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Keywords:

  • Heterogeneous nuclear ribonucleoprotein A1;
  • Stem cells;
  • Smooth muscle cells;
  • Cell differentiation;
  • Serum response factor;
  • Myocyte-specific enhancer factor 2C

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

To investigate the functional involvements of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in smooth muscle cell (SMC) differentiation from stem cells, embryonic stem cells were cultivated on collagen IV-coated plates to allow for SMC differentiation. We found that hnRNPA1 gene and protein expression was upregulated significantly during differentiation and coexpressed with SMC differentiation markers in the stem cell-derived SMCs as well as embryonic SMCs of 12.5 days of mouse embryos. hnRNPA1 knockdown resulted in downregulation of smooth muscle markers and transcription factors, while enforced expression of hnRNPA1 enhanced the expression of these genes. Importantly, knockdown of hnRNPA1 also resulted in impairment of SMC differentiation in vivo. Moreover, we demonstrated that hnRNPA1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Acta2 and Tagln genes using luciferase and chromatin immunoprecipitation assays. We further demonstrated that the binding sites for serum response factor (SRF), a well-investigated SMC transcription factor, within the promoter region of the Acta2 and Tagln genes were responsible for hnRNPA1-mediated Acta2 and Tagln gene expression using in vitro site-specific mutagenesis and luciferase activity analyses. Finally, we also demonstrated that hnRNPA1 upregulated the expression of SRF, myocyte-specific enhancer factor 2c (MEF2c), and myocardin through transcriptional activation and direct binding to promoters of the SRF, MEF2c, and Myocd genes. Our findings demonstrated that hnRNPA1 plays a functional role in SMC differentiation from stem cells in vitro and in vivo. This indicates that hnRNPA1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine. STEM CELLS 2013;31:906–917


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Smooth muscle cells (SMCs) play important roles in embryonic development of various organs/tissues such as the cardiovascular system and hollow organs like the bladder, the uterus, and the gastrointestinal tract. They also play a critical role in the pathogenesis of cardiovascular diseases such as stroke and atherosclerosis. They are the predominant cell component in the neointima and atherosclerotic lesion and produce a large proportion of the extracellular matrix aiding lesion development. Traditionally, the source of neointimal SMCs was thought to be from local migration from the tunica media—the main smooth muscle layer of blood vessels. However, recent studies have shown that stem cells and vascular progenitor cells from the circulation or the vessel wall to be present in lesions and to have the capacity to differentiate into SMCs [1]. Moreover, in vitro differentiated SMCs from stem cells represent an unlimited source of SMCs which can be used to construct autologous human vessels in vitro to replace disease or injured vasculature [2]. Therefore, understanding the molecular mechanisms behind stem cell differentiation into SMCs is important, as this knowledge could potentially be used as treatments, both in the development of drug therapies based on SMC differentiation inhibition and in the repair of vascular damage. Although over the last decade significant advances have been made in understanding the molecular mechanisms of SMC differentiation from stem/progenitor cells by the researchers from other groups [3–9] and within our group [10–15], further research is required to address the precise factors that direct stem cell differentiation toward SMCs.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) belong to the RNA-binding protein family and play important roles in regulating gene expression at both transcriptional and post-transcriptional levels [16]. More than 20 hnRNPs family members have been identified and characterized in human. Among them, hnRNPA1, a nucleocytoplasmic shuttling protein that has been primarily suggested as a key player in mRNA metabolism and biogenesis [17], has been extensively studied. It has been reported that hnRNPA1 regulates mRNA biogenesis or gene expression by binding to nascent pre-mRNA in a sequence-specific manner [18] and promoting the annealing of cRNA strands [19, 20], nuclear export of mature mRNAs [21], and mRNA turnover [22], and translation [23–25]. It has also been shown that hnRNPA1 plays an important role in gene alternative splicing by regulating splice site selection [26–29], exon skipping, or inclusion [30, 31], and in telomere biogenesis/maintenance by directly binding to both single-stranded and structured telomeric DNA repeats [32, 33]. Recently it was reported that in addition to its traditional role in RNA biogenesis, hnRNPA1 could be a key transcriptional regulator of gene expression through direct binding to G-quadruplex and/or other specific DNA sequence, or interaction with other proteins [34–39]. Importantly, it has been reported that hnRNPA1 nucleocytoplasmic shuttling activity is also required for normal myelopoiesis and BCR/ABL leukemogenesis [40]. However, to date, there has been no report of the functional involvement of hnRNPA1 in SMC differentiation and embryonic smooth muscle development. In this study, we first identified an essential role of hnRNPA1 in SMC differentiation from embryonic stem cells (ESCs). We demonstrated that hnRNPA1 regulated the expression of SMC differentiation genes by directly binding to and activating its promoter activity of specific SMC genes such as Acta2 and Tagln and SMC-specific transcription factor genes such as serum response factor (SRF), myocyte-specific enhancer factor 2c (MEF2c), and Myocd. In addition, we further demonstrated that knockdown of hnRNPA1 in stem cells could significantly impair their differentiation capacity toward SMCs in vivo. Our findings highlight the importance of hnRNPA1 for SMC differentiation from stem cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

hnRNPA1 Gene Expression Plasmid, Reporter Gene Plasmids, and Related SRF Binding Site Mutants

Mouse full length hnRNPA1 gene was amplified by RT-PCR from day 3 differentiating ESCs with primer set as shown in Supporting Information Table S1 and cloned into Kpn I/Pst I sites of the pCMV5-HA expression vector, designated as pCMV5-HA-hnRNPA1. Mouse SRF, MEF2c, and Myocd gene promoter DNA were amplified by PCR from mouse genomic DNA with primer set as shown in Supporting Information Table S1 and cloned into Mlu I/Hind III sites of the pGL3-enhancer expression vector (Promega Southampton, UK, http://www.promega.com/b/uk/), designated as pGL3-SRF-Luc, pGL3-MEF2c-Luc, and pGL3-Myocd-Luc, respectively. The reporter vectors pGL3-Tagln-Luc and pGL3-Acta2-Luc were generated in our group as reported previously [41]. Using pGL3-Tagln-Luc and pGL3-cta2A-Luc plasmid as a template, we carried out site-directed mutagenesis to change the SRF binding element to an unrelevant DNA sequence, so the resultant plasmids will not carry the SRF binding site. All vectors were verified by DNA sequencing.

