MCB Accepted Manuscript Posted Online 6 July 2015 Mol. Cell. Biol. doi:10.1128/MCB.00118-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

DYNAMIC REGULATION OF AP-1 TRANSCRIPTIONAL COMPLEXES DIRECTS

2

TROPHOBLAST DIFFERENTIATION

3 4

Kaiyu Kubota§,1, Lindsey N. Kent§,2, M.A. Karim Rumi§, Katherine F. Roby¶, and

5

Michael J. Soares§,3

6 7

Institute for Reproductive Health and Regenerative Medicine, §Department of

8

Pathology and Laboratory Medicine and the ¶Department Anatomy and Cell

9

Biology, University of Kansas Medical Center, Kansas City, Kansas 66160

10 11

Submitted to: Molecular and Cellular Biology

12

Running Head: Regulation of trophoblast differentiation

13

Key Words: hemochorial placentation, AP-1, FOSL1, JUNB, trophoblast

14

differentiation

15 16

1

Supported by American Heart Association and Japan Society for the Promotion of

17

Science postdoctoral fellowships. To whom correspondence may be addressed:

18

Institute of Reproductive Health and Regenerative Medicine, Department of Pathology

19

and Laboratory Medicine, University of Kansas Medical Center, MS3050, 3901 Rainbow

20

Boulevard, Kansas City, KS, USA 66160. Tel: 1-(913)-588-5690; Fax 1-(913)-588-8287;

21

E-mail: [email protected]

22 23

2

Present address: Department of Molecular Virology, Immunology, and Medical

24

Genetics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA

25 26

3

To whom correspondence may be addressed: Institute of Reproductive Health and

27

Regenerative Medicine, Department of Pathology and Laboratory Medicine, University

28

of Kansas Medical Center, MS3050, 3901 Rainbow Boulevard, Kansas City, KS, USA

29

66160. Tel: 1-(913)-588-5691; Fax 1-(913)-588-8287; E-mail: [email protected]

30 31 32

2

33 34

ABSTRACT Placentation is a process that establishes the maternal-fetal interface and is

35

required for successful pregnancy. The epithelial component of the placenta consists of

36

trophoblast cells, which possess the capacity for multi-lineage differentiation and are

37

responsible for placental-specific functions. FOS like antigen 1 (FOSL1), a component

38

of AP-1 transcription factor complexes, contributes to the regulation of placental

39

development. FOSL1 expression is restricted to trophoblast giant cells and invasive

40

trophoblast cells. In the present study, we characterized the FOSL1 regulatory pathway

41

in rat trophoblast cells. Transcriptome profiling in control and FOSL1 knockdown cells

42

identified FOSL1 dependent gene sets linked to endocrine and invasive functions.

43

FOSL1 was shown to occupy AP-1 binding sites within these gene loci, determined by

44

chromatin immunoprecipitation (ChIP). Complementary in vivo experiments using

45

trophoblast specific-lentiviral delivery of FOSL1 shRNAs provided an in vivo validation

46

of FOSL1 targets. FOSL1 actions require a dimerization partner. Co-

47

immunoprecipitation, co-immunolocalization, and ChIP analyses showed that FOSL1

48

interacts with JUNB and to a lesser extent JUN in differentiating trophoblast cells.

49

Knockdown of FOSL1 and JUNB expression inhibited both endocrine and invasive

50

properties of trophoblast cells. In summary, FOSL1 recruits JUNB to form AP-1

51

transcriptional complexes that specifically regulate the endocrine and invasive

52

trophoblast phenotype.

53

3

54 55

INTRODUCTION The placenta is a specialized tissue of pregnancy that permits development of

56

the embryo within the female reproductive tract and effectively facilitates the redirection

57

of resources from the mother to the fetus (1). Placentation is categorized based on the

58

connectivity between maternal and embryonic tissues. In hemochorial placentation, as

59

seen in rodent and most primate species, maternal blood directly bathes specialized

60

extraembryonic cells referred to as trophoblast (2). The trophoblast lineage arises early

61

in embryonic development. As the embryo grows, a subset of totipotent stem cells

62

become committed to the trophoblast cell lineage (3, 4). These cells are situated on the

63

surface of the blastocyst and are called trophectoderm. They give rise to a trophoblast

64

stem (TS) cell population initially apposed to the inner cell mass of the blastocyst and

65

expand into extraembryonic ectoderm (5-7). TS cells differentiate into multiple

66

specialized trophoblast cell types. In the rat, TS cells differentiate into syncytial

67

trophoblast cells, spongiotrophoblast cells, glycogen cells, trophoblast giant cells, and

68

invasive trophoblast cells (8, 9). Each differentiated cell type contributes to a core

69

function of the placenta. Syncytial trophoblast cells specialize in transport,

70

spongiotrophoblast and trophoblast giant cells synthesize and secrete peptide and

71

steroid hormones, glycogen cells are an energy reservoir, and invasive trophoblast cells

72

penetrate into the uterus and modify the uterine vasculature. Regulatory mechanisms

73

controlling the trophoblast lineage have been investigated (10-13).

