Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression
2005; Elsevier BV; Volume: 167; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61236-9
ISSN1525-2191
AutoresLaure Favot, Susan Hall, Sheila G. Haworth, Paul R. Kemp,
Tópico(s)Congenital heart defects research
ResumoImmediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of γ-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized γ-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-α expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension. Immediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of γ-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized γ-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-α expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension. During normal vascular development undifferentiated mesenchymal cells are recruited to an endothelial tube where they differentiate into vascular smooth muscle cells (VSMCs) in response to various stimuli.1Owens GK Regulation of differentiation of vascular smooth-muscle cells.Physiol Rev. 1995; 75: 487-517Crossref PubMed Scopus (1404) Google Scholar Once this process has been completed, the VSMCs in most vessels remain fully differentiated in the absence of pathological stimuli. This is not so, however, in the pulmonary circulation in which pulmonary arterial pressure and resistance fall dramatically after birth as blood flow increases. The pulmonary arterial VSMCs nearest the lumen change shape rapidly and undergo transient dedifferentiation with actin depolymerization and cytoskeletal remodeling, accompanied by a burst of cell replication.2Hall SM Hislop AA Pierce CM Haworth SG Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation.Am J Respir Cell Mol Biol. 2000; 23: 194-203Crossref PubMed Scopus (140) Google Scholar These changes do not occur when there is maintenance of a high vascular resistance due to neonatal hypoxia.3Allen KM Haworth SG Impaired adaptation of pulmonary circulation to extrauterine life in newborn pigs exposed to hypoxia: an ultrastructural study.J Pathol. 1986; 150: 205-212Crossref PubMed Scopus (34) Google Scholar, 4Tulloh RM Hislop AA Boels PJ Deutsch J Haworth SG Chronic hypoxia inhibits postnatal maturation of porcine intrapulmonary artery relaxation.Am J Physiol. 1997; 272: H2436-H2445PubMed Google Scholar, 5Hall SM Gorenflo M Reader J Lawson D Haworth SG Neonatal pulmonary hypertension prevents reorganisation of the pulmonary arterial smooth muscle cytoskeleton after birth.J Anat. 2000; 196: 391-403Crossref PubMed Google Scholar There is an association between the level of expression of genes encoding cytoskeletal proteins and actin polymerization that is not limited to VSMCs. This association occurs in many cell types including other muscle cells and fibroblasts. Indeed, increased actin polymerization and the concomitant reduction of the monomeric, G-actin pool may be important contributors to muscle cell differentiation. Modulation of the activity of the transcription factor serum response factor (SRF) has been implicated in the modulation of gene expression by actin polymerization.6Miralles F Posern G Zaromytidou AI Treisman R Actin dynamics control SRF activity by regulation of its coactivator mal.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar, 8Sotiropoulos A Gineitis D Copeland J Treisman R Signal-regulated activation of serum response factor is mediated by changes in actin dynamics.Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar Actin-dependent changes in SRF activity are regulated by one co-activator (Mal) and one inhibitory transcription factor (yin yang 1, YY1). Miralles and colleagues6Miralles F Posern G Zaromytidou AI Treisman R Actin dynamics control SRF activity by regulation of its coactivator mal.