Determination of Short and Long Tyrosine Hydroxylase Activity by Use of VP16 CREB, PGL3 Luc, and CMV Luc

Many pathways in organisms are regulated to induce a change in the expression of particular genes the organism needs to produce for different cellular functions. It is important to study the different levels and mechanisms of gene regulation for both its real world and laboratory applications.

There are a variety of factors that regulate gene expression and function on the level of transcription. One of these factors is cAMP response element-binding (CREB). CREB induces transcription through protein domains and mechanisms (Brindle et al., 1993). This transcription factor binds to sequences in the DNA to induce a response that can increase or decrease the transcription of particular genes. The cyclic AMP (cAMP) response element-binding protein (CREB) has been demonstrated to be a key mediator of cellular promoter response to cAMP (Muchardt et al., 1990). In previous studies phosphorylated CREB at serine-133 has been shown to be required for its activation by cAMP on the regulation level of transcription (Sabban, 1997).

The activity of tyrosine hydroxylase (TH) regulates the biosynthesis of catecholamines (dopamine, norepinephrine, and epinephrine) (Nagamoto-Combs et al., 1997) by catalyzing the conversion of L-tyrosine to DOPA (Jinkyu et al., 2000). TH activity can be regulated by short-term and medium to long-term regulation of gene expression (Fujisawa & Okuno, 2005). It is reported that TH is phosphorylated by protein kinases including a cyclic AMP-dependent protein kinase (PKA) that results in the activation of TH (Kumer et al., 1996; Fujusawa & Okuno, 2005). Several signaling pathways converge on the same promoter elements to active TH transcription (Sabban, 1997). Subsequently, the levels of cAMP can regulate TH transcription rate. For example, TH expression has been studied using PC12 cells where endogenous TH transcription is modulated by several different stimuli that include cAMP (Lewis-Tuffin, Quinn, Chikaraishi, 2003). PC12 is short-hand for the rat adrenal pheochromocytoma cell line.

In this experiment, gene expression in transfected cells were studied by linking a promoter sequence to a reporter gene (luciferase). Luciferase produces a yellow-green light through the reaction:

ATP (energy) + d-Luciferin (substrate) + O2 + luciferase (enzyme) ? Oxyluciferin + AMP + Pyrophosphate + CO2 + Light (photons) at 562
The amount of activity can be assessed via plasmids that contain promoters in which affect the levels of protein expression. Constitutively active promoters can result in an increase in protein expression. In a previous study by Xu, Zhang, and Kone (2004), CREB-VP16 strongly induced HKalpha(2) promoter-luciferase activity.

In this experiment, activity of both long and short TH with and without VP16 CREB was studied with the pGL3 luc (lacking a promoter) and CMV luc to observe long and short TH expression when introduced to cells containing one of the two vectors.

Experimental Procedures

Total protein was isolated from the PC12 cells to have a luciferase-containing lysate for the luc assay. Each well was washed multiple times with PBS and a total of 120 ul of lysis buffer per well was added. The lysates were allowed to spin in the centrifuge to pellet the cellular debris. The supernatant contained the protein lysate and was transferred to microfuge tubes. Six samples were used for the luciferase assay. The luciferase assay was conducted using a 96 well plate with 140 ul of assay buffer per well. The blank contained lysis buffer and the rest of the wells contained the lysate. The plate was read by the luminometer and KC junior software was used to collect the data.

To determine the overall protein concentration in each sample as well as the luciferase activity, a protein assay was conducted. This included both long and short TH with and without VP16 CREB, pGL3 luc, and CMV luc. The standards were set up in a clear 96 well plate in duplicate. The blank contained water and lysis buffer. Each well contained 9.5 ul od dH202, 10 ul of standard (0.5 ug, 1 ug, 2.5 ug, and 5 ug), and 0.5 ul of lysis buffer. The samples were set up in duplicate with 19.5 ul of dH2o and 0.5 ul of the sample. Bradford reagent was added to the wells. Measurements were taken on a spectrophotometer and the data was collected with KC junior software before exported to Microsoft? Excel.

