1.2 Marco Teórico 1 La autoestima
1.2.1.2 Dimensiones de la autoestima
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Expressed Transcription Factor
Figure 4.4 - The effect of Oct-2 isoforms on the human iNOS promoter in ND7 cells
G raph to show the activity o f a 7.0K b p rom ote r region o f the hum an iN O S prom ote r (p H iN O S -7 .0 - Blue) and a 1.3Kb hum an iNOS p ro m o te r region (pH iN O S -3.0 - Y ellow ) in ND7 cells. P rom oter regions w e re linked to a luciferase re p o rte r gene and co -tra n sfe cte d w ith eithe r an em pty expression vector (pJ7) or pJ7 into w h ich a selected O ct-2 isoform cD N A has been cloned. A ctivities w e re d eterm ined by assays fo r the luciferase reporte r gene after 72 hours and equate d fo r plasm id uptake as described (see 2.7.4 and 2.7.6). Data are the result o f three separate transfection expe rim e nts and are presented as m ean ± SD.
4.2.5 - Investigation into the ability of octamer-like motif from the iNOS gene promoter to bind Oct-2
Characterisation of the iNOS gene promoter revealed an octamer-like DMA binding motif which has been shown to be protected by binding proteins during induction of iNOS gene expression in murine macrophages. The data presented above argues that all three tested isoforms of Oct-2 are able to activate iNOS gene expression. In order to determine the involvement of the octamer-like motif in this activation DNA mobility shift assay experiments were performed. The annealed primers Oct-Nos 1 and Oct-Nos 2, which contain the octamer-like motif from the
iNOS promoter, were radiolabelled and incubated with in vitro translated
Oct-2 proteins as described (see 2.9.3 and 2.9.4). For each of the three Oct-2 isoforms used three reactions were performed. Firstly, a binding reaction with no competitor species added. Secondly, a binding reaction
with a 1 0 0 fold excess of a specific competitor, un-radiolabelled annealed
Oct-Nos 1 and Oct-Nos 2. Thirdly, a binding reaction with a 100 fold excess of a non-specific competitor, the ORE binding site.
Figure 4.5 shows the weak binding ofjOct-2.1 (Lane 1), and the stronger binding of Oct-2.4 and Oct-2.5 (Lanes 4 and 7 respectively) to the octamer-like motif in the iNOS promoter. The binding of each isoform is
inhibited by specific competition (Lanes 2,5 and 8 ) but not by non-specific
competition (Lanes 3,6 and 9).
The specific binding shown for each of the Oct-2 isoforms is strong evidence that the octamer-like motif plays a role in the mediation of iNOS gene regulation by Oct-2. Mutation of the octamer and a repetition of the transient transfection experiments that indicated that Oct-2 does indeed activate iNOS gene expression may further assess the importance of this motif in iNOS regulation.
8
<== Bound i v t Oct-2
Figure 4.5 - The binding of Oct-2 isoforms to the iNOS octamer-like motif
Binding of in vitro translated Oct-2.1, Oct-2.4 and Oct-2.5 to annealed and
radiolabelled Oct-Nos 1 and Oct-Nos 2 primers (lanes 1,4 and 7 respectively). Specific competition against binding by 100 fold excess
unlabelled Oct-Nos 1 and Oct-Nos 2 (lanes 2,5 and 8 respectively). Non
specific competition against binding by 100 fold excess of unlabelled ORE binding site (lanes 3,6 and 9).
The octamer-like sequence (ATGCAAAA) found in the 1755bp luciferase reporter construct pGLH/H2 was changed by site directed mutagenesis to ATGCGCAA and provided by our collaborator, Dr. W.J. Murphy. This new
construct was named pGLOctMut. The mutation used has been
previously shown to abolish binding and activation of immunoglobulin genes in B-lymphocytes (Ballard and Bothwell, 1986).
4.2.6 - The role of the octamer-like motif in mediating the weak repression of the iNOS gene promoter by Oct-1
Before studies were made on the effects of changing the octamer-like motif on the activation of the iNOS gene by Oct-2, the repression of iNOS by Oct-1 was studied. Thus each iNOS promoter luciferase reporter construct (pGLH/H2 and pGLOct/Mut) was co-transfected into BHK and ND7 cells along with either the empty pJ7 expression vector or pJ7 into which the Oct-1 cDNAs had been cloned. Transiently transfected cells were left for 72 hours and luciferase activity was then assayed as described (see 2.7.6).
