells were incubated in the mentioned media minus FCS. Normal cell culture media was replaced with serum-free media for 12hrs prior to the start of stimulation, the cells were washed once with 1XPBS before being incubated in the absence and presence of the B1 receptor agonist desArg10KD (Sigma Aldrich) and LPS (Sigma Aldrich) as described in the figure legends.

3.3.2 RT-PCR

Total RNA was extracted from l6HBE and H2126 cells using RNeasy mini kits as described by thr manufacturer (QIAGEN) and checked by denaturing agarose gel electrophoresis and ethidium bromide staining. Samples with sharp 28S and 18S ribosomal bands were used for first-strand cDNA synthesis. Single-stranded cDNA was generated using Omniscript reverse transcriptase (100 U; QIAGEN) in a 20-μl reaction mixture containing reaction buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol), 0.5 mM dNTP, 0.5 μg oligo(dT)12-18 (Invitrogen), 10 U rRNasin (Promega, Madison, WI), and 2 μg of total RNA. The reaction was carried out for 1 h at 37°C. Amplification of cDNA by PCR was performed using oligonucleotide primer pairs (GeneWorks, Australia) for the human B1 receptor and SOD1. The reactions were carried out using a RoboCycler (Stratagene, La Jolla, CA) in a 25-μl reaction mixture containing reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2), 0.2 mM dNTP, 2.5 U Taq polymerase (QIAGEN), and 1-2 μl of cDNA. Each primer was added at a final concentration of 0.2 μM. PCR was for 30 to 35 cycles, each cycle consisting of 30 s denaturation at 94°C, annealing at 60°C for 20 s, and extension at 72°C for 50 s. PCR reaction products were separated on 2% agarose gels containing 50 μg/ml ethidium bromide and visualized under UV light.

3.3.3 Construction of Reporter Plasmids

AP-Luc, CP-AP-Luc and CP-Luc was constructed forward and reverse primers (Table 3), digested with BglII/HindIII (AP-Luc) and MluI/NheI (CP-AP-Luc and CP-Luc) and cloned into their respective sites of pGL3 Basic vector (Promega). To generate the deletion constructs, various regions of B1R alternative promoter were amplified using AP-Luc as a template. All of the PCR fragments are flanked by an XhoI site at one end and HindIII site at the other end. Following XhoI/HindIII digestion, fragments were introduced into the corresponding restriction sites of AP-Luc and CP-Luc vector.

3.3.4 Transfection and Luciferase Assays

Twenty-four hours before transfection, 16HBE and H2126 were seeded at a concentration that would achieve 70% confluence in 96-well tissue culture plates. Liposome-mediate co-transfection using a mixture of 400 μl of Opti-MEM, 4.8 μl of LipofectAMINE 2000 (Invitrogen), 0.6 μg of the reporter construct, and 0.2 μg of pRL-TK (Promega, Madison, WI) (as a control for measuring transfection efficiency) per well was carried out as recommended by the manufacturer (Life Technologies, Inc.). The transfection mixture was replaced with normal growth medium 6 h later. Forty-eight hours after transfection, the cells were harvested.

Dual-luciferase assay was performed with the dual-luciferase reporter assay system (Promega). Cells were harvested after transfection at time points indicated in the experiments. The cells were washed with 1x PBS and were dissolved in 30 µl of 1x passive lysis buffer. The cells were rocked at room temperature for 5 min and transferred to -80°C immediately until required. Twenty microlitres of cell extract was transferred to a solid plate containing 50µl of luciferase assay reagent II (LARII) and the firefly luciferase emission was measured using a luminometer (Machine?). Next, Stop & Glo Reagent (50 µl) was placed into the plate to initiate renilla luciferase activity. The ratio of firefly luciferase activity to renilla luciferase activity was calculated to obtain luciferase activity normalised to an internal control.