74 75

Activator protein-1 (AP-1) consists of a family of basic leucine-zipper transcription

76

factors inducible in response to a variety of extracellular stimuli (14). The composition of

4

77

AP-1 is best characterized as heterodimers of FOS family (FOS, FOSB, FOSL1,

78

FOSL2) and JUN family (JUN, JUNB, JUND) proteins or as JUN family homodimers (15,

79

16). The AP-1 family plays an important role in the regulation of fundamental cellular

80

processes including cell proliferation, differentiation, motility, and invasion (14-16).

81

There is a remarkable specificity of the actions of AP-1, which is determined by the

82

composition of its constituent proteins (15, 16).

83 84

FOS and JUN family transcription factors are expressed in rodent and human

85

trophoblast cells (17-21) and have been implicated in the regulation of transcription of

86

an assortment of genes expressed in trophoblast cells (22-28). Mouse mutagenesis

87

has demonstrated roles for FOSL1 and JUNB in placental development (29, 30). Null

88

mutations at either Fosl1 or Junb loci result in early embryonic death. The initial

89

phenotypic descriptions suggested that FOSL1 and JUNB contributed to the regulation

90

of vascularization of the labyrinth zone of the mouse placenta (29, 20). FOSL1 is

91

prominently expressed in trophoblast giant cells and in endovascular invasive

92

trophoblast cells, placing it in a position to potentially regulate the transcription of genes

93

involved in hormone biosynthesis and in vascular remodeling, respectively (20). In rat

94

TS cells, FOSL1 expression is prominently increased during trophoblast differentiation

95

correlating with acquisition of both endocrine and invasive properties (20, 31).

96

Furthermore, FOSL1 was identified as a downstream mediator of a phosphatidylinositol

97

3-kinase/AKT signaling pathway promoting trophoblast invasion and vascular

98

remodeling (20). In vivo disruption of FOSL1 using trophoblast-specific lentiviral

99

delivery of Fosl1 shRNAs inhibited the depth of endovascular trophoblast cell invasion

5

100

(20). These actions of FOSL1 on the invasive trophoblast cell phenotype are conserved

101

in rat and human trophoblast cells (20, 21).

102 103

In this study, we delve deeper into the actions of FOSL1 on trophoblast cell

104

differentiation. Targets for FOSL1 action and FOSL1 dimerization partners are

105

identified in differentiating trophoblast cells. In vitro and in vivo research strategies were

106

performed utilizing TS cells and lentiviral trophoblast-specific gene manipulation,

107

respectively. Our experimental findings demonstrate a cooperative role for FOSL1 and

108

JUNB in regulating trophoblast cell invasive and endocrine phenotypes.

109

6

110 111

EXPERIMENTAL PROCEDURES Animals – Holtzman Sprague-Dawley rats were obtained from Harlan

112

Laboratories (Indianapolis, IN). Animals were housed in an environmentally controlled

113

facility with lights on from 0600 to 2000 h and were allowed free access to food and

114

water. The presence of a seminal plug or sperm in the vaginal smear was designated

115

day 0.5 of pregnancy. Tissues for histological analysis were frozen in dry-ice cooled

116

heptane and stored at -80°C until processed. Tissue samples for RNA or protein were

117

frozen in liquid nitrogen and stored at -80°C until processed. Female rats were made

118

pseudopregnant by mating with vasectomized males. The presence of a seminal plug

119

was designated day 0.5 of pseudopregnancy. The University of Kansas Animal Care

120

and Use Committee approved protocols for the care and use of animals.

121 122

Rcho-1 TS cells and rat blastocyst-derived TS cells – Rcho-1 TS cells were

123

maintained in stem state medium [RPMI-1640 culture medium (Life Technologies,

124

Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Sigma-Aldrich, St.

125

Louis, MO), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 1 mM sodium pyruvate (Life

126

Technologies), 100 μM penicillin, and 100 U/ml streptomycin (Life Technologies)] and

127

passaged with trypsin-EDTA (Fisher, Pittsburgh, PA) (32, 33). Differentiation was

128

induced by replacing stem state medium with differentiation promoting medium [NCTC-

129

135 medium (Sigma-Aldrich) supplemented with 1% horse serum (Life technologies), 50

130

μM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES, 4-(2-hydroxyethyl)-1-

131

piperazineethanesulfonic acid, 38 mM sodium bicarbonate, 100 μM penicillin, and 100

132

U/ml streptomycin (Fisher)]. Rat blastocyst-derived TS cells were grown in stem state

7

133

medium as described above, supplemented with fibroblast growth factor 4 (FGF4, 37.5

134

ng/ml; Sigma-Aldrich), heparin (1.5 μg/ml; Sigma-Aldrich), and rat embryonic fibroblast-

135

conditioned medium (70% of the final volume). Differentiation was induced by removal

136

of FGF4, heparin, and rat embryonic fibroblast-conditioned medium (34). Stem state

137

cells were collected within 48 h of subculture and differentiated cells were maintained

138

for 8 days in differentiation medium prior to harvesting.

