NF-κB and CREB are required for angiotensin II type 1 receptor upregulation in neurons.

Nuclear factor kappa B (NF-κB) and the Ets like gene-1 (Elk-1) are two transcription factors that have been previously established to contribute to the Angiotensin II mediated upregulation of Angiotensin II type 1 receptor (AT1R) in neurons. The cAMP response element binding protein (CREB) is another transcription factor that has also been implicated in AT1R gene transcription. The goal of the current study was to determine if NF-κB and CREB association was required for AT1R upregulation. We hypothesized that the transcription of the AT1R gene occurs via an orchestration of transcription factor interactions including NF-κB, CREB, and Elk-1. The synergistic role of CREB and NFκB in promoting AT1R gene expression was determined using siRNA-mediated silencing of CREB. Electrophorectic Mobility Shift Assay studies employing CREB and NF-κB demonstrated increased protein - DNA binding as a result of Ang II stimulation which was blunted by siRNA silencing of CREB. Upstream inhibition of p38 mitogen activated protein kinase (p38 MAPK) with SB203580 or inhibition of the calmodulin kinase (CAMK) pathway using KN-62 blunted changes in CREB and NF-κB expression. These findings suggest that Ang II may activate multiple signaling pathways involving p38 MAPK leading to the activation of NF-κB and CREB, which feed back to upregulate the AT1R gene. This study provides insight into the molecular mechanisms involving multiple transcription factor activation in a coordinated fashion which may be partially responsible for sympathoexcitation in clinical conditions associated with increased activation of the renin angiotensin system

CREB contributes to Ang II-mediated increases in AT₁R, NF-κB, active Elk-1, c-fos.

<p>Knockdown of CREB by siRNA in CATH.a cells. (A) RT-PCR and Western blotting (lower panel) showing efficient suppression of CREB mRNA in CATH.a cells. (B) Inhibitory effect of CREB siRNA on Ang II-induced p65 NF-κB activation. siRNA transfected cells were stimulated with Ang II (100 nM) for 1 hour. p65 NF-κB levels were detected by western blotting. (C) Effect of CREB siRNA on p65 NF-κB nuclear translocation. Cells were transfected with CREB siRNA and subjected to Ang II activation over 1 hour period. Following nuclear/cytoplasmic fractionation, the lysates were immunoblotted for p-CREB and p65 NF-κB levels by western blotting in both nuclear and cytoplasmic fractions. (D) CATH.a cells were transfected with anti-CREB siRNA followed by Ang II stimulation for 8 hours. The cells were then probed with specific antibody against pElk-1 and (E) p-cFos. (F) Cells were either mock transfected with vehicle or anti-CREB siRNA and subsequently stimulated with Ang II (100 nM). Following the specified time course, the cells were probed for AT₁R message (lower panel) and protein levels. Values are expressed as mean ± SEM. *p<0.05 vs control, #p<0.05 vs Ang II, †p<0.05 vs corresponding untreated nuclear fraction. n = 3–4 per treatment.</p

Ang II increases CREB and NF-κB activity in an AT1R and CREB-dependent manner.

<p>CATH.a neurons were transfected with CREB and NF-κB reporter constructs, consisting of a mixture of CREB and NF-κB - responsive luciferase constructs and a constitutively expressing Renilla construct. (A) NF-κB relative luciferase activity was quantified under several experimental conditions with Ang II treatment alone or pretreatment with losartan, anti-CREB siRNA or mock transfected cells. (B) CREB luciferase activity was measured under the similar experimental conditions. Values are expressed as mean ± SEM. *p<0.05. n = 3.</p

Ang II stimulates CREB and NF-κB binding to DNA.

<p>(A) CREB-DNA binding activity was determined under different treatment conditions using a CREB specific labeled probe. (Lane 1) No nuclear extract (negative control). (Lane 2) Ang II (100 nM). (Lane 3) Losartan (1 µM)+ Ang II (100 nM). (Lane 4) SB203580 (20 µM) + Ang II (100 nM) at 1 hour. (Lane 5) KN62 (10 µM)+ Ang II (100 nM). (Lane 6) SB203580 (20 µM) + Ang II (100 nM) at 8 hour. (Lane 7) Non-transfected cells (Ang II (100 nM). (Lane 8) Transfected cells (CREB siRNA)+ Ang II (100 nM). (<b>B</b>) To study the synergistic actions of CREB and NF-κB, we conducted EMSA reactions, using NF-κB specific labeled probes under different conditions. (Lane 1) Ang II (100 nM). (Lane 2) Losartan (1 µM)+ Ang II (100 nM). (Lane 3) Transfected cells (CREB siRNA)+ Ang II (100 nM). (Lane 4) Ang II (100 nM). (Lane 5) Transfected cells (CREB siRNA)+ Ang II (100 nM). (C) To confirm the specificity of the CREB-DNA binding bands we did competitive (Lane 1) and supershift assay (Lane 3) using anti CREB antibody, compared to normal CREB-DNA binding (Lane 2). (D) To confirm specificity of the NF-κB -DNA bands, we similarly performed competitive assay (Lane 3) and supershift assay using anti-p65 antibody (Lane 4). Normal binding and negative control using no nuclear lysate are also included (Lane 2 and Lane 1, respectively).</p

CREB and p65 NF-κB do not physically associate and CBP binds to both CREB and p65 NF-κB.

<p>All representative blots are in the following order: Lanes 1–4: immunoprecipitated cell lysate, no ligand, Ang II, Los, Ang II+Los; Lanes 5–8: Total lysate, no ligand, Ang II, Los, Ang II+Los. (<b>A</b>). CREB (C) and p65 NF-κB do not associate. As shown in the first four co-IP lanes, there is no detectable signal but total p65 NF-κB was still detected. (<b>B</b>). CBP and p65 NF-κB physically associate as shown in the first four lanes. (<b>C</b>). CBP and CREB physically associate.</p

CREB is phosphorylated at Ser 133 by Angiotensin II.

<p>(A) CATH.a cells were stimulated with Ang II (100 nM) for specified time periods as indicated and immunoblotted for total and phosphorylated CREB protein using specific antibodies for total CREB and Ser 133 phosphorylation. (B) Effect of Ang II stimulation on CBP protein kinetics in CATH.a cells. The cells were stimulated with Ang II over a specified time course and the CBP concentration in the cell lysates was determined by western blot. Representative blots shown. Values are expressed as mean ± SEM. *p<0.05 vs control. n = 3–4.</p

A proposed model of Ang II-mediated AT₁R upregulation.

<p>In response to an Ang II stimulus, NF-κB disengages from IkB and enters the nucleus to interact with the CBP and binds to the promoter region of Elk-1. In addition, CREB and CBP complex formation facilitates the interaction between p65 and the Elk-1 promoter. This sets up a downstream cascade mechanism involving the increased transcription of Elk-1 and the the c-Fos component of AP-1 eventually leading to the upregulation of AT1 receptor.</p