3.3.5 Identification of B1R transcription start site/s

5'-Rapid Amplification of cDNA Ends (5′-RACE) PCR was performed on RNA isolated from 16HBE and H2126 using the GeneracerTM kit (Invitrogen). 5'-RACE PCR was performed as per manufacturer's instructions (Invitrogen). Sequences for B1R specific forward primer and reverse (dT)17-adaptor primer are shown in Table 3 .The nested PCR product was digested with EcoRI and XbaI, and ligated into the EcoRI/XbaI sites of the XXXXXXXXX. Nucleotide sequences were determined by sequencing using 3730 ABI DNA Sequencer.

3.3.6 Sequence and statistical analysis

In silico sequence analysis of B1R alternative promoter was performed using Transcription Factor Search (Copyright 1994-2000, Yutaka Akiyama-need proper ref).

Sequences were aligned using the program Clustal W (http://align.genome.jp/).

Statistical analyses were performed using paired one-tailed Student's t-test (GraphPad Prism 5.0) and two-way Anova with Bonferonni post-hoc test to determine significance. P<0.05 were considered to be statistically significant.

Table3. List of primers used in this chapter

Primer

Description

Sequence (5'  3')

B1R Fow2a

B1R Rev 1

B1R Fow

B1R Rev

Sod1 Fow

Sod1 Rev

BglII Fow

HindIII Rev

MluI Fow B1R core

NheI Rev B1R core

B1R HindIII -21R

B1R XhoI -766F

B1R XhoI -410F

GR 5'Primer

GR 5'Nested

RT Rev 3

RT Rev 2

Forward primer for B1R detection in RT-PCR

Reverse primer for B1R detection in RT-PCR

Forward primer for AP-Luc cloning

Reverse primer for AP-Luc cloning

Forward primer for CP-Luc and CP-AP-Luc cloning

Forward primer for CP-Luc and CP-AP-Luc cloning

Reverse primer for AP ∆410-Luc, AP ∆766-Luc, CP-AP ∆410-Luc and CP-AP ∆766-Luc cloning

Forward primer for AP ∆766-Luc and CP-AP ∆766-Luc cloning

Forward primer for AP ∆410-Luc and CP-AP ∆410-Luc cloning

Primers supplied from Invitrogen for 5' RACE PCR that binds to the RNA oligo

Reverse primer for 5' RACE PCR

Reverse nested primer for 5' RACE PCR

Table4. Summary of reporter vectors used in this chapter

Luciferase reporter vector

Description

pGL3 Basic

pGL3 Control

pRL TK

AP-Luc

CP-Luc

CP-AP-Luc

AP ∆766-Luc

AP ∆410-Luc

CP-AP ∆766-Luc

CP-AP ∆410-Luc

Does not contain any promoter or enhancer

Contains SV40 promoter and enhancer

Contains Renilla

Contains intron II and exon II of B1R upstream of luciferase gene in pGL3 Basic

Contains 5' flanking promoter upstream/downstream? of luciferase gene in pGL3 Basic

Contains alternative promoter sandwiched between core promoter flanking upstream and luciferase gene downstream in pGL3 Basic

Contains a 766bp fragment upstream of B1R ATG? Or Exon 3

Contains a 410bp fragment upstream of B1R ATG? Or Exon 3

Contains core promoter upstream of 766bp fragment

Contains core promoter upstream of 410bp fragment

Fig1. Schematic diagram of plasmid constructs used in this chapter. The genomic structure of B1R at the top with the arrow indicating the translation start site. 1500bp of the 5' flanking region relative to exon I is the core promoter while 1020bp upstream of exon 3 is known as the alternative promoter.

3.4 RESULTS

3.4.1 Human lung cell lines express varying levels of basal kinin B1 receptor mRNA.

Six lung cell lines were investigated for basal mRNA expression of kinin B1 receptor (Table 5).