139 140

Short hairpin RNA (shRNA) constructs and production of lentiviral particles

141

– Two Fosl1 shRNAs were designed and subcloned into pLKO.1 using AgeI and EcoRI

142

restriction sites (20). Jun, Junb, Jund, Grhl1 shRNAs constructs subcloned in pLKO.1

143

were obtained from Sigma-Aldrich (St. Louis, MO). A control shRNA that does not target

144

any known mammalian gene, pLKO.1-shSCR (plasmid 1864), was obtained from

145

Addgene (Cambridge, MA). Sequences representing the sense target site for each of

146

the shRNAs used in the analyses are provided in Table 1. Second-generation lentiviral

147

packaging vectors (Addgene) were used to produce lentiviral particles. Culture

148

supernatants containing lentiviral particles were harvested every 24 h for two to three

149

days, centrifuged to remove cell debris, filter sterilized, concentrated by

150

ultracentrifugation, and stored at −80°C until used. Lentiviral vector titers were

151

determined by measurement of p24 gag antigen by enzyme-linked immunosorbent

152

assay (Advanced Bioscience Laboratories, Kensington, MD).

153 154 155

In vitro lentiviral transduction – Rcho-1 TS cells and rat blastocyst-derived TS cells were exposed to lentiviral particles, selected with puromycin dihydrochloride

8

156

(Sigma-Aldrich; 2 μg/μl) for two days, and then maintained in a lower concentration of

157

the antibiotic (1 μg/μl). The puromycin selective pressure was removed during in vitro

158

differentiation.

159 160

In vivo lentiviral transduction – Rat embryos were transduced with lentiviral

161

particles as previously described (20, 35). Briefly, blastocysts collected on gestation day

162

4.5 were treated with Pronase (Sigma-Aldrich; 10 mg/ml for 10 min) to remove zonae

163

pellucidae and incubated with concentrated lentiviral particles (750 ng of p24/ml) for 4.5

164

h. Transduced blastocysts were transferred to uteri of day 3.5 pseudopregnant rats for

165

subsequent evaluation of gene knockdown and placentation site phenotypes on

166

gestation day 11.5.

167 168

DNA microarray analysis – DNA microarray analysis was performed by the

169

University of Kansas Medical Center Biotechnology Support Facility, as previously

170

described (31). Briefly, Affymetrix 230 2.0 DNA microarray chips (Affymetrix, Santa

171

Clara, CA) were probed with cDNAs generated from differentiated Rcho-1 TS cells

172

expressing control or Fosl1 shRNAs. Each experimental group was analyzed in

173

triplicate. Hybridization signals were normalized with internal controls using the MAS5

174

algorithm in the Expression Console (Affymetrix) and fold change computed. Significant

175

differences were determined by paired two-tailed Student t-tests. Microarray data was

176

processed for functional analysis using Ingenuity Pathway Analysis (Qiagen, Redwood

177

City, CA). Probe sets included in the analysis were restricted to those changing at least

9

178

two-fold between group comparisons with signal strengths of ≥ 500 for the maximal

179

value.

180 181

Quantitative reverse transcription-PCR (qRT-PCR) – RNA was extracted

182

using TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNAs

183

were reverse transcribed from RNA by using cDNA synthesis reagents from Applied

184

Biosystems (Foster City, CA) according to the manufacturer's instructions. Power SYBR

185

Green PCR Master Mix (Applied Biosystems) was used in the PCR. Reactions were

186

processed using a 7500 Real-Time PCR system (Applied Biosystems). Conditions

187

included an initial holding stage (50°C for 2 min and 95°C for 10 min) and 40 cycles

188

(95°C for 15 s and 60°C for 1 min) followed by a dissociation stage (95°C for 15 s, 60°C

189

for 1 min, and then 95°C for 15 s). Primers used in the analyses are provided in Table 2.

190

The comparative cycle threshold method (ΔΔCT) was used for relative quantification of

191

the amount of mRNA normalized to 18S RNA. Values are presented relative to controls

192

for each gene.

193 194

Primary antibodies – Antibodies to FOSL1 (SC605) and JUNB (SC-8051 for all

195

applications except SC-73 was used for chromatin immunoprecipitation, ChIP) were

196

obtained from Santa Cruz Biotechnology (Santa Cruz, CA). We obtained antibodies to

197

GRHL1 (HPA005798) and pan-cytokeratin (F3418) from Sigma-Aldrich. Antibodies to

198

JUN (610326), JUND (5226-1), GAPDH (AB2302), and Histone H3 (ab1790) were

199

obtained from BD Biosciences (Franklin Lakes, NJ), Epitomics (Burlingame, CA),

200

Millipore (Billerica, MA), and Abcam (Cambridge, MA), respectively. Antibodies for

10

201

PRL3D1 and CYP11A1 were previously generated and characterized by our laboratory

202

(36, 37).