A detailed description of the rest of the materials and methods is provided in Supporting Information, including ESC culture and SMC differentiation, immunoblotting analysis, indirect immunofluorescent staining for cells or frozen sections, real-time quantitative PCR (RT-qPCR), nucleofection, small interfering RNA (siRNA) experiments, chromatin immunoprecipitation (CHIP) assay, transient transfection and luciferase assay, hnRNPA1 knockdown in ESCs and in vivo SMC differentiation, and coimmunoprecipitation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Nuclear Protein hnRNPA1 Is Upregulated During SMC Differentiation from ESCs

In our previous nuclear proteomics analysis (data not shown), we observed that the nuclear protein expression levels of hnRNPA1, one of the several upregulated nuclear proteins found during early SMC differentiation, were significantly increased in the differentiating ESCs. To further confirm this finding, ESCs were seeded on mouse collagen IV-coated flasks and cultured in SMC differentiation medium in the absence of leukemia inhibitory factor (LIF) for 3–7 days to allow SMC differentiation as described previously [10–15, 41, 42]. As shown in Figure 1A, gene expression levels of SMC differentiation-specific markers, smooth muscle α-actin (Acta2), and smooth muscle myosin heavy chain (SM-MHC) (Myh11), were significantly induced in our stem cell differentiation model. In parallel with SMC-specific gene inductions, the expression levels of hnRNPA1 were transiently elevated during SMC differentiation. The levels of hnRNPA1 expression peaked at day 3 and displayed a sustained signal until day 7 of SMC differentiation (Fig. 1B, 1C). However, no significant changes were observed for hnRNPA1 gene expression during endothelial cell differentiation from ESCs (unpublished data), suggesting that hnRNPA1 may play a role during ESC differentiation to SMCs.

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Figure 1. hnRNPA1 is upregulated during smooth muscle cell (SMC) differentiation. (A): SMC-specific genes were activated during stem cell differentiation toward SMCs. Undifferentiated embryonic stem cells (ESCs) were plated onto dishes coated with 5 μg/ml of collagen IV and cultured in differentiation medium to allow for SMC differentiation. Total RNA from undifferentiated ESCs (day 0) or differentiating ESCs at day 3 (d3), 5 (d5), and 7 (d7) was harvested and subjected to real-time quantitative PCR analysis with primers specific for Acta2 and Myh11. (B): hnRNPA1 mRNA was upregulated during SMC differentiation. (C): Nuclear protein from undifferentiated ESCs (day 0) or differentiating ESCs at the indicated time points were harvested and subjected to Western blot analysis with antibodies against hnRNPA1 and H4. The data presented here are representative images or mean ± SEM of three independent experiments. Significant difference from control, *, p < .05. Abbreviations: hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; H4, histone 4.

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Nuclear Protein hnRNPA1 Expression Pattern in ES-Derived SMCs and Embryonic and Adult SMCs

To detect the expression pattern of hnRNPA1 in the ES-derived SMCs, double immunofluorescence staining with antibodies against hnRNPA1 and the smooth-muscle-specific markers (smooth muscle alpha actin [SMαA] and SM-MHC) was performed on day 7 differentiated SMCs. As expected, hnRNPA1 expression was colocalized with the smooth muscle markers (Fig. 2A). Importantly, immunofluorescent staining analyses using confocal microscopy further confirmed the nuclear or perinuclear location of hnRNPA1 proteins in ES-derived SMCs (Fig. 2A). To investigate the involvement of hnRNPA1 during cardiovascular development, double (hnRNPA1 and SM-MHC) immunohistochemical stainings were performed in the cross-sections of mouse embryos (day 9.5 and day 12.5), heart, aorta, small intestines, and skeletal muscle. hnRNPA1 was expressed in some cell clusters which were negative for SMC marker SM-MHC in the day 9.5 of mouse embryos (Fig. 2B), while its expression level was more profound in cells transformed/differentiated into SM-MHC-positive cells (cells with double positive for hnRNPA1 and SM-MHC, white arrows) in the day 12.5 of mouse embryos (Fig. 2C). Importantly, hnRNPA1 was also expressed in aortic SMCs (Fig. 2D) and cardiomyocytes (Fig. 2E), but not or weakly if any, expressed in small intestines (Fig. 2F) and skeletal muscle tissues (Fig. 2G). Notably, hnRNPA1 was localized in the nuclear, perinuclear as well as cytoplasm in the embryonic and aortic SMCs, but mainly expressed in the cytoplasm of cardiac cells, which is consistent with its nucleocytoplasmic shuttling activity. The expression patterns of hnRNPA1 in ES-derived SMCs, mouse embryos, and various adult tissues such as heart, aorta, small intestines, and skeletal muscles strongly suggest that hnRNPA1 might play a role in SMC differentiation in vitro and cardiovascular development in vivo.