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar showed that Mal acted as a SRF co-activator and was localized primarily in the cytoplasm of cells in which actin was depolymerized, but in the presence of F-actin it translocated to the nucleus and increased SRF activity at its binding site (the CArG box).6Miralles F Posern G Zaromytidou AI Treisman R Actin dynamics control SRF activity by regulation of its coactivator mal.Cell. 2003; 113: 329-342Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar We previously showed that in VSMCs and C2C12 muscle cells the activation of the SM22α promoter by increased actin polymerization required an intact SRF binding site and an intact inhibitory YY1 binding site.7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar Increased actin polymerization resulted in reduced YY1 DNA binding activity and increased SRF DNA binding in the same cells. Taken together these data imply a dual control mechanism for the regulation of SRF activity at CArG boxes present in the promoters of genes encoding cytoskeletal proteins by actin polymerization. YY1 is an important inhibitor of muscle cell differentiation and expression of muscle-specific genes, including smooth muscle-specific genes7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar, 9MacLellan WR Lee TC Schwartz RJ Schneider MD Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1.J Biol Chem. 1994; 269: 16754-16760Abstract Full Text PDF PubMed Google Scholar, 10Chen CY Schwartz RJ Competition between negative acting YY1 versus positive acting serum response factor and tinman homologue Nkx-2.5 regulates cardiac alpha-actin promoter activity.Mol Endocrinol. 1997; 11: 812-822PubMed Google Scholar, 11Martin KA Gualberto A Kolman MF Lowry J Walsh K A competitive mechanism of carg element regulation by YY1 and SRF: implications for assessment of Phox1/Mhox transcription factor interactions at CArG elements.DNA Cell Biol. 1997; 16: 653-661Crossref PubMed Scopus (25) Google Scholar, 12Liu T Wu J He F Evolution of cis-acting elements in 5′ flanking regions of vertebrate actin genes.J Mol Evol. 2000; 50: 22-30Crossref PubMed Scopus (17) Google Scholar that acts primarily through the inhibition of SRF activity at CArG boxes. Furthermore, the proteolysis of YY1 appears to be important in muscle cell differentiation.13Walowitz JL Bradley ME Chen S Lee T Proteolytic regulation of the zinc finger transcription factor YY1, a repressor of muscle-restricted gene expression.J Biol Chem. 1998; 273: 6656-6661Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar YY1 has also been shown to be an important activator of a number of genes associated with the cell cycle, including histone genes14Wu F Lee AS Yy1 as a regulator of replication-dependent hamster histone h3.2 promoter and an interactive partner of ap-2.J Biol Chem. 2001; 276: 28-34Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar and the c-myc gene.15Riggs KJ Saleque S Wong KK Merrell KT Lee JS Shi Y Calame K Yin-yang 1 activates the c-myc promoter.Mol Cell Biol. 1993; 13: 7487-7495Crossref PubMed Scopus (163) Google Scholar, 16Lee TC Zhang Y Schwartz RJ Bifunctional transcriptional properties of YY1 in regulating muscle actin and c-myc gene expression during myogenesis.Oncogene. 1994; 9: 1047-1052PubMed Google Scholar This dual role of YY1 as an inhibitor of muscle cell differentiation and activator of proliferation makes it a good candidate regulator of the changes in pulmonary VSMC phenotype observed during early postnatal life. We therefore determined the temporal and spatial expression of YY1 in the pulmonary arteries of normal and pulmonary hypertensive piglets by immunohistochemistry, and studied the localization of YY1 in cultured muscle cells. These experiments suggested that the localization of YY1 is regulated by the actin cytoskeleton. We therefore studied the effect of agents that alter actin polymerization on the localization of YY1 in muscle cells in vitro. We also determined the effect of YY1 and agents that modify the cytoskeleton on the c-myc promoter. These studies implicate YY1 in the remodeling of the pulmonary vasculature immediately after birth. To generate reporter vectors for the Myc promoter, a 540-bp fragment of the c-myc promoter (comprising bases 2081 to 2618 of HSMYCC) was amplified from human genomic DNA using the primers 5′-ATAAAG-CTTAGCAAAAGAAAATGGTATTCGCGCGTA-3′ and 5′-ATATCTAGAAAAGCCCCCTATTCGCTCCGGATCTC-3′. This fragment was cloned into pGEM-T Easy (Promega, Madison, WI), sequenced, and then subcloned into pCAT-basic and pGL3-basic plasmids (Promega). The expression vectors pCYY1 and pLIMK have been described previously.7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar The vector pEGFP-YY1 was generated by PCR amplification of EGFP from pCAGGS-EGFP using primers 5′-GGCAAAGAATTCCGCCACCA-3′ and 5′-GATATCC-TTGTACAGCTCGTCATGCCGTGAGTG-3′ and amplification of YY1 using primers 