203 204

Western blotting – Whole-cell lysates were prepared in lysate buffer (Cell

205

Signaling Technology, Danvers, MA). Nuclear lysates were prepared with NE-PER

206

Nuclear Protein Extraction Kit (Thermo Fisher scientific, Rockford, IL). Lysates were

207

fractionated by SDS-PAGE. Separated proteins were electrophoretically transferred to

208

PVDF membranes. Blots were probed with the designated antibodies overnight at 4°C,

209

followed by incubation with horseradish peroxidase-conjugated secondary antibodies to

210

rabbit (Cell Signaling Technology) or mouse (Sigma-Aldrich) IgG for 1 h at room

211

temperature. Reaction products were visualized by incubation with Luminata Western

212

HRP Substrates according to the manufacturer's instructions (Millipore). Lysates from

213

293FT cells (Invitrogen, Eugene, OR) transfected with rat Jund cDNA subcloned into

214

pCMV SPORT6 (GE Healthcare, Lafayette, CO) were used as a positive control for

215

JUND immunoblots.

216 217

Immunolocalization – Ten-micrometer cryosections of placentation sites were

218

prepared and stored at −80°C until used. Tissue sections mounted on glass slides or

219

Rcho-1 TS cells plated on chamber slides were fixed in 4% paraformaldehyde, washed

220

with phosphate buffered saline (PBS), and permeabilized in PBS containing 0.25%

221

Triton X100. Following a blocking step in 10% normal goat serum for 30 min slides

222

containing the specimens were incubated with the designated primary antibodies

223

overnight at 4°C and subsequently with secondary antibodies for an additional 30 min at

11

224

room temperature. We used a secondary goat anti-rabbit antibody conjugated with

225

cyanine 3 bis-N-hydroxysuccinimide (NHS) ester (Cy3; Jackson ImmunoResearch

226

Laboratories, Inc., West Grove, PA), a secondary goat anti-rabbit antibody conjugated

227

with Alexa Fluor® 488 (Invitrogen), or a secondary goat anti-mouse antibody conjugated

228

with fluorescein isothiocyanate (FITC; Sigma-Aldrich). Nuclei were visualized with 4′,6-

229

diamidino-2-phenylindole (DAPI; Molecular Probes, Carlsbad, CA). Trophoblast cells

230

were visualized by immunostaining with pan-cytokeratin antibodies. Fluorescence

231

images were captured using a Leica DMI 4000 microscope equipped with a charged-

232

coupled device camera (Leica Microsystems GMbH, Welzlar, Germany).

233 234

Analysis of DNA content – DNA content was estimated by flow cytometry as

235

previously described (31). Cells were trypsinized and fixed in 70% ethanol and then

236

stained with propidium iodine.

237 238

Chromatin immunoprecipitation (ChIP) – ChIP analysis was performed

239

according to a previously published procedure (20). Briefly, Rcho-1 TS cells stably

240

transduced with control, Fosl1 or Junb shRNA-expressing lentiviruses were grown to

241

confluence in 150-mm dishes and differentiated for 8 days. Cells were then fixed with

242

1% formaldehyde, and purified nuclear lysates were sonicated on ice to prepare DNA

243

fragments at a size of approximately 500 bp. Lysates were immunoprecipitated with 5

244

μg of FOSL1 or JUNB antibodies and collected on protein A agarose beads (Sigma-

245

Aldrich). Rabbit IgG (BD Biosciences) was used as a nonspecific control.

246

Immunoprecipitated chromatin fragments were washed and eluted from protein A

12

247

agarose beads. DNA-protein interactions were reverse cross-linked and purified using a

248

QiaQuick PCR purification kit (Qiagen). Purified DNA fragments were characterized by

249

quantitative PCR (qPCR) using primers listed in Table 3. Putative AP-1 response

250

elements (TGAGTCA) were targeted in the ChIP analysis and identified by searching

251

specific gene loci, including 10 kbp 5’ and 3’ flanking the locus, using rVista 2.0 (38;

252

http://rvista.dcode.org/). Occupancy/enrichment was normalized to input samples by

253

use of the ΔΔCT method and presented relative to IgG controls.

254 255

Co-immunoprecipitation – Rcho-1 TS cells or rat blastocyst-derived TS cells

256

were washed with ice-cold PBS and extracted in cell lysis buffer (Epitomics). Lysates

257

were immunoprecipitated using the designated antibodies and collected on protein-A

258

agarose beads. Rabbit IgG or mouse IgG (BD Biosciences) were used as a nonspecific

259

control. Immunoprecipitated protein complexes were washed with PBS, eluted with cell

260

lysate buffer, separated by SDS-PAGE, and processed for western blot analysis using

261

the Clean blot IP detection reagent (Thermo).

262 263

Matrigel invasion assay – In vitro cellular invasive activities were measured as

264

previously described (39). Rcho-1 TS cells were differentiated for 8 days, trypsinized,

265

and placed on Transwell inserts (6.5-mm diameter, 8-μM pore; BD Biosciences) coated

266

with 400 μg/ml phenol red-free Matrigel (BD Biosciences) at a density of 3 × 104 cells

267

per insert. Cells were initially plated in stem state medium overnight and then switched

268

to differentiation medium and allowed to invade for 72 h. Invaded cells situated on the

13

269

under surface of the membrane were stained with Diff-Quik (Dade Behring, Newark,

270

DE), visualized under stereomicroscopy, and counted.