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Figure 2. Nuclear protein hnRNPA1 is expressed in differentiated SMCs, embryos, aorta, and heart, and colocalizes with SMC-specific markers. (A): Nuclear protein hnRNPA1 is colocalized with SMC-specific markers in differentiating ESCs. Double immunofluorescence staining was conducted on day 7 differentiated SMCs with antibodies against hnRNPA1 and SMαA or SM-MHC. (B–G): Sections prepared from day 9.5 (B) and 12.5 embryos (C), aorta (D), heart (E), small intestines (F), and skeletal muscle tissues (G) were subjected to double immunostaining of hnRNPA1 (red) together with SM-MHC (green). Nuclei were counter-stained with DAPI (blue). Red arrows in (B) indicate hnRNPA1-positive cell cluster. White arrows in (C) indicate double-positive cells for hnRNPA1 and SM-MHC. The data presented here are representative images of three independent experiments/tissues. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; SMC, smooth muscle cell; SM-MHC, smooth muscle myosin heavy chain.

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An Essential Role of hnRNPA1 in SMC Differentiation from ESCs

In order to determine the role of hnRNPA1 in SMC differentiation, the effects of endogenous hnRNPA1 knockdown on the expression of SMC-specific genes were examined. Knockdown of endogenous hnRNPA1 gene expression significantly decreased mRNA and protein expression of multiple SMC-specific differentiation genes (Acta2, Tagln, Cnn1, and Myh11) and transcription factors (SRF, Myocd, and MEF2c) (Fig. 3A). To further investigate whether hnRNPA1 gene activation can promote SMC differentiation, we generated hnRNPA1 expression plasmids. hnRNPA1 overexpression was conducted in differentiating ESCs using different amounts of hnRNPA1 plasmids. RT-qPCR and Western blot analyses revealed that enforced hnRNPA1 expression in differentiating ESCs significantly increased SMC gene expressions at both mRNA (Fig. 3B1, 3B2) and protein (Fig. 3B4) levels in a dose-dependent manner. Importantly, gene expression levels of SMC transcription factors were also dramatically increased in hnRNPA1 overexpressing cells (Fig. 3B3, 3B4). To quantify the effects of hnRNPA1 overexpression on SMC differentiation more accurately, flow cytometry analysis was conducted to detect SMαA and calponin expression in the cells overexpressing hnRNPA1. We observed that enforced hnRNPA1 expression in the day 5 differentiating cells induced more SMC differentiation from stem cells (81. 23% ± 5.33% vs. 41.55% ± 6.3% for SMαA expression; 73.9% ± 6.69% vs. 35.33% ± 5.2% for calponin expression, in the hnRNPA1 overexpressing and control cells, respectively). Taken together, the above findings strongly suggest that hnRNPA1 gene expression is essential for SMC differentiation from stem cells.

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Figure 3. hnRNPA1 is essential for smooth muscle cell (SMC) differentiation from embryonic stem cells (ESCs). (A): hnRNPA1 knockdown downregulated SMC-specific transcription factors and SMC markers. hnRNPA1-specific small interfering RNA (siRNA) and random siRNA control were transfected into day 3 differentiating ESCs; after an additional 2–3 days of culture, total RNA and protein were harvested and subjected to real-time quantitative PCR (RT-qPCR) (left panel) and Western blot analyses (right panel). (B): hnRNPA1 overexpression promotes SMC differentiation. Undifferentiated ESCs were nucleofected by nucleofector II with different amounts of hnRNPA1 expression plasmids phnRNPA1. Nucleofected cells were plated on dishes coated with 5 μg/ml of collagen IV and cultured for 3–4 days in differentiation medium. Total RNA and protein were harvested and subjected to RT-qPCR analysis for SMC gene expression (B1 and B2), transcription factors (B3), and Western blot analysis (B4). An appropriate amount of empty vector pCMV5 was included as plasmid amount compensation. Protein (40 μg) was loaded into each well, blotted, and probed with specific antibodies for hnRNPA1, SMαA, SRF, MEF2c, and SM-MHC. α-Tubulin was included as internal control. *, p < .05 versus control. The data presented here were representative images or mean ± SEM of three independent experiments. *, p < .05 versus control. Numbers under the hnRNPA1 bands in the panel B4 indicate the average relative protein expression levels of hnRNPA1 from three independent experiments. Abbreviations: hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; MEF2c, myocyte-specific enhancer factor 2c; SM-MHC, smooth muscle myosin heavy chain; SRF, serum response factor.

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To further test whether hnRNPA1 gene regulation is specific for SMC differentiation in our system, hnRNPA1 gene overexpression and knockdown experiments in differentiating ESCs were performed. The chosen cell lineage markers based on our previous findings as they displayed upregulation during ESC differentiation on collagen IV [15] were analyzed with real-time RT-PCR. Interestingly, we observed that none of the examined genes which are specific for other cell lineages was found to be significantly influenced by hnRNPA1 gene overexpressing (Supporting Information Fig. S1A) or silencing (Supporting Information Fig. S1B), indicating that although hnRNPA1 is expressed in cardiac cells, it plays a role likely specifically in SMC differentiation.

