INDUCED PLURIPOTENT STEM CELLS

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Nitric Oxide Determines Mesodermic Differentiation of Mouse Embryonic Stem Cells by Activating Cla...
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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Nitric Oxide Determines Mesodermic Differentiation of Mouse Embryonic Stem Cells by Activating Class IIa Histone Deacetylases: Potential Therapeutic Implications in a Mouse Model of Hindlimb Ischemia FRANCESCO SPALLOTTA,a JESSICA ROSATI,a STEFANIA STRAINO,a SIMONA NANNI,b ANNALISA GRASSELLI,c,d VALERIA AMBROSINO,a DANTE ROTILI,e SERGIO VALENTE,e ANTONELLA FARSETTI,c ANTONELLO MAI,e MAURIZIO C. CAPOGROSSI,a CARLO GAETANO,a BARBARA ILLIf a

Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’ Immacolata - Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; bIstituto di Patologia Medica, Universita` Cattolica del Sacro Cuore, Rome, Italy; cIstituto di Neurobiologia e Medicina Molecolare, Centro Nazionale delle Ricerche, Rome Italy; dIstituto Nazionale Ricovero e Cura Anziani–Istituto di Ricovero e Cura a Carattere Scientifico, Ancona, Italy; eIstituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento Studi Farmaceutici Universita` degli Studi di Roma ‘‘La Sapienza,’’ Rome, Italy; fLaboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico Monzino–Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy Key Words. Nitric oxide • Stem cells • Histone deacetylases • Chromatin • Differentiation

ABSTRACT In human endothelial cells, nitric oxide (NO) results in class IIa histone deacetylases (HDACs) activation and marked histone deacetylation. It is unknown whether similar epigenetic events occur in embryonic stem cells (ESC) exposed to NO and how this treatment could influence ESC therapeutic potential during tissue regeneration. This study reports that the NO-dependent class IIa HDACs subcellular localization and activity decreases the global acetylation level of H3 histones in ESC and that this phenomenon is associated with the inhibition of Oct4, Nanog, and KLF4 expression. Further, a NO-induced formation of macromolecular complexes including HDAC3, 4, 7, and protein phosphatase 2A (PP2A) have been detected. These processes correlated with the expression of the mesodermal-

specific protein brachyury (Bry) and the appearance of several vascular and skeletal muscle differentiation markers. These events were abolished by the class IIa-specific inhibitor MC1568 and by HDAC4 or HDAC7 short interfering RNA (siRNA). The ability of NO to induce mesodermic/ cardiovascular gene expression prompted us to evaluate the regenerative potential of these cells in a mouse model of hindlimb ischemia. We found that NO-treated ESCs injected into the cardiac left ventricle selectively localized in the ischemic hindlimb and contributed to the regeneration of muscular and vascular structures. These findings establish a key role for NO and class IIa HDACs modulation in ESC mesodermal commitment and enhanced regenerative potential in vivo. STEM CELLS 2010;28:431–442

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Pluripotent embryonic stem cells (ESCs) are useful to study early developmental processes and may be considered a potential therapeutic tool in cell therapy and regenerative medicine. In the presence of leukemia inhibitory factor (LIF), ESCs express a combination of transcription factors associated with proliferation and stemness, including Oct4, Nanog,

Sox2, and proteins of the Kruppel-like factor (KLF) family [1], and maintain self-renewing properties. Upon LIF removal, ESCs undergo spontaneous differentiation into primary germ layers and, if injected into a blastocyst, can give rise to all mammalian cell types [2, 3]. During development, tissue-specific genes are timely expressed by superimposed chromatin remodelling events which regulate RNAPol-II complex binding or detachment from enhancer promoter sequences. DNA methylation and the

Author contributions: F.S.: Conception and design, collection and assembly of data; J.R., S.S., S.N., A.G., V.A.: Collection and assembly of data; D.R., S.V., A.M.: Provision of study material; A.F.: Data analysis; M.C.C.: Final approval of manuscript; C.G.: Conception and design, writing of manuscript; B.I.: Conception and design, data analysis and interpretation, writing of manuscript. F.S. and J.R. contributed equally to this manuscript. Correspondence: Carlo Gaetano, M.D., Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, Via dei Monti di Creta 104, 00167 Rome, Italy. Telephone: +39-06-66462431; Fax: +39-0666462430; e-mail: [email protected]. Barbara Illi, PhD., Laboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy. Telephone: +39-06-66462428; Fax: +39-0666462430, e-mail: [email protected]; [email protected] Received August 9, 2009; accepted for C AlphaMed Press 1066-5099/2009/ publication December 29, 2009; first published online in STEM CELLS EXPRESS January 13, 2010. V $30.00/0 doi: 10.1002/stem.300