271 272

Progesterone radioimmunoassay (RIA) – Measurements of progesterone in

273

medium conditioned by Rcho-1 TS cells were performed using RIA as previously

274

reported (40). Progesterone concentrations were normalized to cellular DNA content

275

with E.Z.N.A Tissue DNA kit (Omega Bio-Tek, Norcross, GA).

276 277

Statistical analyses – Values are expressed as the mean ± the standard error of

278

the mean (SEM). All experiments were conducted at least in triplicate and were

279

replicated two to three times. Statistical comparisons between two means were

280

evaluated with Student’s t test. Analysis of variance and Tukey’s post hoc tests were

281

used in assessing differences among three or more means using GraphPad Prism

282

(GraphPad Software Inc, La Jolla, CA).

283

14

284 285

RESULTS FOSL1 and the trophoblast lineage – The hemochorial placenta consists of

286

multiple trophoblast lineages. These cell types differentiate from TS cells and include

287

trophoblast giant cells, spongiotrophoblast cells, glycogen cells, invasive trophoblast

288

cells, and syncytial trophoblast (8, 9). The activation of specific sets of genes defines

289

each lineage. Monitoring expression of Prl3d1, Tpbpa, Prl5a1, Gcm1, and Pcdh12

290

provide insights into the development of trophoblast giant cells, spongiotrophoblast cells,

291

invasive trophoblast cells, syncytial trophoblast, and glycogen trophoblast cells (32, 34,

292

41). To evaluate mechanisms of trophoblast cell differentiation, we utilized Rcho-1 TS

293

cells, which can be induced to differentiate (32).

294

Expression of FOSL1 was low in the stem state Rcho-1 TS cells, as we reported

295

previously (Fig. 1A,B; 20, 31). After differentiation, FOSL1 expression was dramatically

296

increased, localized to nuclei, and correlated with elevated expression of trophoblast

297

cell lineage specific markers, Prl3d1, Tpbpa, Prl5a1, Gcm1, and Pcdh12 (Fig. 1A; 20,

298

31). We next evaluated a role for FOSL1 in trophoblast differentiation following genetic

299

manipulation by lentiviral-transduced Fosl1 shRNAs. FOSL1 expression was efficiently

300

inhibited in the differentiated trophoblast cells expressing Fosl1 shRNAs (Fig. 1C,D,E).

301

Knockdown of FOSL1 did not adversely affect trophoblast giant cell formation but did

302

significantly decrease trophoblast giant cell DNA content (Fig. 1F). Similar effects on

303

ploidy have been observed in Rcho-1 TS cells following inhibition of phosphatidylinositol

304

3 kinase (31). Please note that Rcho-1 TS cells are tetraploid (33). Disruption of

305

FOSL1 also compromised expression of trophoblast cell differentiation-associated

306

transcripts, Prl3d1 and Prl5a1, but not Gcm1 or Pcdh12 (Fig. 1G). Tpbpa transcript

15

307

levels significantly increased following FOSL1 knockdown, suggesting that FOSL1

308

represses Tpbpa expression and/or the presence of Tpbpa-expressing trophoblast cells.

309

These findings further support the involvement of FOSL1 as a regulator of the

310

differentiation of specific trophoblast cell lineages.

311 312

FOSL1 targets in differentiating trophoblast cells – Since FOSL1 is known to

313

interact with DNA and regulate transcription we next sought to identify components of

314

the transcriptome affected by FOSL1. A DNA microarray was performed in

315

differentiated trophoblast cells under control conditions and following FOSL1

316

knockdown. The array results have been deposited in the GEO database (Accession No.

317

GSE68272; http://www.ncbi.nlm.nih.gov/geo/). Transcripts regulated by FOSL1 were

318

functionally categorized into pathways regulating specific cellular functions (Fig. 1H,

319

Tables 4, 5, 6). A subset of the FOSL1-dependent transcripts was selected for further

320

analyses by qRT-PCR. This subset of transcripts each exhibited an increase in their

321

expression during trophoblast cell differentiation and a dependence on FOSL1 (Fig.

322

2A,B). We next determined whether FOSL1 interacted with putative AP1 binding motifs

323

located in the vicinity of these target genes. FOSL1 ChIP analysis was performed in the

324

differentiated trophoblast cells and demonstrated FOSL1 occupancy at consensus AP-1

325

binding sites within these target gene loci (Fig. 2C). Collectively, these observations

326

are consistent with a role for FOSL1 in the transcriptional regulation of trophoblast

327

differentiation.

328 329

We subsequently evaluated an in vivo role for FOSL1 using a trophoblast lineage specific gene knockdown strategy (20, 35). FOSL1 was strongly expressed in nuclei of

16

330

trophoblast giant cells within the uteroplacental interface at E11.5 and significantly

331

decreased following FOSL1 knockdown (Fig. 3A,B,C), similar to our previous

332

observations (20). In vivo FOSL1 knockdown significantly attenuated the expression of

333

Prl3d1, Grhl1 and Cyp11a1 (Fig. 3D). Immunohistochemical analysis further confirmed

334

the dependence of PRL3D1 and CYP11A1 expression on FOSL1 (Fig. 3E,F).