hnRNPA1 Plays an Important Role in SMC Differentiation In Vivo

To further explore the functional importance of hnRNPA1 in SMC differentiation and development in vivo, hnRNPA1 knockdown and control ESC lines were generated using hnRNPA1 and nontarget shRNAs, respectively. Compared to nontarget shRNA-infected ESCs, hnRNPA1 was significantly inhibited in hnRNPA1 knockdown ESCs (Fig. 4A, day 0). During SMC differentiation, as expected, hnRNPA1 gene expression levels were significantly increased in nontarget shRNAs-infected cells, while only slightly increased in hnRNPA1 shRNAs-infected cells (Fig. 4A, day 5), suggesting that hnRNPA1 gene expressions were successful knockdown in these cells. To investigate whether hnRNPA1 knockdown impairs in vivo SMC differentiation potential, nontarget and hnRNPA1 shRNAs-infected ESCs were subcutaneously injected into C57BL/6J mice with 100 ng/ml of platelet-derived growth factor-BB to promote in vivo SMC differentiation as described in our previous study [10]. Immunofluorescence staining with antibodies against SM-MHC and calponin showed that a large number of SM-MHC- or calponin-positive cells were presented in the implants of nontarget shRNAs-infected ESCs, while much less SM-MHC- or calponin-positive cells were observed in that of hnRNPA1 shRNAs-infected ESCs (Fig. 4B, 4C and Supporting Information Fig. S2). As expected, the majority of cells in the Matrigel implants were β-gal-positive, indicating its exogenous origins (Fig. 4B and Supporting Information Fig. S2). Importantly, real-time PCR analyses showed that hnRNPA1 gene expression levels in the Matrigel implants of hnRNPA1 shRNAs-infected ESCs were significantly lower than that of nontarget shRNAs-infected ESCs (Fig. 4D), further confirming the efficiency of hnRNPA1 knockdown and the importance of hnRNPA1 in SMC differentiation. Taken together, these data clearly suggest that hnRNPA1 plays an important role in SMC differentiation from stem cells in vitro and in vivo.

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Figure 4. hnRNPA1 plays an important role in smooth muscle cell (SMC) differentiation in vivo. (A): hnRNPA1 was successful knocked down during SMC differentiation in vitro. Total RNA samples were harvested from parent ESCs, undifferentiated nontarget and hnRNPA1 shRNAs-infected ESCs (day 0), day 5 differentiating nontarget, and hnRNPA1 shRNAs-infected ESCs (day 5) and subjected to real-time quantitative PCR analysis with hnRNPA1-specific primers. (B, C): Less SMCs were observed in the implants of hnRNPA1 shRNAs-infected ESCs. Matrigel plugs (n = 5 each group) were harvested 13 days after cell implantation. Frozen sections from implants were subjected to double immunofluorescence staining with antibodies against β-galactosidase (β-gal) and SM-MHC. The total numbers of SM-MHC-positive cells per field were counted by two well-trained independent investigators blinded to the treatments, from four random high power fields (×200) in each section, two sections from each implant and five implants for each group. Representative images (B) and quantitative data (C) were presented here. *, p < .05. (D): hnRNPA1 was successful knockdown in vivo. Total RNA samples were extracted from partial Matrigel implants and subjected to real-time PCR analysis. The data presented here were mean ± SEM of five mice. *, p < .05. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; ESC, embryonic stem cell; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; SM-MHC, smooth muscle myosin heavy chain.

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hnRNPA1 Mediates SMC Differentiation Gene Expression Through Transcriptional Activation

Using in vitro and in vivo experiments we have demonstrated that hnRNPA1 plays an important role in SMC differentiation and development through modulating SMC differentiation gene expression. In order to understand how hnRNPA1 regulates SMC gene expression, day 3 differentiating ESCs were transfected with pCMV5 or phnRNPA1 and treated at day 5 with the RNA synthase inhibitor, actinomycin D (1 μg/ml) for 6 hours. Total RNA was harvested and subjected to RT-qPCR analyses to examine Acta2 and Tagln gene expression. We observed that SMC differentiation gene (Acta2 and Tagln) induction by hnRNPA1 overexpression was abolished by actinomycin D treatment (Fig. 5A), suggesting that hnRNPA1 may activate Acta2 and Tagln gene expression at a transcriptional level. Luciferase activity assay and SMC-specific gene reporter were applied to examine whether hnRNPA1 overexpression will activate the specific SMC gene transcription. The reporter gene plasmids used in the luciferase assay, pGL3-Acta2-Luc and pGL3-Tagln-Luc, were previously designed and cloned in our laboratory [41]. Data shown in Figure 5B revealed that the overexpression of hnRNPA1 in differentiating ESCs significantly increased Acta2 or Tagln gene promoter activities, indicating that hnRNPA1 overexpression can activate specific SMC gene promoters. Finally, CHIP assays were conducted using an hnRNPA1 antibody in the differentiating ESCs to further verify if hnRNPA1 activates specific SMC gene transcription through direct binding to their promoters. Data shown in Figure 5C revealed that hnRNPA1 directly bounds to the promoters of Acta2 and Tagln, and such binding was dramatically enhanced by hnRNPA1 overexpression. Taken together, above findings demonstrate for the first time that hnRNPA1 regulates Acta2 and Tagln gene expressions during SMC differentiation through direct binding to the promoter region of specific SMC genes.

