Figure 1.
the expression of palladin, SMA, and SM22 is induced during SMC differentiation.
A. Undifferentiated A404 cells were induced to differentiate into SMCs by treatment with RA (1 µmol/L). Cells were harvested at different time points, and mRNA was isolated. Palladin and SMC marker genes SMA, SM22, and smoothelin were quantified with real time RT-PCR, and normalized to ribosomal18s. Values represent the mean ±SEM of three independent experiments (n = 3). # p<0.01; * p<0.05 B. Representative Western blots show that SMA and palladin are induced at the protein level in A404 cells treated with RA. Undifferentiated subconfluent A404 cells were treated with RA, and cells were lysed with RIPA buffer at different time points. An equal amount of protein was loaded for SDS-PAGE, and blotted with anti-SMA, palladin, and GAPDH antibodies. The protein expression was normalized to GAPDH. Quantification of protein expression is showing in the bar graph. Values represent the mean ±SEM (n = 3). # p<0.01.
Figure 2.
SMA and SM22 genes are down or up regulated by silencing or overexpression of palladin.
A) Undifferentiated A404 cells were transfected with siRNA oligos and then treated with RA for 48 h. The expression of SMA, SM22, and palladin was determined by real time RT-PCR, and normalized to ribosomal 18s. Values represent the mean±SEM, n = 3. * p<0.05. Inset shows overexpressed palladin was knocked down with siRNA. B) Undifferentiated A404 cells were transfected with myc tagged palladin constructs, and cells were harvested 36 h after transfection. The mRNA expression of SMC marker genes was measured by real time RT-PCR using specific primers, and normalized to ribosomal18s. Values represent the mean±SEM (n = 3). #,p<0.01. C) Representative Western blots show that overexpression of full length, but not the amino (N) or carboxyl (C) terminal halves of palladin can induce SMA expression. Smoothelin expression was not changed. Undifferentiated A404 cells were transfected with myc tagged palladin constructs, and harvested 36 h after transfection. The cell lysates were loaded for SDS-PAGE, and blotted with anti-SMA, palladin, and GAPDH antibodies. The protein expression was normalized to GAPDH.
Figure 3.
Palladin induced SMC marker genes transcription is mediated in part through the CArG elements found within the SM22 and SMA promoters.
SM22 and SMA promoter-enhancer-luciferase plasmids were co-transfected with a palladin expression plasmid into undifferentiated A404 cells. Cells were lysed 36 h after transfection, and luciferase assays were performed and normalized to protein contents. A. Palladin significantly induces the transcriptional activity of wild type SMA and SM22. Mutation of CArG in SM22 or CArG A (A) and B (B) in the SMA promoter decreased the response to palladin (p<0.01 compared to wild type; n = 3). B) Palladin had no significant effect on the transcriptional activity of the CArG independent gene, ACLP, while KLF4 activity decreased (n = 3, p>0.05). C) Full length palladin, but neither amino (N) or carboxyl (C) termini of palladin, induced SMC gene transcriptional activity. Values represent the mean±SEM (n = 3). #, p<0.01.
Figure 4.
Chromatin immunoprecipitation assay showing that palladin associates with the SMA promoter in differentiated A404 cells and rat aortic SMCs.
A) Schematic structure showing the SMA promoter region amplified in ChIP assay. B) Undifferentiated A404 cells were differentiated into SMCs with RA. A404 cells were harvested 48 h after RA treatment, and chromatin IP was performed. Subconfluent R518 cells under normal culture condition were harvested and ChIP assays performed with SRF and palladin antibodies. The intensity of SMA promoter was quantified with real time PCR. No antibody was used as negative control. Values represent the mean±SEM (n = 3). *, p<0.05.
Figure 5.
Palladin induces SMC marker gene transcription through the myocardin-MRTF-SRF pathway.
A) down regulation of MRTFs and myocardin attenuates palladin induced SMA activity in A404 cells. siRNA to MRTFs (siMRTF-A and siMRTF-B) and dominant negative myocardin (DN Myo) constructs were cotransfected with SMA promoter luciferase constructs alone or with a palladin expressing plasmid into A404 cells, and luciferase activities were measured. Values represent the mean±SEM (n = 3). *, p<0.05; # p<0.01. B) SMA and SM22 promotor transcriptional activities were decreased in palladin null APSCs. Wild type and palladin null APSCs were transfected with SMA and SM22 luciferase constructs. Luciferase assays were performed 36 h after transfection. Values represent the mean±SEM (n = 3). *, p<0.05. C) palladin, myocardin and MRTFs interdependently regulate SMC marker genes. SMA promoter luciferase construct was cotransfected with MRTF-A, -B, Myocardin, or palladin expressing plasmids into wild type, palladin null, and myocardin null APSCs. Luciferase activities were measured. Values represent mean ±SEM of three independent experiments. *, p<0.05 compared to WT.