335

Discrepancies in FOSL1 targets identified using the in vitro versus in vivo models may

336

be linked to several factors, including: i) impact of distinct environmental factors

337

emanating from the uterine decidua and/or from the embryo affecting the in vivo

338

behavior of trophoblast cells, which are absent in the cell culture; ii) the presence of

339

unique factors in the in vitro culture conditions, which are absent in the in vivo model; iii)

340

distinct compositions of trophoblast lineages in the dissected in vivo specimens versus

341

the Rcho-1 TS cell cultures.

342 343

The findings of these experiments indicate that FOSL1 targets a defined set of genes activated during trophoblast cell differentiation.

344 345

GRHL1 regulation of trophoblast cell differentiation – Among the FOSL1

346

target genes was a transcription factor, GRHL1, an evolutionarily conserved regulator of

347

epithelial cell differentiation (42, 43) and a component of the human trophoblast

348

transcriptome (44, 45). GRHL1 belongs to the mammalian GRHL transcription factor

349

family, consisting of GRHL1, GRHL2 and GRHL3 (42, 43). Expression of Grhl1 was

350

robustly upregulated during trophoblast cell differentiation, whereas Grhl2 exhibited only

351

a modest increase accompanying differentiation (Fig. 4A,B). In contrast, Grhl3 was

352

highly expressed in the stem state and expression declined following differentiation (Fig.

17

353

4A). The differentiation-dependent increase in GRHL1 expression and its dependence

354

on FOSL1 led us to evaluate a possible role for GRHL1 as a mediator of FOSL1 actions

355

on the differentiated trophoblast cell phenotype. GRHL1 expression was successfully

356

inhibited with Grhl1 shRNAs (Fig. 4C,D). Disruption of GRHL1 expression did not

357

significantly affect trophoblast cell migration through a Matrigel extracellular matrix but

358

did inhibit a subset of FOSL1 targets, Prl3d1, Cgm4, and Ppap2b (Fig. 4E,F). These

359

findings suggest that GRHL1 contributes to the downstream actions of FOSL1 on

360

trophoblast cell differentiation.

361

FOSL1 interactions with the JUN family – FOSL1 requires a dimerization

362 363

partner to regulate gene transcription (14-16). This partner is often a member of the

364

JUN family (14-16). This led to an evaluation of potential FOSL1 partners in trophoblast

365

cells. JUN and JUNB were readily detected in nuclei of differentiating trophoblast cells,

366

whereas JUND expression was below the level of sensitivity of the assay (Fig. 5A). Co-

367

immunoprecipitation experiments demonstrated that FOSL1 interacts with JUN and

368

JUNB (Fig. 5B). The relative abundance of the interacting proteins was most

369

impressive between FOSL1 and JUNB. Dual-fluorescence immunohistochemistry

370

analysis demonstrated co-localization of FOSL1 with JUN and JUNB in trophoblast cells

371

from midgestation placentation sites (Fig. 5C,D). These findings suggest that FOSL1

372

heterodimerizes with JUNB and to a lesser extent with JUN in differentiating trophoblast

373

cells.

374

18

375

JUN family regulation of trophoblast cell differentiation – Physical

376

interactions of FOSL1 with JUN family members prompted an investigation of the

377

potential involvement of each JUN family member in FOSL1 guided trophoblast cell

378

differentiation. JUN, JUND, and JUNB expression was silenced in differentiating

379

trophoblast cells using specific sets of shRNAs. FOSL1 targets were evaluated

380

following JUN family member knockdown. Expression of each JUN family member was

381

successfully inhibited using two different shRNAs (Figs. 6A,B,D,E and 7A-C). JUN

382

knockdown showed strong inhibition of Prl3d1 expression and moderate, but significant,

383

inhibition of Crispld2 and Grhl1 expression (Fig. 6C). The expression of other FOSL1

384

target genes (Cgm4, Cyp11a1, Ppap2b, and Mmp9) was not significantly affected by

385

JUN knockdown. JUND knockdown did not affect expression of any of the previously

386

identified FOSL1 target genes (Fig. 6F). In contrast, JUNB knockdown phenocopied

387

FOSL1 knockdown. Each of the FOSL1 target genes investigated was significantly

388

inhibited by JUNB knockdown (Fig. 7D). JUNB ChIP analysis further demonstrated that

389

JUNB occupancy was increased at the target gene loci (Fig. 7E). The findings mirror

390

the co-immunoprecipitation results between FOSL1 and JUN family members in

391

differentiated trophoblast cells. In summary, JUN represents a redundant contributor to

392

the activation of a subset of FOSL1 targets, whereas the evidence indicates that FOSL1

393

and JUNB fully cooperate in the regulation of gene expression during trophoblast cell

394

differentiation.