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Figure 5. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) transcriptionally activates smooth muscle cell (SMC) gene expression through direct binding to the promoters of SMC differentiation genes. (A): Actinomycin D ablated hnRNPA1 overexpression-mediated SMC gene expression. Day 3 differentiating embryonic stem cells (ESCs) transfected with phnRNPA1 or pCMV5 (2.0 μg/1 × 106 cells) were treated with or without actinomycin D (1 μg/ml) for 6 hours and harvested for RT-qPCR analysis of Acta2 and Tagln gene expressions. Dimethyl sulfoxide was included as a control. The data presented here are mean ± SEM of three independent experiments. *, p < .05 (second column vs. first column); **, p < .05 (fourth column vs. second column). (B): hnRNPA1 regulated the promoter activities of SMC differentiation genes. ESCs were cultured on collagen IV and transfected at day 3 with luciferase reporter plasmids pGL3-Acta2-Luc or pGL3-Tagln-Luc (0.15 μg/2.5 × 104 cells) together with phnRNPA1 or pCMV5 (0.2 μg/2.5 × 104 cells). pGL3-Renilla (0.025 μg/2.5 × 104 cells) was included as luciferase plasmid control. Luciferase and Renilla activity assays were detected 48 hours after transfection. Relative luciferase activity was defined as the ratio of Firefly versus Renilla with that of the control (set as 1.0). The data presented here are mean ± SEM of three independent experiments. *, p < .05 (vs. control). (C): hnRNPA1 bounds directly to the promoter regions of SMC differentiation genes. CHIP assays were performed using antibodies against hnRNPA1 and analyzed as described within main text. The data presented here are mean ± SEM of three independent experiments. *, p < .05 (vs. control). Abbreviation: CHIP, chromatin immunoprecipitation.

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hnRNPA1 Regulates SMC Differentiation Gene Expression Through SRF Binding Site Within Their Promoter Region

It has been shown that SRF and its cofactors play crucial roles in SMC differentiation and embryonic cardiovascular system development [43–45]. We wondered if SRF activation is required for hnRNPA1-mediated SMC gene regulation. To this end, control (pCMV5) or hnRNPA1 expression plasmids phnRNPA1 (2 μg/106 cells) and control random siRNA or SRF siRNAs (20 nM) were cotransfected into day 2 differentiating ESCs. Data shown in Figure 6A revealed that while overexpression of hnRNPA1 (second columns) or knockdown of SRF (third columns) alone was significantly upregulated or inhibited SMC-specific gene expression, respectively, knockdown of endogenous SRF gene expression almost abolished SMC-specific gene inductions in hnRNPA1 overexpressing cells (fourth columns), suggesting that SRF gene activation is required for hnRNPA1-mediated SMC-specific gene regulation. Moreover, as a previous study demonstrated that interactions between hnRNPA1 and IκBα are required for maximal activation of NF-κB-dependent transcription [37], we further wondered if hnRNPA1 directly interacts with SMC transcription factor SRF. Coimmunoprecipitation assay using hnRNPA1 antibody showed that hnRNPA1 indeed interacted with SRF, and such interactions were significantly enhanced by hnRNPA1 overexpression (Fig. 6B). It has been demonstrated that SRF binding element (CArG) within promoter region of SMC genes is required for transcriptional activation of SMC genes [46], we further wondered if this is also a case for hnRNPA1-regulated SMC gene transcriptional activation. To this aim, using pGL3-Tagln-Luc and pGL3-Acta2-Luc plasmid as a template, we carried out site-directed mutagenesis to mutate the SRF binding site within gene promoters as illustrated in Supporting Information Figure S3. Data from our luciferase activity assay showed that SRF binding element mutation in pGL3-Tagln-Luc and pGL3-Acta2-Luc resulted in complete loss of their transcriptional activity in response to hnRNPA1 overexpression (Fig. 6C), suggesting that hnRNPA1 regulates SMC differentiation gene expression through SRF binding site within their promoter region.

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Figure 6. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) regulates smooth muscle cell (SMC) differentiation gene expression through SRF binding site within their promoter region. (A): SMC transcription factor SRF gene activation is required for hnRNPA1-mediated SMC differentiation gene expression. Day 2 differentiating embryonic stem cells (ESCs) were cotransfected with control (pCMV5) or hnRNPA1 expression plasmids phnRNPA1 (2 μg/106 cells) and control random siRNA or SRF siRNAs (20 nM) and cultured further 72 hours. Total RNA samples were harvested and subjected to real-time PCR analysis. (B): Coimmunoprecipitation assay showed that hnRNPA1 protein interacts with SRF. (C): SRF binding site mutation abolished SMC differentiation gene promoter activity induced by hnRNPA1 overexpression. The data presented here are representative images or mean ± SEM of three independent experiments, *, p < .05. Abbreviations: siRNA, small interfering RNA; SRF, serum response factor.

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hnRNPA1 Transcriptionally Regulates SRF, MEF2c, and Myocd Gene Expression

Data shown in Figure 3 revealed that hnRNPA1 gene expression also influences the gene expression levels of SMC transcription factors such as SRF, MEF2c, and Myocd, suggesting hnRNPA1 may have a direct role in regulation of these transcription factors during SMC differentiation. To this aim, we first transfected day 3 differentiating ESCs with pCMV5 or phnRNPA1 and treated these transfected cells at day 5 with actinomycin D (1 μg/ml) for 6 hours. Total RNA was harvested and subjected to RT-qPCR analysis to examine SRF, MEF2c, and Myocd gene expression. We found that the effect of hnRNPA1 overexpression on SRF, MEF2c, and Myocd gene expression was completely ablated by actinomycin D (Fig. 7A), which suggested that hnRNPA1 may activate SRF, MEF2c, and Myocd gene expression at a transcriptional level. To further investigate whether hnRNPA1 can regulate SRF, MEF2c, and Myocd gene promoter activity, SRF, MEF2c, and Myocd gene reporter plasmids (pGL3-SRF-Luc, pGL3-MEF2c-Luc, and pGL3-Myocd-Luc) were generated in this study and used in luciferase activity assay. Data shown in Figure 7B revealed that the overexpression of hnRNPA1 in differentiating ESCs significantly increased SRF, MEF2c, or Myocd gene promoter activities, indicating that hnRNPA1 may activate transcriptional activity of these three genes. To further investigate whether hnRNPA1 can directly bind to the promoters of SRF, MEF2c, and Myocd, and its potential binding region(s) of hnRNPA1 within these three gene promoters, we designed a set of specific primers (four pairs) spanning through the respective promoter region of SRF, MEF2c, and Myocd as illustrated in Supporting Information Figure S4 and performed CHIP assay with hnRNPA1 antibody. Data shown in Figure 7C revealed that hnRNPA1 directly binds to the promoter regions between −1,753 and −1,613 of SRF gene, −1,335 and −1,263 of MEF2c gene, or −407 and −262 of Myocd gene, respectively, and such binding activity was significantly increased by hnRNPA1 overexpression. Taken together, these findings strongly suggest that hnRNPA1 transcriptionally regulates SMC transcription factor gene expression during SMC differentiation from stem cells.