Table5. Lung cell lines screened for B1R mRNA expression

Cell lines

Description

HFLF

NHLF

H2126

A549

16HBE

H520

Human fetal lung fibroblasts

Adult human lung fibroblasts

Human lung adenocarcinoma

Human lung adenocarcinoma

SV-40 transformed normal human bronchial epithelial

Human lung squamous cell carcinoma

All cell-lines expressed kinin B1 receptor except for the lung squamous cell line H520 (Fig 2). Expression of B1R was the greatest in the fetal lung fibroblast cell line HFLF (Fig 2).

Fig1. B1R transcript is differentially expressed across a range of cell lines. B1R mRNA mRNA expression of B1R transcripts normalised to housekeeping gene SOD1 in human pulmonary cell lines as quantified by real time PCR. Data represents mean ± SEM from 3 independent experiments.

3.4.2 Cloning of kinin B1 receptor promoters. The promoters of B1R were successfully amplified using random Caucasoid samples and cloned using directional primers (Fig 2A and 2B). The right inserts were confirmed using restriction digest (Fig 2C) and sequencing data from the forward and reverse strand.

C

A

B

Fig2.Repersentative images of successful PCR amplification and restriction digests. PCR amplification of Caucasoid samples produced the expected band size of alternative promoter and exon II of B1R ≈1020bp (A) and 5' flanking core promoter (B). C) Representative of a successful restriction digests confirming right insert (CP-AP-Luc).

3.4.3 The alternative promoter of kinin B1 receptor drives basal activity in lung cancer cell-line while the core promoter drives activity in a lung epithelial cell line.

In the bronchial epithelial cell line 16HBE, both core promoter and alternative promoter constructs were able to significantly drive luciferase expression compared to pGL3 Basic. Luciferase expression was ï‚»2.3-fold higher (p<0.001) while the alternative promoter ï‚»0.7-fold (p=0.037) higher compared with pGL3 Basic expression. This suggests that the core promoter is the promoter of choice in 16HBE (Fig 2).

In the lung adenocarcinoma cell line H2126, both AP-Luc and CP-Luc was able to drive promoter activity. However, the preferred promoter in H2126 is the alternative promoter (AP-Luc) as luciferase activity was increased by ï‚»52-fold (p=0.004) while the core promoter (CP-Luc) increase was only ï‚»15-fold (p=0.017) compared to pGL3 Basic activity (Fig 3). Interestingly, combination of core and alternative promoter within the same construct (CP-AP-Luc) resulted in promoter activity that was lower than the highest single most active promoter activity in each cell line. In 16HBE, CP-AP-Luc was ï‚»1-fold higher (p=0.004) than pGL3 Basic luciferase expression while in H2126, the increase was ï‚»29-fold (p=0.009) compared with pGL3 Basic (Fig 2 & 3).

B

A

Fig3. Cell-type preference of B1R promoters under basal conditions. Basal expression of core promoter, alternative promoter and combined promoters constructs in normal bronchial epithelial cells 16HBE (A) and human lung adenocarcinoma H2126 (B). Activity of construct is indicated by relative luciferase activity obtained by normalising luciferase activity from Firefly to Renilla expression. Data was statistically analysed using Student's t-test on four independent experiments, each performed using at least triplicates. P ≤ 0.05 were considered statistically significant while n.s = not significant. Error bars = SEM

3.4.4 Deletion constructs of the alternative promoter of kinin B1 receptor identified the presence of regulatory elements.

Deletion of the alternative promoter region between -1020bp and -766bp (AP ∆766-Luc) did not alter basal luciferase activity in both 16HBE and H2126 luciferase expression compared to AP-Luc (Fig 4). Further deletion between -766 to -410 (AP ∆410-Luc) increased luciferase activity by 0.7-fold in 16HBE (p=0.02; Fig 2) and 1.5-fold in H2126 (p=0.021; Fig 3) compared to AP ∆766-Luc, suggesting the presence of a negative regulatary element (NRE) that is active in both cell types.