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covalent modification of histones represent the major epigenetic mechanisms regulating gene transcription. Specific enzymatic multiprotein complexes catalyze DNA methylation and a wide variety of post-translational histone modifications, including acetylation, methylation, ubiquitination, sumoylation, and proline isomerization, which modulate chromatin shaping and exert profound effects on gene expression [4, 5]. The importance of epigenetic events during ESC differentiation is demonstrated by the fact that the chromatin profile of undifferentiated ESCs and lineage-committed ESC-derived cells shows striking differences. Furthermore, in undifferentiated ESCs, the promoter region of tissue-specific genes often possess a bivalent epigenetic mark which mediates opposing signals to the transcriptional machinery, retaining tissue-specific genes neither active nor repressed, but rather poised for expression [6, 7]. Acetylation of lysine residues on histone tails is the best known mechanism accounting for chromatin decondensation. Its reversible nature relies on the activity of two families of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs) [3]. Four classes of HDACs are currently known. Class I HDACs (HDAC1, 2, 3, and 8) are nuclear except for HDAC8; these HDACs function as catalytic subunits of large multiprotein complexes mediating transcriptional repression. Class II HDACs are divided into two subclasses, a and b. Class IIa (HDAC4, 5, 7, and 9) perform nuclear/cytoplasmic shuttling and associate to HDAC3 in multienzymatic complexes bearing deacetylase activity; in contrast, class IIb members (HDAC6 and 10) are enriched in the cytosol [8]. Class III HDACs, or sirtuins (Sir-2-like proteins), depend on NADþ to exert their activity mainly on nonhistones proteins [9]. HDAC11 is the only member of class IV, and although its function is basically unknown, it is highly-conserved throughout species [9]. Acetylation of histones H3 and H4 is associated with ESC differentiation in response to LIF withdrawal [10] and other extracellular signals; [11] nevertheless, the role of HDACs during ESC commitment is still poorly characterized and pharmacologic inhibition of histone deacetylase activity has produced conflicting results [12, 13]. Interestingly, members of the class I HDAC family associate to Oct4, contributing to its function in the maintenance of the undifferentiated state [14]. It has been previously shown that mouse ESCs express cardiovascular markers when exposed to laminar shear stress (SS), a phenomenon associated with histone acetylation and formation of transcriptionally active complexes harboring HATs [15, 16]. The production of nitric oxide (NO) is one of the major consequences of the laminar SSdependent regulation of endothelial function [17]. Although NO has been demonstrated to play a pivotal role in the acquisition of the cardiovascular phenotype during development [18, 19] little information is available about the underlying molecular mechanism(s) which may, at least partially, rely on epigenetic processes [20, 21]. Moreover, NO effect on the early differentiation of primary germ layers, namely ectoderm, mesoderm, and endoderm, is currently unknown. In the present study, we show that NO-treated ESCs acquire a predominant mesoderm-like phenotype. This event was associated with decreased expression of the stemness markers Oct4, Nanog, and KLF4, and was paralleled by global histone deacetylation and chromatin remodelling. In this context, NO-induced class-IIa HDACs nuclear retention ‘‘via’’ activation of protein phosphatase 2A (PP2A). Notably, the inhibition of class IIa HDACs activity, either by a selective drug or by short interfering RNA (siRNA), prevented NO-dependent expression of mesodermic markers in

Chromatin Remodelling in Mouse ES Differentiation

the absence of LIF. Finally, in a mouse model of hindlimb ischemia, NO-activated ESCs, injected systemically into the circulation, efficiently localized in the ischemic hindlimb and contributed to vascular and muscle regeneration.

MATERIALS

AND

METHODS

Cell Culture and Treatments Murine ES D3 cells were cultured as described (see supporting Materials and Methods) [15]. Cells were cultured for 24 hours in complete medium containing diethylenetriamine/nitric oxide adduct (DETA/NO 500 lM; (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), trichostatin A (TSA; 32 nmol/l; Sigma), MS27-275 (30 nmol/l; Sigma) or MC1568 (5 lmol/l), either in the presence or absence of LIF. Hypoxia experiments were performed as described [22].

Immunofluorescence Analysis ES cells were plated onto superfibronectin (10 lg/mL; Sigma)coated plates. Immunofluorescence experiments were performed as previously described [15].