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Figure 7. hnRNPA1 transcriptionally regulates SMC transcription factor gene expression through directly binding to its promoters. (A): Actinomycin D ablated hnRNPA1 overexpression-mediated SMC transcription factor gene expression. Day 3 differentiating ESCs transfected with phnRNPA1 or pCMV5 (2.0 μg/1 × 106 cells) were treated with or without actinomycin D (1 μg/ml) for 6 hours and harvested for real-time quantitative PCR analysis of SRF, MEF2c, and Myocd gene expressions. Dimethyl sulfoxide was included as a control. The data presented here are mean ± SEM of three independent experiments. *, p < .05 (second column vs. first column); **, p < .05 (fourth column vs. second column). (B): hnRNPA1 regulated the promoter activities of SMC transcription factor genes. ESCs were cultured on collagen IV and transfected at day 3 with luciferase reporter plasmids pGL3-SRF-Luc, pGL3-MEF2c-Luc, or pGL3-Myocd-Luc (0.15 μg/2.5 × 104 cells) together with phnRNPA1 or pCMV5 (0.2 μg/2.5 × 104 cells). pGL3-Renilla (0.025 μg/2.5 × 104cells) was included as luciferase plasmid control. Luciferase and Renilla activity assays were detected 48 hours after transfection. The data presented here are mean ± SEM of three independent experiments. *, p < .05 (vs. control). (C): hnRNPA1 bounds directly to the promoter regions of SRF, MEF2c, and Myocd genes. CHIP assays were performed using antibodies against hnRNPA1 and analyzed as described before. Precipitated chromatin DNA was used to amplify the promoter regions of SRF, MEF2c, or Myocd genes by real-time PCR with serial-specific primers as illustrated in Supporting Information Figure S4 and shown in Supporting Information Table S1. The data presented here are mean ± SEM of three independent experiments. *, p < .05 (vs. control). (D): Proposed model of hnRNPA1-mediates SMC differentiation. Abbreviations: ESC, embryonic stem cell; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; MEF2c, myocyte-specific enhancer factor 2c; SMC, smooth muscle cell; SRF, serum response factor.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

It is believed that SMC differentiation is a complicated process controlled by a dynamic array of microenvironmental cues and a complicated network of signaling pathways. The identification of such microenvironmental cues and signaling pathways which mediate SMC differentiation from stem/progenitor cells has been more difficult because of our incomplete knowledge of the transcription mechanisms that regulate SMC-specific gene expression. In this study, we have advanced our knowledge of the molecular mechanism mediating SMC differentiation by uncovering an important role of nuclear protein hnRNPA1 in regulating SMC-specific gene expression and SMC differentiation from ESCs. Its functional involvement of hnRNPA1 in SMC differentiation was supported by several lines of evidence. First, we found that the gene and protein expression levels of hnRNPA1 were significantly upregulated during SMC differentiation from stem cells, and observed that hnRNPA1 was strongly expressed in the ES-derived SMCs, embryonic, and adult vascular SMCs. Second, overexpression of hnRNPA1 in the differentiating ESCs significantly upregulated SMC-specific gene and transcription factor expression, while knockdown of hnRNPA1 dramatically inhibited these SMC gene expressions. Third, knockdown of hnRNPA1 in ESCs significantly impairs their SMC differentiation capacity in vivo. Fourth, hnRNPA1 regulates SMC gene transcriptional activation by directly binding to the promoter region of SMC-specific genes. Moreover, we demonstrate that hnRNPA1 can interact with SRF, and the SRF binding site(s) within SMC-specific gene promoters is required for hnRNPA1-mediated SMC gene transcriptional activation. Finally, we also demonstrate for the first time that hnRNPA1 directly regulates SMC transcription factors (SRF, MEF2c, and Myocd) at transcriptional level. This data strongly suggest that hnRNPA1 is a strong SMC differentiation modulator by regulating SMC-specific gene expression at two transcriptional levels: SMC-specific genes and their transcription factors—SRF, MEF2c, and Myocd.

hnRNPA1 and SMC Differentiation

Although the major characterized functions of hnRNP A/B family proteins were involved in telomere biogenesis/maintenance, gene transcription, alternative pre-mRNA splicing, nuclear import and export, cytoplasmic trafficking of mRNA, mRNA stability and turnover, and translation (see review [16]), their functional involvements in stem cell differentiation are largely unknown. Iervolino and coworkers reported that hnRNPA1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis. They found that the survival and differentiation of normal myeloid precursors and colony formation of primary CD34+ stem cells from a patient with chronic myelogenous leukemia in accelerated phase were impaired by expression of a nuclear hnRNPA1 mutant lacking of nucleocytoplasmic shuttling activity, suggesting a role of hnRNPA1 in stem/progenitor cell differentiation. However, no direct evidence has been reported about the functional significance of hnRNPA1 in stem cell differentiation, especially in SMC lineage and embryonic SMC development. In our previous pilot study, hnRNPA1 was revealed using nuclear proteomics as one of several nuclear proteins whose protein levels were significantly upregulated during early SMC differentiation from ESCs (unpublished data), which strongly implies that hnRNPA1 may play a role in SMC differentiation.