In the presence of the core promoter, kinin B1 receptor alternative promoter deletion constructs showed that the removal of the region between -1020bp and -766bp (CP-AP ∆766-Luc) in 16HBE did not alter luciferase expression but increased luciferase activity by 1.3-fold in H2126 (p=0.0045; Fig 4d) compared to both promoter construct, CP-AP-Luc. In the presence of the core promoter, removal of the region between -766 to -410 (CP-AP ∆410-Luc) did nothing to alter luciferase expression in 16HBE (Fig 2) but reduced luciferase activity by 43% in H2126 (p=0.033; Fig 3) compared to CP-AP ∆766-Luc. The complete removal of the alternative promoter increased luciferase activity in 16HBE (p=0.026) but the opposite pattern was observed in H2126 (p=0.034) suggesting that the core promoter (CP-Luc) is more active in 16HBE and the alternative promoter (AP-Luc) is more active in H2126.

3.4.5 Stimulation of promoter-transfected cells with LPS and DAKD did not alter promoter activity in both cell lines

DesArg10kallidin (DAKD) is a specific agonist for B1R and has been shown to increase receptor expression. Stimulation of 16HBE and H2126 cells with 100nM or 1000nM DAKD for 3, 6 and 24h did not significantly change the expression level of any of promoter constructs (CP-AP-Luc, CP-Luc, AP-Luc and promoter deletion constructs) compared with unstimulated cells. General inflammatory stimuli 1ug/mL lipopolysaccharide, also showed no significant change in luciferase activity (data not shown for 16HBE).

B

A

Fig4. Deletion constructs of B1R alternative promoter reveals regulatory regions. Relative luciferase activity of alternative promoter deletion constructs transfected into human bronchial epithelium 16HBE (A) and human lung adenocarcinoma H2126 (B). Data was statistically analysed using Student's t-test on four independent experiments, each performed using at least triplicates. P ≤ 0.05 were considered statistically significant while n.s = not significant. Error bars = SEM

A

B

3.3.5 Stimulation of promoter-transfected cells with LPS and DAKD did not alter promoter activity in both cell lines

DesArg10kallidin (DAKD) is a specific agonist for B1R and has been shown to increase receptor expression. Stimulation of 16HBE and H2126 cells with 100nM or 1000nM DAKD for 3, 6 and 24h did not significantly change the expression level of any of promoter constructs (CP-AP-Luc, CP-Luc, AP-Luc and promoter deletion constructs) compared with unstimulated cells. General inflammatory stimuli 1ug/mL lipopolysaccharide, also showed no significant change in luciferase activity (data not shown).

Fig5. Deletion constructs of B1R alternative promoter in the presence of core promoter revelas regulatory regions. Relative luciferase activity of B1R alternative promoter deletion constructs combined with B1R core promoter transfected into human bronchial epithelial 16HBE (A) and human lung adenocarcinoma H2126 (B). Data was statistically analysed using Student's t-test on four independent experimentss. P ≤ 0.05 were considered statistically significant while n.s = not significant. Error bars = SEM

Fig6. LPS stimulation of H2126 cells does not affect promoter activity. Relative luciferase activity of promoter constructs unstimulated (NT) or stimulated with LPS ([X nM]) at 0, 3, 6, 24hrs in human lung adenocarcinoma H2126. Data was statistically analysed using Data was statistically analysed using Student's t-test on four independent experiments. P ≤ 0.05 were considered statistically significant while n.s = not significant. Error bars = SEM

Fig 7. DAKD stimulation of H2126 cells does not affect promoter activity. Relative luciferase activity of promoter constructs unstimulated (NT) or stimulated with DAKD (100nM and 1000nM) at 0, 3, 6, 24hrs in human lung adenocarcinoma, H2126. Data was statistically analysed using Student's t-test on four independent experiments. P ≤ 0.05 were considered statistically significant while n.s = not significant. Error bars = SEM

3.4.6 Transcription start sites of B1R. 5' RACE PCR was used to determine the transcription start site(s) of B1R in H2126. Based on published B1R sequence, an expected product size would have been approximately 450bp. Instead, at least 5 other distinct products were observed and these PCR products were cloned and sequenced in order to determine whether these unusual banding patters were due to PCR artefacts of if they were in fact due to the presence of alternative B1R transcripts in H2126.

