The actin associated protein, palladin, is a critical structural component of the actin cytoskeleton, and functions as a molecular scaffold interacting with multiple proteins in the actin cytoskeleton 
. Palladin exists as multiple isoforms, and the expression of different isoforms is regulated in a tissue specific manner, suggesting that different palladin variants may be specialized for different functions. Cells lacking palladin have disrupted actin organization and palladin null mice display defective neural tube and ventral closure leading to embryonic death 
. Although it is clear that palladin is required for proper embryonic development, the role of palladin in the development and function of the vasculature or its mechanism of action in SMC is unknown. In the present study, we provide evidence suggesting that palladin plays an important role in the early stage of SMC differentiation. We found that palladin induced the expression of SMC marker genes in vitro
using a SMC differentiation model; that over expression of palladin induced, while down regulation suppressed the expression of SMC marker genes; that palladin activated SMC promoter transcriptional activity through CArG elements by direct or indirect binding to the promoters of SMA or SM22; that palladin translocates and interacts with MRTFs in the nucleus; and that the transcriptional activities of palladin and MRTFs were inter-dependent. Importantly, in palladin deficient mice, the induction of the SM markers, SMA and SM22 was significantly attenuated at E 11.5 in whole embryos and in isolated blood vessels.
The expression of so-called SMC specific markers, such as SMA, SM22 and SM-MHC, is controlled by multiple transcription factors such as SRF and myocardin. Myocardin is exclusively expressed in SMCs and cardiomyocytes and potently induces the transcription of CArG containing SMC differentiation marker genes in the presence of SRF 
. However myocardin does not appear to function in the early stage of induction of SMC differentiation genes, as myocardin null embryonic stem cells were able to differentiate into SMCs in the context of a chimeric knockout mouse 
, and the expression of myocardin unlike palladin was low in the early stages of development of vascular SMCs 
. These studies suggest that there may be alternative molecular mechanisms that contribute to the initiation of SMC differentiation. Palladin presents as early as E8.5 in mice embryos 
and it is most highly expressed at 48 h following RA treatment in A404 () and at 20 days in EB SMC differentiation models
. This precedes the induction of SMC marker genes. The induction of SMC marker transcriptional activity was most significant in undifferentiated A404 cells, whereas the enhancement is moderate in EB derived SMCs and adult aortic SMCs (A404>>>APSCs>
R518). In undifferentiated A404 cells, over-expression of palladin can greatly increase SMA and SM22 promoter activity 600 and 20 fold respectively, while in differentiated SMCs APSC and R518 cells the enhancement is much less. These results suggest that palladin may be a possible candidate in regulating early stage SMC differentiation. One possible mechanism for palladin regulation of expression of SMC marker genes is through MRTFs which are widely expressed, known to be important in regulating expression of SMC specific marker genes, and dependent on the Rho family GTPases and actin dynamics. Palladin's ability to increase the F:G actin would free MRTF from cytosolic G actin. In the luciferase reporter assays, the magnitude of the palladin-enhanced expression of SMC marker genes was markedly attenuated by down regulation of MRTFs and/or myocardin in cultured cells (). This attenuation was greater when both MRTFs and myocardin were down regulated than when individually reduced. This suggests that palladin may function via a SRF-myocardin-MRTF pathway. By using pull down assays, we showed that the C-terminus of palladin directly interacts with the C-termini of MRTFs, which are important for transcriptional activation. This interaction was also confirmed by over-expression experiments that showed that the C-terminus of palladin co-localizes with MRTF-A in the nucleus of rat aortic SMCs and differentiated A404 cells. Palladin binding to MRTFs in the nucleus may promote chromatin remodeling and initiate the MRTF-SRF transcriptional activation of SMCs marker genes. In the reporter and over expression assays, both N-terminal and C-terminal halves of palladin are required for the expression of SMC genes. This plus the fact that the C-terminus of palladin localizes in the nucleus while the N-terminus localizes in the cytoplasm along stress fibers, suggests that nuclear and cytoplasmic distribution of palladin are both necessary for palladin's function. Whether palladin interacts with MRTFs in the cytoplasm and then translocates to nucleus, or they interact in the nucleus is not known.
Another possible mechanism is that palladin can directly interact with the promoters of SMC marker genes and initiate transcription of SMC marker genes. We showed that mutation of CArG elements in the SMC gene promoters significantly decreased the responses to palladin (). In addition, we found that palladin can bind to the SMA promoter within intact chromatin by ChIP assays (). Thus, it is also possible that palladin can shuttle to the nucleus and directly bind to SMC gene promoters to modulate chromatin structure and regulate transcription of SMC marker genes. We detected endogenous palladin in the nuclei of SMCs. We favor the hypothesis that palladin regulates SMC differentiation through both direct and indirect pathways. The possible mechanisms whereby palladin regulates SMC markers are illustrated in . On the one hand, palladin released from the cytoskeleton can translocate to the nucleus and regulate transcription either by binding directly to the promoters of SMC marker genes or by forming a complex with MRTFs and SRF to enhance transcription. In addition palladin's regulation of actin dynamics frees cytosolic MRTFs, which translocate to the nucleus to further enhance transcription of SMC marker genes.
Scheme showing possible mechanisms whereby palladin regulates SMC differentiation.
Palladin deficient mice die by day E15.5 and display multiple defects including defective cranial neural tube closure and fetal liver herniation indicating that palladin plays a critical role in embryonic development. The exact mechanism whereby palladin knockout leads to embryonic lethality remains unclear and the importance of palladin in the development of the vasculature is not clear. However, results of the present studies provide clear evidence that palladin plays an important role in SMC differentiation in in vitro model systems. Moreover, consistent with these results, palladin knockdown embryos E11.5 showed decreased expression of SMA and SM22 protein in the dorsal aorta by immunohistochemstry analysis and in whole embryos by Western blotting (). Finally, a dramatic reduction in SMA, SMA and SM MHC mRNA was found in isolated umbilical vessels at E11.5 (). However, as no visible changes were observed in either the pattern or size of the great vessels or the umbilical vessels, palladin does not appear to contribute to the mechanisms underlying those processes at least at the E11.5 time point. In view of the partial expression of SMA and SM22 in the palladin knockdown vessels, palladin is either not absolutely required for their expression or compensatory mechanisms have been turned on. The decreased expression of the SMC markers at the protein level, in the palladin null embryos is consistent with our earlier finding of decreased force development in palladin null SMCs isolated from EBs. This repressed contractility of the vasculature likely contributes to the palladin null embryonic lethality due to the weakened vessel walls. The death of the embryos at about E15.5 corresponds to the time when the coronary circulation is perfused as it connects to the aorta.
Taken together, our findings demonstrate that palladin plays a key role, through both direct and indirect pathways, in the induction of SMC marker genes during the early stages of SMC differentiation. Further studies are needed to determine mechanisms by which palladin promotes transcriptional activation through binding to SMC promoters, and what genes may compensate, at least in part, for its loss.