Chromatin Immunoprecipitation Chromatin immunoprecipitation were performed as described [23]. Primers were:

mOct4 Forward: 50 -AGACGGGTGGGTAAGCAAGA-30 mOct4 Reverse: 50 -TGAATGTTCGTGTGCCAATTATTT-30 mDesmin Forward: 50 -ACGCGCACCAACGAGAA-30 mDesmin Reverse: 50 -GTAGTTGGCGAAGCGGTCAT-30 mSM22a Forward: 50 -GCTGCCCCCGACAGACT-30 mSM22a Reverse: 50 -CCACACAGGCTCCATATTTGG-30 Flk-1 Forward: 50 -TGCACTTGCAGGCTCCTAATG-30 Flk-1 Reverse: 50 -AGCAACCTGGGAAGCATCAC-30

Total Fractionated Cell Extracts and Western Blots Total extracts were performed as described [24]. Fractionated cellular extracts were performed as described (see supporting Materials and Methods) [20].

HDAC Assay HDAC assays were performed with the HDAC activity assay Kit (Upstate Biotechnology, Lake Placid, NY, USA; http://www. upstatebiotech.com) according to the manufacturer’s instructions.

Nitric Oxide Detection Nitric oxide production was detected as described [25].

Cyclic Guanosine Monophosphate Detection Next, cyclic guanosine monophosphate (cGMP) detection was performed using the direct cGMP enzyme-linked immunosorbent assay kit (Assay Design), according to the manufacturer’s instructions.

Phosphatase Assay PP2A-specific activity was determined by phosphatase assays as previously described [24].

Small Interfering RNA-Mediated Gene Silencing Small interfering RNAs (siRNA) targeting HDAC4, HDAC7, or a scrambled sequence (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) were transfected into ES cells, according to the manufacturer’s instruction (see supporting Materials and Methods).

Spallotta, Rosati, Straino et al.

Immunoprecipitation Cells were washed in phosphate buffered saline solution (PBS) and harvested in 50 mmol/l Tris-HCl (pH 7.5) containing 150 mmol/l NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, supplemented with 1 mmol/l Na3VO4, 5 mmol/l NaF, 1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, and protease inhibitors (Sigma). After preclearing with protein A Sepharose, equal amounts of protein (500 lg) were incubated with 4 lg of primary antibody and 30 ll of 50% slurry of ExactaCruz F matrix (Santa Cruz) overnight at 4 C. Immunocomplexes were washed four times with lysis buffer and resuspended in Laemmli buffer.

mRNA Quantification RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer’s instruction. cDNA synthesis for quantitative real time PCR (qRTPCR) was obtained using the DNA Synthesis In Vitro Transcription Kit (Invitrogen) according to the manufacturer’s protocol. Next, mRNAs levels were analyzed using the SYBR Green qPCR method (Invitrogen) and quantified with the ABI Prism 7,000 SDS (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). For primers sequences, see supporting Materials and Methods.

Confocal Analysis Confocal analyses were performed as described [20].

Shear Stress Experiments Shear stress (SS) experiments were performed as previously described [24]. ES cells were treated for 15 minutes at 37 C with 12 lM Smethylisothiourea (SMT) before exposure to SS in a cone-plate apparatus [24].

Adenoviral Infection ES cells were infected with an adenovirus encoding green fluorescent protein (Ad-GFP; 250 pfu/cell) [26] in serum-free medium for 2 hours and then cultured for an additional 20 hours before further treatment.

Mouse Model of Hindlimb Ischemia C57BL6 male mice (Charles River, Calco, Italy, www.criver.com), 2 months of age and weighing 25-30g, were used for all experiments. Animals were anesthetized with a mixture of 1 g of tribromoethyl alcohol in 1 ml of tert-amyl alcohol (Avertin, Sigma), diluted 1:50 and intraperitoneally injected, 20 ll/g body weight. Hindlimb ischemia and laser Doppler perfusion imaging (LISCA Development AB, Linko¨ping, Sweden) were performed as previously described (for details, see also supporting Materials and Methods) [27]. All experimental procedures were approved by the internal Animal Research Ethical Committee (protocol HH39) according to the Italian Ministry of Health and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Immunoistochemistry At various times after surgery, anesthetized mice were killed and perfused with phosphate-buffered saline (PBS), followed by 10% buffered formalin (10 minutes) at 100 mm/Hg, via the left ventricle. After paraffin embedding, 1 or 3-lm-thick sections were cut from each sample with muscle fibers oriented in the transverse direction. For technical details, see supporting Materials and Methods.

Statistical Analysis Statistical analyses were carried out by analysis of variance. Statistical significance was evaluated by a two-tailed unpaired Student’s t test. A p value

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