A summary of sequence analysis of 20 clones revealed at least 9 different transcriptional start sites (TSS) in H2126. Surprisingly, 5 of the TSS originated from kinin B2 receptor (B2R), a gene located immediately upstream of B1R. In these transcripts, B2R is joined to B1R in a single transcript whereby exon III of B2R and exon I (or exon I and II) of B1R is skipped. Of the remaining 5 transcripts that revealed TSS from B1R, transcripts H and J demonstrated TSS corresponding to the published sequence by Yang and Polgar (1996). However, none corresponded to the TSS of the published B1R mRNA sequence (NM_000710.2). In addition to the alternative start sites, an alternative splice variant which skips exon II of B1R was detected.

600bp

500bp

400bp

300bp

Fig8. 5'RACE PCR analysis of H2126 cDNA reveals multiple products. H2126 cDNA was amplified using the GeneRacer 5'nested primer and RT Rev 2 primer. Expected product size was at 450bp although at least 5 other bands were observed.Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control

E1

E2

E3

+42

E1

E2

E2

E3

+47

E1

E2

E2

E3

+70

+47

E2

E1

E3

E1

E2

E2

E3

+42

E3

E1 11

E2

-12

E1 11

E2

E3

+30

E3

E1 11

E2

-7

E3

E1 11

-4

E3

E1 11

-12

E2

E1

E3

E1 11

E2

E3

ATG

ATG

B1R gene specific primers

Kinin B1 receptor

Kinin B2 receptor

Clones sequenced

Transcript

A

B

C

D

E

F

G

H

I

J

7

1

3

2

1

1

2

2

1

Fig9. Schematic representations of 5' transcription start sites identified in H2126 relative to for B1R (NM_000710) and B2R (NM_000623).

B2R Exon 1

B2R Exon 2

B1R Exon 2

B1R Exon 3

Fig10.

Partial alignment of a representative 5'RACE clone A to B1R (NM_000710) and B2R (NM_000623) reveals novel transcript of hybrid B2R-B1R mRNA. Results show that the sequences were homologous to exons I and II of B2R until the nucleotide indicated by the arrow. Nucleotides downstream the arrow was homologous to exons II and III of B1R. (Aligned using ClustalW)

3.5 DISCUSSION

In this study we have identified important interactions in two promoters of kinin B1 receptor. We found that at basal level the -1600bp region (upstream exon I) of promoter is preferred in 16HBE, and the -1000bp region (upstream exon III) is most active in H2126. When both promoter regions are in the same construct, the luciferase activity level is always lower than when the single most active promoter segment is used suggesting promoter-promoter competition. This study has shown that although both promoters demonstrate cell-type specificity, the alternative promoter also demonstrate regions that are commonly active in the cells. In addition, it is now known that the alternative promoter whether alone or in combination with the 5' flanking core promoter does not respond to stimulation by LPS and desArg10KD in 16HBE and H2126 cell lines. This study has confirmed the similar trend of B1R expression in other cell systems where stimulation of the alternative and core promoter does not respond to stimulation by LPS and DAKD indicating that other regions may be required. In addition, we have also identified alternative TSS of B1R as well as novel kinin receptor transcripts that may play an additional role in kinin receptor gene expression.

The basal cell-specific B1R promoter activity in pulmonary epithelial cells and is in agreement with data obtained from different cell systems including HepG2, rat vascular smooth muscle cells and bovine arterial endothelial cells (Ni et al 1998; Yang & Polgar 1996). Although different cell types were used, the preferential use of the B1R alternative promoter in cancer cell-derived HepG2 (Ni et al 1998) and H2126 from this study may indicate induction of promoter activity by certain factors present in cancerous cells. Evidence of utilizing alternative promoters is increasingly documented, particularly in cancer initiation and progression such as in MYC, CD133, LEF 1 and CYP19 (7-10) For example, CYP19 that encodes for aromatase cytochrome P450 was detected at a significantly higher level in breast adipose tissue of breast cancer patients and the transcripts present indicated a preference for promoter II-specific and exon I.3-specific whereas cancer-free individuals preferably uses promoter I.4 (10). In addition, a recent survey of mammalian promoters showed that human cancer related genes (n=2802) had an average of 2 promoters while the average for other genes was 1.5 (11). BASAL CELL SPEC PROMOTER ACTIVITY