5′-GATATCGCCTCGGGCGA-CACCCTCTACATC-3′ and 5′-TCTAGATCACTGGTTGT-TTTGGCTTTAGCGTGTG-3′. The EGFP cDNA fragment was cloned into pGEM-T Easy, digested with EcoRI and EcoRV, and cloned into EcoRI/EcoRV-digested pCDNA3 to generate pCEGFP. The YY1 fragment was cloned into pCEGFP using EcoRV and XbaI to generate pEGFP-YY1. To make pIRES-YY1 the EGFP-YY1 sequence was removed from pEGFP-YY1 using BamHI and XbaI and was then cloned into SmaI/XbaI-digested pIRES-neo (Clontech, Palo Alto, CA). The cofilin and luciferase sequences were removed from pcDNA3-cofilin and pGL3 using XbaI/EcoRI and NcoI/EcoRI, respectively, and inserted into BamHI-digested pIRES-YY1 using blunt-end ligation. PCR was performed as described previously17Kemp PR Osbourn JK Grainger DJ Metcalfe JC Cloning and analysis of the promoter region of the rat SM22-alpha gene.Biochem J. 1995; 310: 1037-1043Crossref PubMed Scopus (41) Google Scholar using Hi Fidelity Taq polymerase (Roche, Indianapolis, IN). All restriction enzymes were obtained from NEB (Beverly, MA). PAC-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (10% v/v) and were subcultured 1:6 by trypsinization at confluence. C2C12 and P19 cells were cultured as previously described.18Martin K Cooper WN Metcalfe JC Kemp PR Murine BTEB3, a novel transcription factor that activates through GC rich boxes.Biochem J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar C2C12 cells were seeded into 24-well plates at a density of 1.8 × 104 cells/well. After 24 hours the cells were washed with serum-free DMEM and incubated with a mixture of 400 ng of DNA (prepared as indicated in the figure legends) complexed with 2 μl of lipofectamine (Invitrogen, Carlsbad, CA) in OptiMEM (Invitrogen). Cells were incubated with the lipid/DNA mixture for 5 hours before the medium was replaced with DMEM supplemented with 10% fetal calf serum. Forty-eight hours after transfection, Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay (Promega, Madison, WI). Twenty-four hours before transfection, P19 cells were seeded into six-well plates at a density of 2 × 105 cells/well. A 10-μl aliquot of lipofectin (Invitrogen) in 100 μl of OptiMEM (Invitrogen), prepared as described by the manufacturer, was mixed with a total of 2 μg of plasmid (containing 1 μg CAT vector, 0.5 μg pCMVβgal, and 0.5 μg comprised of 0.25 μg of pCDNA3 plus 0.25 μg of test vector or 0.25 μg of each of two test vectors as detailed in the figure legend) in 100 μl of OptiMEM and incubated at room temperature for 15 minutes. The cells were washed with serum-free α-MEM (Invitrogen) before the lipofectin/DNA mixture was added. After 18 hours the medium was changed to α-MEM supplemented with 10% (v/v) fetal calf serum. The cells were harvested 48 hours after transfection, lysed, and assayed for CAT activity and β-galactosidase activity as described previously.17Kemp PR Osbourn JK Grainger DJ Metcalfe JC Cloning and analysis of the promoter region of the rat SM22-alpha gene.Biochem J. 1995; 310: 1037-1043Crossref PubMed Scopus (41) Google Scholar, 18Martin K Cooper WN Metcalfe JC Kemp PR Murine BTEB3, a novel transcription factor that activates through GC rich boxes.Biochem J. 2000; 345: 529-533Crossref PubMed Scopus (34) Google Scholar The pulmonary vasculature of the newborn piglet resembles that of the newborn child and therefore has been used extensively as an experimental model of normal development.19Yam J Roberts RJ Oxygen-induced lung injury in the newborn piglet.Early Hum Dev. 1980; 4: 411-424Crossref PubMed Scopus (21) Google Scholar, 20Perreault T Baribeau J Characterization of endothelin receptors in newborn piglet lung.Am J Physiol. 1995; 268: L607-L614PubMed Google Scholar, 21McComb MA Spurlock ME Expression of stress proteins in porcine tissues: developmental changes and effect of immunological challenge.J Anim Sci. 1997; 75: 195-201Crossref PubMed Scopus (13) Google Scholar, 22Potter CF Dreshaj IA Haxhiu MA Stork EK Chatburn RL Martin RJ Effect of exogenous and endogenous nitric oxide on the airway and tissue components of lung resistance in the newborn piglet.Pediatr Res. 1997; 41: 886-891Crossref PubMed Scopus (44) Google Scholar, 23Levy M Tulloh RM Komai H Stuart-Smith K Haworth SG Maturation of the contractile response and its endothelial modulation in newborn porcine intrapulmonary arteries.Pediatr Res. 1995; 38: 25-29Crossref PubMed Scopus (41) Google Scholar In addition, exposing newborn piglets to chronic hypobaric hypoxia (50.8 kPa) results in maintenance of a high pulmonary vascular resistance and pulmonary hypertension.3Allen KM Haworth SG Impaired adaptation of pulmonary circulation to extrauterine life in newborn pigs exposed to hypoxia: an ultrastructural study.J Pathol. 