TGF-β plays an indisputable role in the development of intimal hyperplasia. Levels of TGF-β increase dramatically following arterial injury, and blocking TGF-β via a number of mechanisms very significantly inhibits the arterial hyperplastic response. Intimal hyperplasia is associated with enhanced VSMC proliferation and migration, posing a conundrum since TGF-β has been shown in vitro
to be an inhibitor of both of these processes23
. Potentially resolving this discrepancy, our recent studies suggest that under circumstances present at the time of arterial injury (specifically in the presence of elevated levels of Smad3), TGF-β significantly enhances proliferation of VSMCs. We, in addition to a number of other investigators, have demonstrated that Smad3 is upregulated following arterial injury in animals.8,41,42,43
Moreover, we have demonstrated that the majority of Smad3 expressing cells also express proliferating marker PCNA.8
These findings have been made by our laboratory in human restenotic lesions as well.44
Thus, the findings of our experiments where we have overexpressed Smad3 both in vitro
and in vivo
, likely have physiological relevance in that a similar state is found following arterial injury. We postulate that enhancement of VSMC proliferation by TGF-β may be the primary mechanism through which this cytokine mediates intimal hyperplasia.
The purpose of the current study is to better understand the signaling mechanism through which TGF-β produces its proliferative effect. Here within we have demonstrated a signaling mechanism through which TGF-β activates ERK MAPK through a pathway involving Smad3. We have demonstrated a protein-protein interaction between Smad3 and activated ERK MAPK. Moreover, we have shown that blocking ERK MAPK decreases TGF-β/Smad3-induced VSMC proliferation. Our in vivo data demonstrate that in injured arteries overexpressing Smad3, there is increased expression of activated ERK MAPK as well as enhanced VSMC proliferation. Together, these data suggest a pathway that involves TGF-β/Smad3/ERK MAPK may have a significant role in the development of intimal hyperplasia in response to TGF-β.
In our initial exploration of the signaling pathways through which TGF-β mediates SMC proliferation, we studied the cyclin-dependent kinase inhibitor p27.8
Nuclear p27 acts as a potent cell cycle inhibitor whereas phosphorylation of p27 results in translocation of this protein out of the nucleus allowing the cell cycle to progress. We found that TGF-β and Smad3 were able to significantly enhance VSMC proliferation by promoting nuclear exportation of p27. Phosphorylation of p27 is a very downstream event in the cellular pathways that lead to proliferation. Thus in evaluating ERK MAPK, we searched for a connection between Smad3 and p27. MAPK has been found to down-regulate p27 expression, increase the degradation of p27, and enhance cytoplasmic sequestration of p27. The effect of all of these events is to eliminate p27’s nuclear inhibition of cell cycle proteins resulting in enhancement of proliferation. Thus, the identification of a role for ERK MAPK in TGF-β induced VSMC proliferation reveals ERK MAPK to be the likely connection between TGF-β/Smad3 and p27.
The role of ERK MAPK in cellular proliferation, specifically VSMC proliferation, is well established. ERK MAPK activation occurs in response to a variety of cytokines and growth factors leading to both the passage of cell cycle checkpoints as well as the activation of transcription factors related to cell proliferation.21,22
We and others have shown that in cultured VSMCs, ERK MAPK plays a critical role in both proliferation and migration.23,24
studies have demonstrated the significance of ERK MAPK in the development of intimal hyperplasia. Hu et al. showed that in a rat carotid balloon injury model there was increased expression of activated ERK MAPK peaking at 5 minutes with elevated levels sustained for up to 7 days post injury.25
Furthermore, extraluminal application of the chemical inhibitor to ERK MAPK PD98059, which was used in our experiments, has been shown to decrease the development of intimal hyperplasia.26
Whether ERK MAPK is the sole pathway through which TGF-β enhances VSMC proliferation is not clear. Recent studies from our laboratory suggest that other members of the MAPK family, specifically p38, may act as intermediates between TGF-β/Smad3 and VSMC proliferation. These studies reveal a completely distinct pathway from ERK MAPK for proliferation involving p38 as well as Akt (unpublished data). Thus it may well be that TGF-β produces its proliferative effect through a number of parallel pathways.
Our data reveal a pathway involving ERK MAPK activation that is downstream from Smad3. There are examples in other cell types where TGF-β has been found to modulate MAPK through a Smad-dependent pathway. Simeone et al. found in pancreatic acinar cells that TGF-β activates ERK MAPK through a pathway that involves Smad4. Moreover, Smad4 has been found to directly affect MAPK in other cell types.27,28
TGF-β is also known to signal through independent pathways. Smad-independent pathways utilized by TGF-β include JNK, PKA, PKC and PI3K/Akt. These pathways are thought to modulate the effects of TGF-β that require rapid activation. This is contrary to the Smad pathways that are typically involved in transcriptional regulation of genes.29
Accordingly, a relationship between TGF-β and ERK MAPK that is independent of Smads has been previously described in a number of cell types. In VSMCs TGF-β through a Smad-independent pathway can enhance production of collagen as well as biglycans.30,31
TGF-β, also in a Smad-independent manner can activate ERK MAPK in chondrocytes, epithelial cells, and hepatic stellate cells.32,33
Although the exact molecular mechanism by which TGF-β activates ERK MAPK in these cells has not been clearly defined, the Ras/Raf signaling pathway may act as an intermediate. Despite the existence of Smad independent signaling pathways however, our data fairly conclusively show that when vascular smooth muscle cell proliferation is the endpoint, TGF-β activated ERK MAPK through a Smad-dependent versus a Smad-independent pathway.
In another variation in the relationship between these two signaling proteins, ERK MAPK has been found to act upstream rather than downstream of the Smad proteins.34,35
Studies have shown that ERK MAPK, can enhance Smad activation and translocation into the nucleus.30,36
In VSMCs, for example, ERK MAPK has been shown to phosphorylate Smad2 at its linker region.30
A similar observation has been made in rat mesangial cells leading to an increase in collagen IV expression.37
There are examples of MAPK being upstream from other members of the TGF-β superfamily including bone morphogenetic proteins (BMP) in human umbilical vein endothelial cells (HUVECs).38,39
These data, along with our new observation that Smad3 can activate MAPK, suggests the potential for a positive feedback loop between these two proteins. This positive feedback loop, if it exists, might have the potential to greatly enhance TGF-β’s effect on VSMC proliferation.
Smads have been traditionally thought to influence cell function through gene regulation. In fact, Aoki et al. has demonstrated that in pancreatic stellate cells, TGF-β through Smad3 induces secretion of IL-1β which in turn signals through ERK MAPK to further enhance cell secretion.40
However, since stimulation of VSMCs with TGF-β results in phosphorylation of ERK MAPK as early as 15 minutes and since Smad3 is the intermediate, these findings raise the possibility that Smad3 may immediately and directly interact with ERK MAPK leading to its activation. Further supporting this hypothesis is the fact that our immunoprecipitation studies reveal a direct protein-protein interaction between Smad3 and ERK MAPK.
In conclusion, we demonstrate a mechanism in VSMCs by which TGF-β activates Smad3, which in turn activates ERK MAPK leading to VSMC proliferation. Moreover, our in vitro findings are supported by in vivo studies using a rat carotid injury model. Although the mechanism by which TGF-β enhances intimal hyperplasia has not been fully elucidated, TGF-β-induced VSMC proliferation through this signaling pathway is a likely contributor. Manipulation of TGF-β or the various components of its signaling pathway may prove useful in creating targets that allow inhibition of the devastating and ubiquitous process that leads to restenosis.