At basal level, deletion constructs of the alternative promoter did not identify any regulatory element between -1020bp and -766bp in both our pulmonary cell lines. This differs from Chai et al (1996) who observed enhancer-like activity between -1842 to -812bp at basal level in HepG2 cell line. However, we did observe a NRE between -766 and -410bp that was active in both pulmonary cell lines. This NRE observation is in agreement with data from Chai et al (1996) who observed NRE between -800 and -380bp. It is not known why the PRE observed by Chai and colleagues (1996) was not seen in our study but there may be quite a few reasons for it. Firstly, the region analysed in their study was longer by approximately 820bp compared to our construct. This large region may include a PRE that is responsible for their observation. Secondly, the difference may be due to the difference of cells studied. It is not surprising to observe that cell-specific regions are active as B1R promoters are known to be active in different cell lines (Yang and Polgar 1996, Ni et al 1998).

Table2. Summary of results from deletion construct luciferase assay. The top table summarises the luciferase activity of alternative promoter deletion constructs in both cell lines while the bottom table summarises luciferase activity of deletion constructs of alternative promoter in presence of 1500bp of core promoter. (PRE=positive regulatory element, NRE= negative regulatory element)

Cell line/Promoter construct

(-1020 to -766bp)

(-766 to -410bp)

16HBE

-

NRE

H2126

-

NRE

Cell line/Promoter construct

CP + (-1020 to -766bp)

CP + (-766 to -410bp)

16HBE

-

-

H2126

NRE

PRE

In the context of promoter regulation, the transcriptional activity of a gene is subjected to combinatorial regulation (15). To further understand the consequence of promoter interaction, the core promoter was combined with the alternative promoter deletion constructs. A combination of core promoter with -1020 to -766bp of the alternative promoter increased activity in H2126 cells but did not alter luciferase activity in 16HBE suggesting that this region acts as a NRE only in H2126 cells and may be responsible for cell specific activity. Therefore, this NRE may only be active in cell lines where the alternative promoter is the preferred choice. In the presence of the core promoter, the region between -766 and -410bp had a positive effect on luciferase activity in only H2126 cells again suggesting cell specific region activity. The final removal of 410bp of the alternative promoter in H2126 further reduced luciferase activity as expected as the alternative promoter is the active promoter in H2126. The opposite was seen in 16HBE where luciferase activity was increased, most likely due to removal of promoter competition from the alternative promoter. With the addition of core promoter to the alternative promoter deletion constructs, two unexpected observations were made. Firstly, the NRE activity between -766 to -410bp in 16HBE was lost when the core promoter was added. The reason is not clear but perhaps promoter competition has come into play when a cell preferred (16HBE preference for core promoter) was added. Promoter competition has been observed within a gene as well as between genes in close proximity (13, 14)(Xie et al 2003). In the genome, promoter competition between some adjacent genes is prevented by a specialised sequence of DNA, known as insulator, to which proteins bind to and provide a barrier between the genes (15). However, in this instance, the promoter is within the gene and therefore it is highly possible that promoter competition occurred. The second interesting observation made was that the NRE observed in H2126 between -766 to -410bp became a PRE when the core promoter was added. It is possible that the presence of a weaker promoter (H2126 preference for alternative promoter) resulted in activity of the region to increase the strength of the promoter and therefore leaning the favour towards the alternative promoter. DELETION CONSTRUCTS SINGLE and COMBINATION