1986; 150: 205-212Crossref PubMed Scopus (34) Google Scholar, 4Tulloh RM Hislop AA Boels PJ Deutsch J Haworth SG Chronic hypoxia inhibits postnatal maturation of porcine intrapulmonary artery relaxation.Am J Physiol. 1997; 272: H2436-H2445PubMed Google Scholar, 24Tulloh RM Hislop AA Haworth SG Role of NO in recovery from neonatal hypoxic pulmonary hypertension.Thorax. 1999; 54: 796-804Crossref PubMed Scopus (9) Google Scholar In the present study, normal Large White piglets were sacrificed within 5 minutes of birth and at 3, 6, or 14 days of age (n = 5 at each age). Other animals were exposed to chronic hypoxia from 3 days to 6 (6-day hypoxic) or 14 (14-day hypoxic) days of age (n = 4 for each time point) and sacrificed immediately after removal from the chamber. All piglets were sacrificed by an intraperitoneal injection of sodium pentobarbital (100 mg/kg). All animals received humane care in compliance with the British Home Office Regulations and the Principles of Laboratory Animal Care formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Blocks of tissue were taken from the mid-lung region of all animals and processed for histology, 5-μm sections were cut, and adjacent sections were stained with antibodies to YY1 (sc-281; Santa Cruz Biotechnology, Santa Cruz, CA) or γ-smooth muscle-specific actin (γ-SM actin) (Dr. J. Lessard, University of Cincinnati, Cincinnati, OH). After incubation with biotin-conjugated goat anti-mouse antibody, binding was visualized by diaminobenzidine (StrepABComplex; DakoCytomation, Ely, UK) before light counterstaining with Mayer's Hemalum. Nuclear staining with YY1 was assessed in duplicate sections without counterstaining. Control sections were incubated with 4% goat serum instead of primary antibody. C2C12 or PAC-1 cells were seeded into four-well Lab-Tek chamber slides (Nunc, Naperville, IL) at a cell density of 1.5 × 104 cells/well 24 hours before transfection. Cells were transfected with 470 ng of DNA (pYY1-luc or pYY1-cof) and with 2 μl of lipofectamine as described above and incubated overnight in DMEM supplemented with 10% fetal calf serum. The cells were treated with cytochalasin D (Calbiochem, Nottingham, UK) latrunculin B, or jasplakinolide (Molecular Probes, Eugene, OR) for 6 hours, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes, and stained with 0.5 μg/ml of phalloidin TRITC (Sigma-Aldrich, St. Louis, MO) and 300 nmol/L DAPI (Molecular Probes). Images were generated using an Olympus TX 70 inverted microscope coupled to an Ultraview LCI confocal imaging system (Perkin Elmer). C2C12 or PAC-1 cells were seeded into four-well Lab-Tek chamber slides (Nunc) at a cell density of 1.5 × 104 cells/well 24 hours before transfection. The cells were washed with serum-free DMEM and incubated with a mixture of 470 ng of DNA comprised of 350 ng EGFP-YY1-cofilin vector and 120 ng of pEF-FLAG actin expression plasmids (as indicated in the figure legends) complexed with 2 μl of lipofectamine (Invitrogen) in OptiMEM (Invitrogen), according to the manufacturer's instructions. Forty-eight hours after transfection the cells were fixed with 4% paraformaldehyde in PBS for 15 minutes and permeabilized for 10 minutes in 0.3% Triton X-100 in PBS. Cells were incubated for 30 minutes in PBS containing 1% bovine serum albumin, 1 hour with anti-M2 flag antibody (Sigma-Aldrich), and 1 hour with anti-rabbit-Cy3 antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) according to the manufacturer's instructions. Images were generated as described above. The actin and mutant actin expression plasmids were a gift from Prof. R. Treisman, Cancer Research UK London Research Institute, London, UK. The YY1-specific siRNA (siY1) used in this study has been described previously.25Kurisaki K Kurisaki A Valcourt U Terentiev AA Pardali K Ten Dijke P Heldin CH Ericsson J Moustakas A Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation.Mol Cell Biol. 2003; 23: 4494-4510Crossref PubMed Scopus (134) Google Scholar The control siRNA (siY2) was a double-stranded 20-mer designed in the YY1 noncoding sequence and corresponds to the nucleotide sequence: 5′-TTCCAAGTGTGCATATTGTA-3′. Both double-stranded RNAs were made by Dharmacon Research, Inc. (Lafayette, CO). The siRNA duplexes were transfected into C2C12 cells using lipofectamine as described below. For Western blot and proliferation studies, 18,000 C2C12 cells were seeded into each well of a 24-well plate and cultured to 60% confluency. Cells were transfected as described above using 2 μl of lipofectamine per well and 150 ng of siRNA. For RNA extraction, 5 × 105 cells were seeded into 100-mm Petri dishes and cultured to 60% confluency. Cells were transfected as described above using 32 μl of lipofectamine per dish and 4.6 μg of siRNA. Cells were harvested 48 hours after transfection and lysed for 5 minutes at 4°C in sample buffer (2% sodium dodecyl sulfate, 2 mmol/L ethylenediamine tetraacetic acid, 20% glycerol, and 100 mmol/L Tris, pH 7.5). The protein concentration was determined26Lowry OH Rosenbrough NJ Farr AL Randall RJ Protein measurement with the folin phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar and dithiothreitol (25 mmol/L) was added. Protein samples (30 μg) were denatured and solubilized by heating for 5 minutes at 95°C, electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes as described previously.7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar After transfection cells were allowed to proliferate for 48 hours, and cell number was determined by a colorimetric assay using the CellTiter 96 AQueous One Solution cell proliferation assay (Promega). Total RNA was extracted from C2C12 cells with the RNeasy kit (Qiagen, Valencia, CA). RNA concentration was quantified fluorimetrically using Ribogreen RNA-binding dye (Molecular Probes), according to the manufacturer's protocol. cDNA was synthesized from 250 ng of RNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. RNA from C2C12 cells was reverse-transcribed as described above. PCR was performed in a total volume of 25 μl with 12.5 μl of SYBR Green PCR master mix (Applied Biosystems, Foster City, CA), 2 μl of cDNA, and 2.5 μl of each primer (10 pmol/μl; forward: 5′-GCTGTGACCAAAAACGATGGA-3′; reverse: 5′-GGCTGTCTGTGAAGTCCCTCTTA-3′). To assay 18S rRNA levels, real-time PCR was performed using a VIC-labeled TaqMan probe and TaqMan real-time PCR chemistry using an 18S rRNA detection kit (Applied Biosystems, Foster City, CA). Reactions were performed in an ABI-Prism 7000 sequence detector (Applied Biosystems) in triplicate, initiated with hot-start by heating to 95°C for 10 minutes, and amplified using the following conditions: 95°C for 30 seconds and 60°C for 1 minute for 40 cycles. The final cycle was followed by an additional extension time of 10 minutes at 60°C. Analysis was performed after normalization of samples to their 18S rRNA expression levels as described previously.27Ellis PD Smith CW Kemp P Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice.J Biol Chem. 2004; 279: 36660-36669Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar In normal vessels γ-SM actin expression in the inner medial smooth muscle layers of the elastic arteries of 6-day-old piglets was reduced compared to the levels observed at birth (Figure 1). By day 14 both the distribution and staining intensity of γ-SM actin was similar to that seen at birth. In 6-day-old piglets exposed to chronic hypoxia from 3 days of age (3 days hypoxia) there was no reduction in γ-SM actin in inner medial layers, and staining was uniform across the media and of similar intensity to that seen at birth. In 14-day-old piglets exposed to chronic hypoxia from 3 days of age (11 days hypoxia), staining for γ-SM actin was uniform across the vessel wall and was consistently more intense than that seen in 14-day-old normal piglets. This increase in staining intensity is consistent with previous ultrastructural studies showing increased myofilament density in hypoxic animals.3Allen KM Haworth SG Impaired adaptation of pulmonary circulation to extrauterine life in newborn pigs exposed to hypoxia: an ultrastructural study.J Pathol. 