Using the antibodies specific for hnRNPA1 and SM-MHC, one of the most specific SMC markers, our double immunofluorescent staining showed that hnRNPA1 is strongly expressed and colocalized with SM-MHC in the ES-derived SMCs, embryonic, and adult aortic SMCs. Notably, hnRNPA1 was shown to localize in the nuclear, perinuclear as well as cytoplasm in the embryonic and aortic SMCs, but mainly expressed in the cytoplasm of cardiac cells, which is consistent with its nucleocytoplasmic shuttling activity. These findings strongly suggest that hnRNPA1 may play a role in SMC differentiation. Subsequently, the importance of hnRNPA1 in SMC differentiation from ESCs was further demonstrated using gain and loss of hnRNPA1 gene functions in differentiating ESCs. We have demonstrated that enhanced hnRNPA1 gene expression significantly promotes SMC differentiation, while knockdown of endogenous hnRNPA1 gene expression dramatically inhibits SMC differentiation. No such effects were observed in differentiating ESCs on other cell lineage gene expression (Supporting Information Fig. S1). Furthermore, we also demonstrate that knockdown of hnRNPA1 in ESCs significantly inhibits their ability to differentiate into SMCs in vivo as we observed less SM-MHC- or calponin-positive SMCs in the Matrigel plugs implanted with hnRNPA1 knockdown ESCs (Fig. 4 and Supporting Information Fig. S2). Taken together, our findings indicate that hnRNPA1 plays a critical role in mediating SMC differentiation from ESCs in vitro and in vivo.

hnRNPA1 Regulates SMC Differentiation Gene Expression Through a Transcriptional Mechanism and SRF Binding Sites Within Promoter Regions of SMC-Specific Genes

One of the novel findings in this study is that we clearly demonstrate for the first time that nuclear protein hnRNPA1 can regulate SMC differentiation through a transcriptional mechanism. Although huge amount of evidence showed that the main functions of hnRNPA1 in regulating gene expression are involved in mRNA biogenesis and mRNA transport [15, 16], recent research also strongly suggests that hnRNPA1 can regulate gene expression via a transcriptional mechanism. It was reported that hnRNPA1 regulates various gene expression through binding to the gene promoter regions [34, 47–50] or functioning as a component of the gene transcription complex [51]. In consistent with these findings, we provided strong evidence in this study which firmly demonstrated that hnRNPA1 is a transcription activator in regulation of SMC-specific gene expression (Acta2 and Tagln) during SMC differentiation from ESCs. In the presence of actinomycin D (RNA synthesis inhibitor), hnRNPA1 overexpression no longer increases SMC-specific gene (Acta2 and Tagln) expression, suggesting that hnRNPA1 regulates SMC gene expression at transcriptional level. Using SMC-specific gene reporters, we further confirmed that SMC gene promoter activity was significantly enhanced by enforced expression of hnRNPA1 in the differentiating ESCs. Importantly, data from CHIP assay (Fig. 5C) also provide strong evidence to show that hnRNPA1 can directly bind to the promoter DNA of SMC-specific genes, Acta2 and Tagln. All these data strongly suggest that hnRNPA1 is an important player in SMC-specific gene transcriptional regulation.

The SRF-CArG interaction is believed to be a critical convergence point for signals that either activates SMC gene expression to promote SMC differentiation under physiological environments or represses SMC gene expression during pathophysiological conditions [46]. By supporting this notion, we first observed that endogenous SRF activation is required for hnRNPA1-mediated SMC gene expression, and found that hnRNPA1 protein directly interacts with SRF, suggesting a close partnership between nuclear protein hnRNPA1 and SMC transcription factor SRF in the regulation of SMC-specific gene expression. Most straightforward and powerful evidence to confirm the importance of such partnership in regulation of SMC gene transcriptional activation was obtained from our SRF binding sites mutagenesis experiments, in which we observed that Acta2 and Tagln gene reporters with mutated SRF binding sites almost lost its response to hnRNPA1 overexpression. All together, we demonstrate that hnRNPA1 regulates SMC gene expression either through its directly binding to SMC gene promoter DNA or via its interaction with SRF, or both.