395 396 397

FOSL1 and JUNB regulation of the trophoblast phenotype – Trophoblast cells exhibit a number of specialized functions, including their capacity to invade

19

398

extracellular matrices and produce steroid hormones. Trophoblast cell invasion is

399

critical for remodeling the uterine interface (46) and steroid hormone production,

400

especially progesterone, a hormone indispensible for the maintenance of pregnancy

401

(47). In experiments shown above (Figs. 2 and 7), FOSL1 and JUNB were directly

402

linked to the regulation of expression of Mmp9, which encodes a matrix

403

metalloproteinase implicated in trophoblast cell invasion (48, 49), and Cyp11a1, which

404

encodes the rate-limiting enzyme controlling progesterone biosynthesis (50).

405

Consequently, the effects of FOSL1 and JUNB knockdown on trophoblast cell invasion

406

and progesterone production were investigated. Disruption of either FOSL1 or JUNB

407

similarly inhibited the movement of trophoblast cells through Matrigel (Fig. 8A,B), an in

408

vitro measure of their invasive potential. Additionally, knockdown of either FOSL1 or

409

JUNB inhibited trophoblast cell CYP11A1 protein concentrations and progesterone

410

production (Fig. 8C,D). Since we obtained similar phenotypes when either FOSL1 or

411

JUNB were disrupted we examined potential roles of each transcription factor on the

412

expression of its partner. FOSL1 knockdown inhibited FOSL1 expression but not JUNB,

413

whereas JUNB knockdown inhibited both JUNB and FOSL1 expression (Fig. 8E,F).

414

ChIP analysis further demonstrated that both FOSL1 and JUNB occupancy within the

415

Fosl1 gene locus (Fig. 8G). Thus FOSL1 and JUNB also contribute to a positive

416

feedback regulation of FOSL1 expression during trophoblast differentiation.

417 418

FOSL1 and JUNB partnership in blastocyst-derived rat TS cell

419

differentiation – Roles for FOSL1 and JUNB were next investigated using blastocyst-

420

derived rat TS cells (Fig. 9A-D). Blastocyst-derived rat TS cells exhibit many

20

421

similarities to Rcho-1 TS cells but also some differences, including their dependence on

422

FGF4 for proliferation and capacity for differentiation (34). Similar to Rcho-1 TS cells,

423

rat blastocyst-derived TS cells prominently expressed JUN and JUNB, whereas JUND

424

expression was below the level of detection (Fig. 9A). Additionally, FOSL1 was shown

425

to specifically interact with JUN and JUNB (Fig. 9B) as was demonstrated in Rcho-1 TS

426

cells (Fig. 4B). Each of the FOSL1 and JUNB targets identified in Rcho-1 TS cells were

427

also validated for blastocyst-derived rat TS cells (Fig. 9C,D). Some differences in the

428

expression of JUN and JUNB were noted for rat TS cells, which included differentiation-

429

dependent increases in both JUN and JUNB expression (Fig. 9A). These trophoblast

430

cell-associated JUN and JUNB expression patterns may reflect the disparate conditions

431

used to culture Rcho-1 TS cells versus rat blastocyst-derived TS cells or alternatively

432

intrinsic differences in their developmental states (34).

433

Collectively, our findings indicate that FOSL1 and JUNB cooperate to control

434

elements of trophoblast cell differentiation, including acquisition of invasive and

435

endocrine properties.

436 437

21

438 439

DISCUSSION Elucidation of signaling pathways controlling trophoblast cell differentiation is a

440

key to understanding the process of placentation. In the present study, we utilized

441

Rcho-1 TS cells, rat blastocyst-derived TS cells, and developing rat placentation sites to

442

characterize the involvement of FOSL1 and JUNB in the regulation of trophoblast cell

443

differentiation. FOSL1 and JUNB cooperate in differentiating trophoblast cells to

444

regulate target genes dictating endocrine and invasive trophoblast lineages.

445 446

The expression pattern of FOSL1 was helpful in identifying it as a candidate

447

regulator of trophoblast cell differentiation. In the TS cell stem state, FOSL1 is

448

expressed at very low levels and does not possess a recognized function. Thus, stable

449

TS cells were readily established expressing shRNAs to Fosl1. These Fosl1 shRNAs

450

effectively inhibited the differentiation-dependent upregulation of FOSL1, altering the

451

differentiated trophoblast cell transcriptome, and the ability of trophoblast cells to invade

452

extracellular matrices and produce peptide and steroid hormones. FOSL1 targets such

453

as GRHL1 may in turn regulate elements of the FOSL1-directed differentiation pathway.

454

The actions of FOSL1 could be tracked to its occupancy at regions containing AP-1

455

binding motifs for several target genes identified by transcriptome profiling. This does

456

not represent the full breadth of FOSL1 involvement in regulating the differentiated

457

trophoblast cell genome. FOSL1 can be viewed as an entry point into a regulatory

458

network controlling trophoblast cell differentiation. Identification of its genome-wide

459

binding sites via ChIP sequencing and its binding partners will provide additional insight

460

into the regulation of trophoblast cell differentiation.