Discussion

A standard curve represented in Figure 1 was constructed in order to find the appropriate protein concentrations of the all of the vectors and long/short TH involved in the experiment. The luminometer reached its maximum during the high sensitivity readings. The sensitivity was adjusted for the rest of the three transfections and the data is labeled accordingly in Figures 2 and 3. The pGL3 luc and CMV luc were used as baselines to compare the activity of the long and short TH with and with out the VP16- CREB. A two- fold increase was chosen to present the data in order to compare and contrast the average activity within the two different sensitivities.

The short TH promoter was more active than the long TH promoter under high sensitivity using the pGL3 luc vector (Figure 3). The two-fold increase of the long TH promoter was approximately 2053 average luc activity/ug of protein. The two-fold increase of the short TH promoter was approximately 3568 average luc activity/ug of protein (Figure 3). The long TH possibly has silencers in which the short TH does not have because it is shorter than the long TH. Transcriptional silencers are thought to be the reason of decrease in expression of the long TH compared to the short TH. In a study by Popescu and Tumer (2004), tobacco plants transformed with an hpRNA vector harboring the full-length RPL3B cDNA exhibited efficient silencing of both RPL3A and RPL3B genes studied and reduced L3 levels. L3 deficiency due to silencing led to a reduction in cell number. Although in plants, a similar mechanism of silencing for the TH gene causing a reduction in activity could explain the decreased expression in long TH.

The CMV luc vector contained a promoter from the cytomegalovirus that is constitutively active. This was used as a positive control (Figure 2). The long TH + VP16-CREB showed an increase in activity compared to the short TH + VP16-CREB. The long TH + V16-CREB average luc activity/ug of protein with a two fold increase was 2463 and 1540 for the short TH + V16-CREB. As hypothesized earlier, the long TH probably has silencers; however, there is a possibility that the long TH, due to its size being larger than the short TH, also has more CRE sites than the short TH. This difference can in turn cause an activation level that "overcomes" the silencers it has by showing an increase in activity compared to the short TH + VP16-CREB activity.

In a study by Lewis-Tuffin, Quinn, & Chikaraishi that examined TH gene expression from TH promoter mutants, cAMP CREB site mutants, and transcriptional interference with dominant negative transcription factors, induced transcription was regulated primarily by activity at the CRE (2003).
A CRE linked to an enhancerless TH promoter fully supported expression in the study by Lazaroff et al. (1995). CRE from its normal position that resulted in full activity indicated that the CRE is critical for TH transcription (Lazaroff et al., 1995). This finding can help put the results of the long TH + VP16-CREB in perspective because the long TH would show an increase in expression if it had more CRE sites.

Working with cells with plasma allows for the introduction of vectors, in a fairly simple method, and the introduction of proteins. This, in turn, enables the ability to express proteins or a form of them (dominate/negative) where the results can be analyzed and interpreted. The findings within this experiment help to illustrate that CREB is important in various levels of protein regulation especially transcription. The conclusion of the study by Reusch, Colton, and Klemm asserts that CREB acts as a primary regulator of adipogenesis and the authors suggest that CREB may play similar roles in other cells and tissues (2000). This further supports the idea presented that CREB is indeed a regulator and holds an important role within different cells.

The experiment should be repeated under the same sensitivities using the same grade of luciferase in order to increase and expand the ability to compare the results. Future research should include experiments that take the long TH + VP16-CREB and mutate CRE sites in order to see what the affect on expression is. The analysis of deletions and mutations in the upstream 2400-base pair region of the rat TH gene by using transient transfection demonstrated that CRE was essential for both cAMP-mediated induction and basal transcription of the TH gene (Kwang Soo et al., 1993). Another suggestion would be to try to manipulate the transcription factors and CRE-bind factors for the short and long TH in this experiment. Jinkyu et al. identified and characterized transcription factors involved in the recognition and activation of the CRE of the TH gene and they suggested that CRE-binding factors interact with the CRE to regulate transcriptional activity of TH in conjunction with CBP (2000). Their suggestion could be built upon using materials from the current experiment to further analyze TH expression.

References

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Popescu, S.C. and N.E. Tumer. 2004. Silencing of ribosomal protein L3 genes in N. tabacum reveals coordinate expression and significant alterations in plant growth, development and ribosome biogenesis. Plant J. 39, 29-44.

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Xu, X., Zhang, W., Kone, B.C. 2004. CREB trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene. Am J Physiol Cell Physiol. 287, C903-11.