Initial studies described in this paper confirmed that CREB depletion in vascular SMCs elicits changes consistent with those observed in SMCs from pathologically remodeled arteries in vivo. Such changes include changes in the expression of SMC markers and contractile factors such as SM myosin, calponin, and fibronectin; increases in proliferation and proliferation-related factors such as cyclin D1; and increases in extracellular matrix production. The data support previous experiments showing that inhibition of CREB activity with dominant negative forms of CREB enhances SMC proliferation and migratory behavior (33
). These studies further establish CREB as a key regulator of the SMC phenotype.
Several reports have suggested that CREB is required for mitogen-induced SMC proliferation rather than acting as an inhibitor of growth (18
). These results differ from ours likely because they were obtained by studies in which cells were exposed to mitogenic stimulus for brief periods of time (minutes to hours), rather than the 48- to 72-h time periods we have used. We too observe rapid CREB phosphorylation when cells are treated with PDGF-BB for periods from 10 min to 24 h. However, the rapid and transient activation of CREB is likely a braking mechanism by which cells attempt to prevent uncontrolled proliferation. We would also argue that vascular remodeling is a protracted process, one in which SMCs are subjected to various mitogens and cytokines over a period of days to weeks or months. Thus, our experiments, which assess changes in CREB content and activity over several days, may more accurately reflect what happens to CREB and its impact on SMCs in the clinically relevant in vivo situation.
Differences between acute and chronic receptor-mediated intracellular signaling are common in many biological systems. Differences in gene expression following acute versus chronic exposure to ethanol (63
), antipsychotic drugs (2
), and various analgesics (45
) are well-known examples. Likewise, differential activation of type 1 and 2 tumor necrosis factor alpha receptors has been reported in acute versus chronic inflammatory conditions (26
). The PI3-kinase/Akt pathway in particular exhibits differential regulation in response to acute or chronic activation by a number of extracellular stimuli, including insulin, insulin-like growth factor-1, angiotensin II, and PDGF (1
). Kaplan-Albuquerque and colleagues (32
) have reported that agents which promote SMC differentiation, such as thrombin, produce transient activation of PI3-kinase/Akt and sustained activation of MAPK cascades, while agents that promote SMC proliferation, such as PDGF, elicit sustained activation of PI3-kinase/Akt and transient MAPK activation. Interestingly, chronic activation of Akt has been shown to diminish CREB levels in PC12 cells, although the mechanism was not determined (68
). Thus, there is substantial precedent in the literature to support a model in which sustained activation of PI3-kinase/Akt signaling by PDGF leads to decreased CREB levels in SMCs.
The mechanisms that link chronic Akt activation to CREB depletion are the focus of ongoing studies. Although Du and Montminy (12
) have reported that Akt can phosphorylate CREB at serine 119, we have been unable to replicate these results, and there is no evidence that Akt can phosphorylate CREB serines 103 and 107. These sites are substrates for CKII in vitro, but their phosphorylation by CKII in vivo has not been firmly established. Moreover, CKII is generally unregulated by extracellular stimuli and thus has constitutive kinase activity, making its participation in the depletion of CREB in response to PDGF unlikely. We have attempted to block PDGF-induced CREB loss in SMCs with pharmacological inhibitors of CKII with no success (unpublished data). Another possibility is that phosphorylation of CREB serines 103 and 107 is regulated by protein phosphatases. Taylor et al. (57
) have reported that these sites are recognized by protein phosphatase 1. However, this phosphatase is not known to be regulated by PI3-kinase or Akt. Thus, the mechanism(s) that couples Akt signaling to CREB nuclear export, ubiquitination, and proteasomal degradation remains unresolved but is the focus of ongoing studies in our laboratory.
Our data also show that SMC proliferation in response to PDGF-BB is mediated by a combination of ERK, JNK, and PI3-kinase/Akt signaling, while CREB loss is regulated by PI3-kinase/Akt alone. We envision a model in which concomitant activation of ERK, JNK (either via ERK or via MEK3/6), and PI3-kinase/Akt signaling pathways is required for SMC proliferation (Fig. ). Loss of any one pathway by pharmacological blockade is sufficient for the attenuation of SMC growth. PI3-kinase/Akt signaling contributes to proliferation via numerous mechanisms, at least one of which is the downregulation of CREB content. How then does depletion of CREB alone (via siRNA) induce SMC proliferation, which is governed by multiple signaling pathways? The answer may lie in the ability of other signaling pathways, such as ERK, to regulate CREB activity without causing CREB loss. These pathways have been implicated in regulating CREB activity in other cell types (65
). The coordinate control of CREB phosphorylation/activity and CREB content underscores the importance of CREB as a regulatory target and a central control element in the regulation of the SMC phenotype.
FIG. 10. Model of PDGF-induced SMC proliferation and CREB depletion. PDGF stimulates SMC proliferation via activation of ERK, JNK, and PI3-kinase signaling pathways. Inhibition of any one of these pathways blocks PDGF's mitogenic impact. PDGF-induced CREB loss (more ...)