1986; 150: 205-212Crossref PubMed Scopus (34) Google Scholar YY1 protein was expressed in the VSMCs of all pulmonary arteries of all normal and hypertensive piglets (Figure 1) but with spatial and temporal differences. In the elastic arteries of normal newborn, 6- and 14-day-old piglets YY1 was strongly and uniformly expressed in the nuclei of all of the VSMCs in all medial layers across the vessel wall (Figure 1), but expression in the VSMC cytoplasm varied with age. In newborns this staining was weak and uniformly expressed across all medial layers. At 6 days the intensity of cytoplasmic immunostaining for YY1 appeared less than at birth in the inner medial layers. By 14 days it had increased in all medial layers and was now particularly strong in the outer layers. In the elastic arteries of 6-day-old hypoxic animals, YY1 was uniformly expressed in the cytoplasm of all medial layers. Similarly, a uniform cytoplasmic staining for YY1 was observed in the elastic arteries of 14-day-old hypoxic piglets, but in these arteries there was a striking absence of immunostaining in many of the nuclei in the outer medial layers. In the smaller muscular pulmonary arteries, YY1 protein was present in both the nuclei and cytoplasm of all VSMCs in both normal and hypertensive animals (Figure 1). In all animals the bronchial SMCs showed strong and uniform cytoplasmic and nuclear expression of YY1 (Figure 1). Comparison of YY1 cytoplasmic staining with that of γ-SM actin, in adjacent sections, revealed close spatial similarities in the intensity of staining (Figure 1). Thus in newborn and 14-day-old piglets, both γ-SM actin and YY1 were uniformly expressed across the elastic media whereas at 6 days of staining of both was weaker in the inner media. In the hypoxic, hypertensive 6-day-old animals, there was no loss of either γ-SM actin or cytoplasmic YY1 staining in the inner medial layers. These experiments indicate an association between the expression of SM γ-actin and the presence of cytoplasmic YY1 in remodeling pulmonary VSM. C2C12 cells provide a readily manipulable muscle cell background in which changes in actin polymerization alter the activity of the promoters of genes that encode contractile proteins.7Ellis PD Martin KM Rickman C Metcalfe JC Kemp PR Increased actin polymerization reduces the inhibition of serum response factor activity by yin yang 1.Biochem J. 2002; 364: 547-554Crossref PubMed Scopus (30) Google Scholar To determine whether YY1 localization in muscle cells in vitro was restricted to the nucleus, C2C12 cells were immunostained for YY1 protein. Fluorescent staining for YY1 was readily detectable in both the nucleus and cytoplasm (Figure 2A). These data indicate that unlike many transcription factors, YY1 does not exist in the nucleus by default, suggesting that YY1 localization is regulated. To study the factors regulating the localization of YY1 in muscle cells in vitro we transfected C2C12 cells with an expression vector for an EGFP-YY1 fusion protein (pEGFP-YY1). Transfection of these cells with pEGFP-YY1 showed that like the native YY1 protein, the EGFP-YY1 fusion protein was distributed throughout the cell in both the nucleus and cytoplasm (data not shown). One possible mechanism regulating the localization of YY1 was thought to be via the actin cytoskeleton. Therefore, cells were co-transfected with pEGFP-YY1 and a cofilin expression vector. The primary function of cofilin is to facilitate actin depolymerization, making it an important regulator of the cytoskeleton, and to mediate nuclear localization of actin.28Pendleton A Pope B Weeds A Koffer A Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells.J Biol Chem. 2003; 278: 14394-14400Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar In these experiments all of the cytoplasmic EGFP-YY1 protein relocated to the nucleus in the majority of the EGFP-YY1-expressing cells (data not shown). However, it was not possible to know whether or not the cells in which EGFP-YY1 remained detectable in the cytoplasm had also been transfected with the cofilin expression vector. Therefore, we cloned the EGFP-YY1 fusion protein under the control of an IRES with either luciferase (pYY1-luc) or cofilin (pYY1-cof) on the same mRNA, transfected the C2C12 cells, and found that luciferase did not modify the localization of EGFP-YY1 compared to transfection with pEGFP-YY1 alone (Figure 2B). However, in the presence of cofilin, all of the cytoplasmic EGFP-YY1 became localized to the nucleus (Figure
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