To determine whether the alternative promoter could be involved in B1R inducibility, 16HBE and H2126 cells were stimulated with LPS and DAKD. LPS stimulation did not affect the luciferase activity of any constructs (CP-Luc, AP-Luc, CP-AP-Luc) at 3hr, 6hr and 24 hr. The lack of response from LPS induction on the AP-Luc construct is in accordance to an early study from Ni et al (1998) where the authors observed that the LPS and IL-1β could not induce activity of 1900bp of alternative promoter in human HepG2 and rat VSMCs. The induction of the alternative promoter is still not known as the activity of the 1900bp construct mentioned above was not altered by LPS, TNF-α or IL-1β (6). In silico sequence analysis shows possible motifs for transcription factors such as SP-1, CAAT-box, AP-2 and NF-1 (35). This suggests that the alternative promoter might not be receptive to general stimulants such as LPS and IL-1β or that other multiple regions are required. However, the combined promoter constructs CP-AP-Luc in 16HBE and H2126 was also non-responsive to LPS stimulation. This lack of response could mean that 1) other regulatory elements outside of both promoters are responsible for B1R inducibility by LPS, 2) the LPS dose was insufficient to trigger a response and 3) the upregulation by LPS could not be captured using the time points 3, 6 and 24 hrs. Previous studies have shown that LPS stimulation for only 3hr was sufficient for a significant upregulation on B1R gene expression (12). Although previous studies have successfully used the dose and stimulation time used in this chapter to capture B1R upregulation, each cell type is different and is subjected to differential regulation, especially in immortalised cell lines. Participation of more than one regulatory element for LPS induction has been demonstrated in some studies. For example the maximal LPS induction of the TNF-α promoter is mediated by conjunctive action of at least two separate cis-acting regulatory elements (13). To determine if the promoters may respond to a more specific stimulus, DAKD was used. As mentioned in Chapter 1 DAKD is a natural B1R ligand that is able to stimulate receptor production in a positive feedback mechanism (14-16). A previous study has shown B1R upregulation post-DAKD stimulation at 100nM in IMR-90 cell line (15). In this study, 100nM and 1000nM of DAKD stimulation at 3, 6 and 24 hr post-transfection did not induce upregulation from basal level of any promoter constructs in both 16HBE and H2126. The lack of inducibility is again unsurprising as no consensus has been achieved from contrasting results from previous studies (indicate refs). The dose and the time course used in this study cover the expression of B1R mRNA expression by DAKD stimulation. This highlights the tight and delicate balance of which B1R is regulated at the promoter level and is an indication that other regions outside of core promoter, exon II and intron II of B1R may play a role in the upregulation of B1R by LPS and DAKD. In an attempt to locate the domains involved, Yang et al (2001) constructed a human B1R minigene that consist of 1.8kb of core promoter, exon I, 1.5kb of intron I, exon 2, intron 2 and luciferase gene. This minigene exhibited promoter activity with LPS and desArg10KD stimulation which was abolished with the replacement of minigene with 1.8kb 5' core promoter construct. Co-transfection of minigene with c-Jun also increased luciferase activity in a dose-dependent manner although the sequence of promoter responsible in unknown. This study shown that region/s outside the promoters may be necessary for induction in 16HBE and H2126. STIMULATION

Multiple factors determine promoter selection and activation. Fundamentally, regulation of cell-type or tissue-type specific gene expression is believed to be achieved by combinatorial control of factors including diverse sequence structure and composition of core promoter elements and also cell-type specific components of the core transcription machinery that recognise the diverse sequence (11, 17). For example, an octamer motif in B-cell specific B29 promoter provides binding sites for uB/LyFl that is critical for promoter expression in pre-B cells but not in B cells (18). In addition, local epigenetic regulation including DNA methylation, histone modification and chromatin remodelling (19-21) contributes to the diversity of gene expression. It is interesting to note that HepG2 (12) and H2126 from this study are both derived from cancerous cells and displayed a preference for the alternative promoter of B1R. Evidence of utilizing alternative promoters is increasingly documented, particularly in cancer initiation and progression such as in MYC, CD133, LEF 1 and CYP19 (7-10). For example, CYP19 that encodes for aromatase cytochrome P450 was detected at a significantly higher level in breast adipose tissue of breast cancer patients and the transcripts present indicated a preference for promoter II-specific and exon I.3-specific whereas cancer-free individuals preferably uses promoter I.4 (10). In addition, a recent survey of mammalian promoters showed that human cancer related genes (n=2802) had an average of 2 promoters while the average for other genes was 1.5 (11).