Figure 6.
Carboxyl terminus of palladin interacts with MRTFs in SMCs.
A) Full length, amino (N), or carboxyl (C) terminus of myc tagged palladin constructs were cotransfected with flag tagged MRTF-A or B into HEK-293 cells. Co-IP assays were performed, and proteins were detected with myc and flag antibodies. Note that different regions of the gels are shown for full length, and the N- and C-termini, which as expected, run differently, as shown in Fig. 2C. B) in vitro pull down assays showed that MRTF-A interacts with palladin directly. Recombinant GST tagged palladin was purified from E.Coli and MRTF-A and –B proteins were translated in vitro. C) MRTF-A interaction with palladin is not via the amino terminal RPEL domain. The two RPEL motifs (mut1: R33A and P34A and mut2: L39A and V40A) mutants and an amino terminal 100 aa deletion of MRTF-A (ΔN) were cotransfected with myc tagged palladin into HEK-293 cells, and co-IP was performed 36 h after transfection with anti-myc antibody and blotted with anti-myc or flag antibodies.
Figure 7.
Confocal images demonstrating nuclear localization of full length endogenous palladin and of C terminal but not N terminal palladin.
A) Endogenous palladin was detected in nuclei of mature SMCs (rat aortic SMCs). Cultured subconfluent R518 cells were fixed with cold methanol and stained with a palladin polyclonal antibody, and with Alexa fluor conjugated secondary anti-rabbit antibody. Inset shows the Western blot results of palladin in a nuclear fraction. B) Carboxyl (C) terminus of palladin co-localizes with MRTF-A in the nucleus. Rat aortic SMCs were transfected with myc tagged C-terminus of palladin. The C-terminus of palladin was detected with an anti-myc epitope antibody, and the endogenous MRTF-A was detected with an anti-MRTF antibody and secondary Alexa fluor conjugated anti-rabbit antibody. C) Expressed carboxyl (C) terminus but not amino (N) terminus of palladin localizes in the nucleus of R518 cells. Myc tagged N- or C- terminal palladin constructs were transfected into cultured R518 cells by electroporation. Cells were fixed after 72 h transfection, and proteins were detected with myc antibody. Secondary antibody labeling alone, under identical conditions showed no detectable fluorescence. Scale bar, 10 µm. D) endogenous palladin was accumulated in the nucleus by leptomycin B (LMB). Cultured subconfluent R518 cells were trypsinized and subjected to nucleus-cytosolic fractionation. Equal amounts of protein were loaded for Western bloting. Leptomycin B 20 and 50 nM increased the palladin expression level in the nucleus fraction. Tubulin was used as a marker for the cytosolic fraction.
Figure 8.
SMA and SM22 expression was attenuated in homogenates of palladin E11.5 knockdown embryos, in isolated umbilical vessels and in dorsal aortae.
A) Representative images show that the expression of SMA and SM22 was attenuated in dorsal aortae at E11.5 embryos by immunohistochemistry analysis. Embryos were fixed and embedded for sectioning. Frontal sections were stained with palladin, SMA, and SM22 antibodies and corresponding secondary antibodies conjugated with biotin. The signals were developed with DAB and photographed on a Axioskop 2 Zeiss microscope. Scale bar, 50 µm. B) Western blots showing a significant decrease in the protein expression of SMA and SM22 in homogenates of whole embryos with its quantification shown in C. The whole embryos were dissected genotyped, and homogenized in RIPA buffer. An equal amount of protein was loaded for SDS-PAGE, and blotted with SMA, SM22, palladin, SMC specific α-actinin, and GAPDH antibodies. The signals were normalized to GAPDH. Values represent the mean±SEM (n = 3). *, p<0.05. D) The expression of SMA, SM22, and SM MHC mRNA is markedly decreased in umbilical vessels isolated from E11.5 palladin +/− and −/− mice compared to Wt. Umbilical vessels were dissected from E11.5 wt, het, and knockdown E11.5 embryos. RNA was extracted and SMA, SM22 and SM MHC quantitated by real time RT-PCR. Data was normalized to 18s. Values represent the mean±SEM (n = 3). *, p<0.05.
Figure 9.
Scheme showing possible mechanisms whereby palladin regulates SMC differentiation.
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 or their associated proteins. In addition palladin's regulation of actin dynamics frees cytosolic MRTFs, from G actin which translocate to the nucleus to further enhance transcription of SMC marker genes.