Figure 4.6A shows that the decrease in iNOS promoter activity in BHK cells caused by the Oct-1 transcription factor is not dependent upon the presence of a functional octamer-like motif. Figure 4.6B likewise shows
that in ND7 cells the functional octamer motif is not required. The
presence of other octamer binding proteins in ND7 cells may explain the weaker repression seen by Oct-1 on pGLH/H2 in ND7 cells when compared to that seen in BHK cells. Once the octamer motif is mutated, however, the binding of not only Oct-1 but also these other octamer binding proteins is inhibited. As a result the weak repression seen in ND7 cells becomes stronger more in line with that seen in BHK cells.
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Oct-1 0 Vector pGLH/H2 pGLOctMutiNOS Reporter Construct
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Q. O 0: 150 i= 100 o 50 0 - X . □ Oct-1 ^ Vector pGLH/H2 pGLOctMutINOS Reporter Construct
Figure 4.6 - The role of the octamer-like motif in mediating the repression of the iNOS gene promoter by Oct-1
Graphs to show the activity of a 1755bp region of the murine iNOS promoter (pGLH/H2 - Blue) and the same region in which the octamer-like
motif has been altered (pGLOctMut - Red) in (A) BHK cells and (B) ND7
cells. Promoter regions were linked to a luciferase reporter gene and co transfected with either an empty expression vector (pJ7) or pJ7 into which an Oct-1 cDNA had been cloned. Activities were determined as described (see 2.7.4 and 2.7.6). Data are the result of three separate transfection experiments and are presented as mean ± SD.
4.2.7 - The role of the octamer-like motif in mediating the activation of the murine iNOS gene promoter by Oct-2
Having demonstrated that selected Oct-2 isoforms are able to weakly activate the iNOS gene promoter (see 4.2.2 and 4.2.3) the role of the
octamer-like motif was investigated. Transient transfections were
performed in BHK cells using the pGLOctMut luciferase reporter construct and selected Oct-2 cDNA expression vectors. After 72 hours transient transfection the luciferase activity was assayed as described (see 2.7.6).
Figure 4.7A shows that the ability of all three tested Oct-2 isoforms to activate the iNOS promoter in BHK cells is dependent upon the presence of a functional octamer-like motif. The weak 1.5 fold activation originally attributed to Oct-2.1 is lessened to a non-significant level by the mutation
of the octamer-like motif (f=1.651, p=0.087). The initially stronger
activation by Oct-2.4 and Oct-2.5 respectively are totally abolished by the mutation and each isoform shows repression of iNOS gene transcription.
In order to investigate the role of a functional octamer-like element in iNOS regulation in ND7 cells, which express endogenous Oct-2, the same transient transfections were repeated in this cell line.
Figure 4.7B shows that the ability of all three tested Oct-2 isoforms to activate the iNOS promoter in ND7 cells is partially dependent upon the presence of a functional octamer-like motif. As previously described, the predominantly B-cell Oct-2.1 isoform causes an approximate four fold activation of the native murine iNOS promoter. The data presented here shows that this activation is almost cut in half by the disruption of the functional octamer-like motif. The weak activation effected by Oct-2.4 on the native promoter is lost. The insignificant activation mediated by Oct- 2.5 remains statistically insignificant when the octamer-like motif is altered (Oct-2.5 on pGLH/H2: f=0.985, p=0.190. Oct-2.5 on pGLOctMut: f=2.418,
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a (D oc X m PGLH/H2 Ü pGLOctMutOct-2.1 Oct-2.4 Oct-2.5 Vector
Expressed Transcription Factor
B < 2
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a Q) a: 500 400 300 200 100 0 □ pGLH/H2 0 pGLOctMut n .Oct-2.1 Oct-2.4 Oct-2.5 Vector
Expressed Transcription Factor
Figure 4.7 - The role of the octamer-like motif in mediating the activation of the iNOS promoter by Oct-2 isoforms
Graphs to show the activity of a 1755bp region of the murine iNOS promoter (pGLH/H2 - Blue) and the same region in which the octamer-like
motif has been altered (pGLOctMut - Red) in (A) BHK cells and (B) ND7
cells. Promoter regions were linked to a luciferase reporter gene and co transfected with either an empty expression vector (pJ7) or pJ7 into which
a selected Oct-2 isoform cDNA had been cloned. Activities were
determined as described (see 2.7.4 and 2.7.6). Data are the result of three separate transfection experiments and are presented as mean ± SD.