This study has identified regions in the alternative promoter that acts in a cell-specific manner but the trans-factor that recognises this sequence in still unknown. In silico analysis of B1R alternative promoter sequence identifies an array of putative elements for transcription factor binding (Fig 17). Of interest are the AP-1, p300 and TATA elements. AP-1 transcription factor is involved in a number of activities including cell proliferation, differentiation, transformation, apoptosis (22-24) and has been shown to be activated post-B1R agonist application (25-27). Two AP-1 sites in the core promoter of B1R were also shown to be responsive to c-Jun in IMR-90 cells (28). p300, also known as p300/CBP is a transcriptional activator involved in DNA-binding of up to hundreds of transcription factors including basal factors such as TATA box binding protein and TFIIB (29-31). The mechanism of transcriptional activation is due to the ability of p300/CBP to acetylate lysines at the amino end of histones and decondense the chromatin (32, 33). The TATA box binds to TATA box-binding protein (TBP), which is a part of the TFIID basal transcription machinery in eukaryotes (34, 35).

The selection and regulation of transcription start site is a complex process that is still not fully understood. The basic mechanism of selecting transcription start site is thought to involve cis- and trans- elements of enhancer and repressor that interact with the basal transcription machinery at the core promoter to influence transcription (36-40). Therefore, regulation of transcription start site reasonably involves DNA sequence elements such as TATA box which recruits TFIID to form the pre-initiation complex (38, 41, 42). However, accumulating data show that multiple factors contribute to the selection of the transcription start site including DNA methylation and presence of tissue-specific transcription factors (indicate ref).These factors ultimately contribute to the diversity of transcription start site usage as observed in a large-scale in vivo study that fine mapped transcriptional start sites of 276 human genes (43). In this study, multiple alternative TSS was observed although only two agreed with any published sequence (Yang and Polgar 1996) and suggests subtle differences in the use of TSS in different systems. It has been shown using large array studies that TSS of mammalian genes are not limited to one single site but can occur over multiple sites in a cluster and that the selection of TSS is highly dependent on tissue specificity (Kawaji et al 2006). In addition to multiple TSS, two other interesting observation/discoveries were made i) a splice variant of B1R with exon II skipping and ii) hybrid transcript consisting of partial sequences from B2R and B1R was detected . These transcripts could be amplified with consistency from different cell lines as well as using different gene-specific primers strongly suggest that the results from 5'RACE are true. The discovery of B1R splice variant and hybrid transcripts from two different genes is not only novel for kinin receptors but may also influence the regulation of kinn receptor gene expression and therefore merits further investigation. Further confirmation and studies of these discoveries will be followed on in Chapter 4 and 5, respectively. TRANSCRIPTION START SITE

In conclusion, this chapter has determined that in bronchial epithelial cells 16HBE the B1R core promoter is more active while in lung adenocarcinoma H2126 the alternative promoter is preferred. This chapter has also shown regions in the alternative promoter that contains general and cell-specific regulatory elements in pulmonary bronchial epithelial cells 16HBE and H2126 although further studies will be required to determine the trans-factors that regulate each region. In addition, it is now known that the alternative promoter either alone or in combination with the 5' flanking core promoter does not respond to stimulation by LPS and desArg10KD in pulmonary epithelial cell lines 16HBE and H2126 suggesting that element/s outside both promoters is necessary for induction. This work has also highlighted an importance of understanding promoter-promoter interplay within a gene that contains multiple promoters. CONCLUSION

Fig 17. In silico analysis of putative transcription factors that may bind to the alternative promoter.

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