The importance of PDGF, PI3-kinase/Akt, and CREB in pulmonary vascular biology is underscored by recent reports showing that blockade of PDGF receptor signaling with imatinib mesylate (Gleevec) blocks the development of pulmonary hypertension and attenuates pulmonary arterial remodeling in rodents and humans (20
). In recent studies, we tested the abilities of the PI3-kinase inhibitor LY294002 and the Akt inhibitor triciribine to prevent pulmonary artery remodeling in a rat model of chronic hypoxic pulmonary hypertension. We found that these agents effectively block pulmonary artery remodeling, inhibit SMC proliferation, and prevent loss of CREB in arterial SMCs (unpublished data). Studies to measure pulmonary arterial remodeling and the development of pulmonary hypertension in mice overexpressing CREB or in which CREB is diminished in vascular SMCs are under way. These studies should confirm the role of CREB in maintaining normal vascular structure.
Our results are also the first to demonstrate that both ubiquitination/degradation and nuclear export are involved in the regulation of CREB content in SMCs (Fig. ). Ubiquitination and degradation of CREB in epithelial cells have been previously reported to occur in response to hypoxia (57
). The loss of CREB in these studies required phosphorylation of CREB at putative CKII sites, since phosphorylated but not unphosphorylated decoy peptides corresponding to these sites could block hypoxia-induced CREB depletion. In our studies, mutation of these same sites also blocked PDGF-induced CREB depletion. Stevenson and colleagues (55
) reported nuclear translocation of CREB in SMCs in response to membrane depolarization or PDGF-BB. These studies were conducted over a period of only a few hours, and the ability of PDGF to stimulate nuclear export over longer time periods was not examined. The rapid nuclear import of CREB in response to PDGF or membrane depolarization was contemporaneous with CREB phosphorylation, although it remains unclear whether phosphorylation of CREB was required for nuclear translocation. Our observations support these previous studies and underscore the importance of regulatory mechanisms other than direct phosphorylation/dephosphorylation in the regulation of CREB activity. They also highlight the numerous mechanisms that can target a single factor to bring about a particular response.
PDGF induces leptomycin-sensitive nuclear export and lactacystin-sensitive degradation of CREB in PA SMCs. Large oval, cell membrane; smaller circle, nuclear membrane; small, solid circles, CREB.
The PI3-kinase/Akt signaling pathway and its downstream effector, GSK-3, have been shown to regulate the intracellular location and degradation of a number of other factors. For example, PI3-kinase/Akt signaling leads to the self-ubiquitination of the ubiquitin ligase Mdm2 (16
). PI3-kinase/Akt inhibition of GSK-3 results in the stabilization of hypoxia-inducible factor 1α (38
) but increases proteasomal degradation of the basic helix-loop-helix protein p8 (21
). In other studies, Akt has been shown to promote nuclear exclusion of the forkhead transcription factor FKHR1 (4
) and block nuclear import of another forkhead protein, AFX (7
). Beals and colleagues (3
) have shown that GSK-3 stimulates nuclear export of the transcription factor NFATc. The concomitant ubiquitination and nuclear export of CREB more closely resemble the effect of the ubiquitin ligase Mdm2 on p53. Here, activation of Mdm2 leads to C-terminal ubiquitination of p53 (36
), which in turn reveals a nuclear export sequence in p53 (24
). As noted earlier, Mdm2 is a target for the PI3-kinase/Akt pathway and perhaps could function as a CREB ubiquitin ligase. Whether Mdm2 ubiquitinates CREB and whether ubiquitination of CREB alters its conformation to reveal a nuclear export signal will be the focus of future studies.
One issue left unresolved by these studies is whether proteasomal degradation of CREB occurs in the nucleus, in the cytosol, or in both locations and whether nuclear export occurs prior to or simultaneously with degradation. Both leptomycin B (nuclear export blockade) and lactacystin (proteasome inhibition) individually restore nuclear CREB content in PDGF-treated cells. If degradation occurs in the nucleus, then leptomycin B should have no effect on CREB loss, while lactacystin should block nuclear degradation. Since leptomycin B alone restores nuclear CREB levels, proteasomal degradation in the nucleus is unlikely. A second possibility is that CREB is exported from the nucleus and degraded in the cytosol. Here, leptomycin B would maintain nuclear CREB levels by preventing export and degradation. However, lactacystin would be expected to protect CREB from degradation, but CREB would accumulate in the cytosol, since nuclear export would be unaffected. Since significant levels of CREB were never detected in the cytosol by immunocytochemistry or by Western blotting in our studies, CREB must be reimported into the nucleus when it cannot be degraded. Stevenson and colleagues (55
) have reported the Ran-dependent nuclear import of CREB in vascular SMCs, making this the most parsimonious explanation of our results.
In conclusion, depletion of CREB in PA SMCs produces phenotypic changes consistent with changes observed in pathologically remodeled vessel walls in vivo. PDGF-BB, a mitogen released in response to vascular damage and during vascular remodeling, leads to a depletion of CREB in SMCs. This depletion is mediated by PI3-kinase/Akt signaling but does not involve GSK-3 or mTOR. CREB loss in response to PDGF is not due to changes in CREB gene transcription but rather to nuclear export and proteasomal degradation. The loss of CREB with PDGF treatment appears to be dependent on CREB phosphorylation at putative CKII sites.