4.2.8 - Investigation into the expression of iNOS protein in the neuronally derived ND7 cell line
Using an antisense method Deans et al. (1996) produced a number of ND7 cell derived stable cell lines that expressed Oct-2 at reduced levels when compared to the wild type. Screening these cells with the nNOS antibody initially indicated raised levels of nNOS protein, an observation which was later confirmed by western blotting. An increase in mRNA levels was also detected in these cell lines by a PCR method. A direct role for Oct-2 in the regulation of nNOS expression in neuronal cells was proposed following transient transfection experiments which identified the 5.1 nNOS promoter to be regulated by Oct-2.
Once the regulation of nNOS by Oct-2 in neuronal cells had been determined the knockout cell lines were screened for both endothelial NOS and inducible NOS expression. Neither ecNOS nor iNOS protein was detected in these experiments (Z.C. Deans - unpublished observation). ecNOS has only been detected in the blood vessels of the developing brain and has also been detected at negligible levels in the
adult brain (Galea et al. 1995; Miyawaki et al. 1995). It is unlikely
therefore that ecNOS is expressed in the sensory neuronal ND7 cell line.
iNOS expression, however, has been detected in many cells including neuronal cells, both in the peripheral nervous system (Young et al. 1992) and in the central nervous system (Bredt et al. 1990). Since the ND7 cells line was derived from the fusion of rat dorsal root ganglia neurons with cells from the mouse neuroblastoma N18Tg2 expression of iNOS may be expected. Furthermore, the presence of neuroblastoma characteristics in ND7 cells may promote iNOS expression since iNOS has tumourigenic properties and has been detected in tumour cells (Pfeilschifter and Vosbeck, 1991). Nevertheless to date iNOS expression has not been detected in ND7 cells. However, ND7 cells may posses the ability to
In order to attempt to detect the expression of IN O S in N D 7 cells, cell cultures were exposed to a number of stimuli which have been shown to
induce iN O S expression in other cells. Thus 1 x 10® N D 7 cells were
exposed to the following stimuli by direct addition to the cell culture media.
(A) No stimuli. (B) lOOU/ml INF-y. (C) lOOU/ml IN F -y and 400}iM
deferoxamine. (D ) Hypoxia. (E) Hypoxia, 100 U/ml IN F -y and 400|aM
deferoxamine. Cells were harvested after 24 hours and levels of iN O S
protein were detected by western blotting, as described (see 2.8.3). A
titration of different concentrations of an iN O S protein positive control
(obtained from Transduction Laboratories) was also subjected to western
blot to ensure that the antibody used was able to detect iN O S .
Figure 4.8 shows that the iNOS antibody used is able to detect iNOS protein to levels as low as 0.31 ^g. However, none of the selected stimuli were able to activate iNOS expression in ND7 cells.
Performance of the iNOS western blot on treated ND7 cell extracts demonstrated that iNOS protein was not detectable in these cells. This may be because ND7 cells do not express iNOS in agreement with the findings of Z.C. Deans (unpublished observation). Alternatively the stimuli chosen in these experiments may not activate iNOS expression in these cells, perhaps because ND7 cells do not have cell surface receptors for stimuli such as LPS and INF-y. This is in keeping with the findings that
different cells require different specific stimuli to express iNOS. For
example murine macrophages express high levels of iNOS in response to lipopolysaccharide (LPS) and interferon-y (INF-y) (Stuehr and Marietta,
1985). Vascular smooth muscle cells respond to interleukin-1 (IL-1)
(Busse and Mulsch, 1990) whilst hepatocytes require simultaneous exposure to IL-1, INF-y, LPS and tumour necrosis factor (TNF-a) for high levels of iNOS expression (Curran et al, 1990; Geller et al. 1993b). Thus a specific combination of stimuli, only deducible by empirical methods, may be required to activate iNOS gene expression in the ND7 cell line.
M A B D
135KDa iNOS ^
97KDa
Figure 4.8 - Western Blot to show the ability of a range of stimuli to induce iNOS gene expression in ND7 cells.
Lanes 1 - 5 : Dilution series (0.31 i^g - 5pg) of iNOS positive control. Treatment of ND7 cells
as follows: A - No stimuli. B - lOOU/ml INF-y. C - 100U/ml INF-y and 400pM deferoxamine.