hnRNPA1: A Potential Transcriptional Regulator of SMC Transcription Factors

Another important finding of this study is that we further demonstrate for the first time that hnRNPA1 acts as a potential transcriptional regulator of SMC transcription factors. Transcription factors SRF, MEF2c, and myocardin play critical roles in SMC differentiation and their interactions with its respective binding elements within promoter regions are essential for muscle-specific gene transcription. It was reported that SRF-CArG box interactions confer context-dependent and signal-responsive control of muscle-specific gene transcription, which is accomplished at multiple levels of regulation including control of SRF expression, cytoplasmic to nuclear translocation, alternative splicing, and post-translational modifications of SRF itself [46]. We have previously demonstrated that SRF phosphorylation and nuclear translocalization are required for NAPDH oxidase 4 (Nox4)-mediated SMC differentiation [15], and the SRF-CArG box interaction and its modulation are essential for SMC gene transcriptional regulation mediated by other signals or molecules [10, 12, 13]. Apart from SRF, growing evidence also suggests that transcription factors MEF2c and myocardin play essential roles in regulation of SMC differentiation gene expression. Myocardin is exclusively expressed on vascular and visceral SMC as well as cardiomyocytes and is a critical SRF coactivator in the transcriptional program regulating SMC differentiation [44, 52–54]. Moreover, a recent study suggests that myocardin overexpression is sufficient for promoting the formation and maturation of SMC-like cells in differentiating human embryoid bodies [55]. Conversely, MEF2c has a specific temporal and spatial expression in the embryo and has been reported to participate in vascular development because mice lacking MEF2c have no differentiated SMCs in the vasculature [56]. Importantly, it has been shown that myocardin is a direct transcriptional target of MEF2c [57] and MEF2c mediates SMC-specific gene expression through direct regulation of myocardin gene expression. All the evidence firmly suggests that MEF2c is a key transcription factor for SMC differentiation. However, much less is known about the regulation of MEF2c gene expression during stem cell differentiation. In this study, we provide strong evidence that SRF, MEF2c, and Myocd gene expressions are transcriptionally regulated by hnRNPA1. Our data from CHIP assays also revealed that hnRNPA1 directly binds to the promoter regions of SRF, MEF2c, and Myocd genes (Fig. 7C). Several different oligonucleotide motifs including the “ATTT” motif, the “TGCTCTC” box, and G-quartets have been identified as the potential hnRNPA1 binding elements in the transcriptional regulatory regions of various genes [16]. However, there is no such consensus motifs present in the examined gene promoter region, suggesting that hnRNPA1 may also bind to other element/s yet to be identified. To define the exact binding site/motif, additional in vitro binding assays, deletion constructs containing different promoter length, and direct site mutational analyses of these gene promoters will be required.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Although we have provided some direct and strong evidence to show that hnRNPA1 regulates SMC-specific gene and transcription factor gene transcriptional activity through direct binding to the promoter regions of these genes (Acta2, Tagln, SRF, Myocd, and MEF2c) (Figs. 5–7), the minimal essential binding elements of hnRNPA1 or exactly hnRNPA1 binding motifs within these gene promoter regions remain to be further identified in our future study. Moreover, whether other gene regulatory functions of hnRNPA1 such as mRNA biogenesis, alternative splicing selection, mRNA transportation, and its nucleo-cytoplasmic shutting activity also play a role in SMC differentiation from stem cells remain to be elucidated. Nonetheless, based on the findings from the previous and current studies, we proposed that in response to SMC differentiation stimuli such as LIF withdrawn, endoplasmic reticulum stress, oxidative stress, collagen I/IV stimulation, and autocrine/paracrine SMC differentiation growth factors or cytokines (transforming growth factor beta or platelet-derived growth factor-BB, etc.), the gene and protein levels of hnRNPA1 are upregulated. Upon activation, hnRNPA1 upregulates SMC transcription factors and/or coactivators gene expression through transcriptional activation, resulting in SMC differentiation gene expression (Fig. 7D). Meanwhile, upregulated hnRNPA1 can also directly regulate SMC differentiation gene expression through SRF binding elements within gene promoter region. Therefore, we have successfully uncovered a novel role of hnRNPA1 in SMC differentiation from stem cells in vitro and in vivo. Our findings will significantly enrich our understanding of the molecular mechanisms in SMC differentiation and benefit future application in engineering tissue vessels.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by British Heart Foundation (FS/09/044/28007 and PG/11/40/28891) and National Natural Science Foundation of China (Nos: 81270001 and 81270180). Dr Qingzhong Xiao is a recipient of British Heart Foundation Intermediate Basic Science Research Fellowship (FS/09/044/28007) and the principal investigator of British Heart Foundation project grant (PG/11/40/28891). This work forms part of the research themes contributing to the translational research portfolio of Barts and the London Cardiovascular Biomedical Research Unit which is supported and funded by the National Institute of Health Research.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0845_sm_SupplTabe1.pdf18KSupplementary Table 1
sc-12-0845_sm_SupplText.pdf107KSupplementary Data
sc-12-0845_sm_SupplFigure1.TIF1588KFigure S1. hnRNPA1 is specific for SMC differentiation from ES cells on collagen IV. ES cells cultured on collagen IV were transfected with pCMV-hnRNPA1 or empty vector control pCMV5 (1μg/106 cells) (A), or random control siRNA or hnRNPA1 siRNA (B), respectively. The cells were harvested and subjected to real-time RT-PCR analysis with sets of primers specific for different cell lineages genes: Flt-1 and CD144 for endothelial, Ddr2 and Thy1 for cardiac fibroblast, Tnnc 1 and actc1 for cardiac myocytes, Alcam and CD133 for hematopoietic progenitor, Dlx3 and Tpbg for trophoblast, CD29 and CD44 for mesenchymal, nestin and Gap43 for neural. The data are means±S.E.M of three independent experiments.
sc-12-0845_sm_SupplFigure2.TIF1911KFigure S2. Knockdown hnRNPA1 reduces calponin-positive SMCs derived from stem cells in vivo. Frozen sections from implants were subjected to double immunofluorescence staining with antibodies against beta-galactosidase (β-gal) and SMC marker calponin. The total numbers of Calponin-positive cells per field were counted by two well-trained independent investigators blinded to the treatments, from four random high power fields (200x) in each section, two sections from each implant and five implants for each group. Representative images (top panels) and quantitative data (bar graphs) were presented here. *p< 0.05.
sc-12-0845_sm_SupplFigure3.TIF1366KFigure S3 Schematic illustration of Acta2 (A) and Tagln (B) gene promoter regions.
sc-12-0845_sm_SupplFigure4.TIF1538KFigure S4 Schematic illustration of SRF (A), MEF2c (B) and Myocd (C) gene promoter regions. The number within the promoter region was defined according to its position with the start condon (‘A’: +1) of respective genes. Black region in respective gene represents the promoter we used in this study.

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