22

461 462

We utilized a candidate approach to identify FOSL1 interacting proteins. FOSL1

463

is known to bind to JUN family proteins (14-16). Both JUN and JUNB were shown to

464

bind to FOSL1 and participate in some of its actions. Among the FOSL1 targets and

465

cellular responses examined in this report JUNB knockdown phenocopied FOSL1

466

knockdown. FOSL1 and JUNB have been independently implicated as regulators of

467

placental development through mouse mutagenesis experiments (29, 30). Furthermore,

468

disruption of the Ppap2b gene, a target for FOSL1 action (present study), also leads to

469

abnormalities in placentation resembling the phenotypes of the Fosl1 and Junb null

470

mouse models (51). Mutagenesis of the Jun gene results in embryonic death due to

471

liver pathologies and no overt placental abnormalities (52), implying that its role in the

472

placenta may overlap with those of other genes. JUN and JUNB exhibit an increase in

473

abundance accompanying differentiation of rat blastocyst-derived TS cells, whereas,

474

Rcho-1 TS cells express JUN and JUNB similarly in stem and differentiated states.

475

Disrupting JUN or JUNB expression in the Rcho-1 TS cell stem state slowed cell

476

proliferation, suggesting that JUN and JUNB could be acting as homodimers or

477

interacting with other JUN or FOS family members or some other protein partner(s).

478

Furthermore, the implication is that the differentiation-dependent increase in FOSL1

479

redirects JUN and JUNB to a new set of targets characteristic of the differentiated

480

trophoblast cell phenotype. JUND was not identified as a participant in the actions of

481

FOSL1. Nonetheless, JUND is a contributor to the regulation of placentation. JUND is a

482

positive regulator of glial cell missing 1 (GCM1) in developing trophoblast cells (53).

483

GCM1 is a transcription factor and is critical for development of the syncytial trophoblast

23

484

lineage and bidirectional transport of nutrients and wastes between maternal and fetal

485

compartments (54, 55). Thus, at least in part, FOSL1 and JUND expression patterns

486

are segregated and contribute to distinct interfaces – trophoblast-maternal versus

487

trophoblast-fetal, respectively. Rcho-1 TS cells and rat blastocyst-derived TS cells do

488

not readily differentiate into syncytial trophoblast (32-34), which is consistent with our

489

inability to demonstrate the involvement of JUND in the regulation of trophoblast

490

differentiation. In addition to the JUN family, FOSL1 is known to interact with several

491

other regulatory proteins (56); however, the nature of these and other potential protein

492

interactions to FOSL1 regulation of trophoblast differentiation remains to be determined.

493

In summary, FOSL1 and JUNB play a fundamental role in regulating trophoblast

494

differentiation. They exhibit overlapping expression patterns in trophoblast cells, they

495

interact with each other, and share target genes. Target genes for FOSL1 and JUNB

496

include genes encoding proteins contributing to cell motility, invasion and endocrine

497

functions of trophoblast cells. We conclude that FOSL1 recruitment of JUNB to specific

498

targets within the genome during differentiation is a key regulatory step in the

499

acquisition of trophoblast cell invasive and endocrine phenotypes.

500

24

501

ACKNOWLEDGEMENTS

502

This work was supported by the National Institutes of Health (HD020676,

503

HD079363, GM103418). We thank Dr. Dong-soo Lee and Dr. Adam Krieg (University

504

of Kansas Medical Center, Kansas City, KS) for assistance and advice with the in vivo

505

lentiviral knockdown and ChIP analyses, respectively. We also appreciate the

506

comments and advice of Dr. Michael W. Wolfe, KUMC, during various stages of this

507

project and thank Stacy McClure, Lesley Shriver, and Martin Graham for administrative

508

assistance.

509

25

510 511 512 513 514 515 516 517 518 519 520 521

REFERENCES 1. Amoroso, E.C. (1968) The evolution of viviparity. Proc. R. Soc. Med. 61, 11881200 2. Wooding, F.B.P. and Burton, G.J. (2008) Comparative Placentation – Structures, Functions, and Evolution. Springer-Verlag, Berlin, Heidelberg 3. Rossant, J. (2001) Stem cells from the mammalian blastocyst. Stem Cells 19, 477-482 4. Cockburn, K. and Rossant, J. (2010) Making the blastocyst: lessons from the mouse. J. Clin. Invest. 120, 995-1003 5. Gardner, R.L. and Beddington, R.S. (1988) Multi-lineage 'stem' cells in the mammalian embryo. J. Cell Sci. 10 (Suppl),11-27 6. Tanaka, S., Kunath, T., Hadjantonakis, A.K., Nagy, A., and Rossant, J. (1998)

522

Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072-

523

2075

524

7. Guzman-Ayala, M., Ben-Haim, N., Beck, S., and Constam, D.B. (2004) Nodal

525

protein processing and fibroblast growth factor 4 synergize to maintain a

526

trophoblast stem cell microenvironment. Proc. Natl. Acad. Sci. USA 101, 15656-

527

15660

528

8. Soares, M.J., Chapman, B.M., Rasmussen, C.A., Dai, G., Kamei, T., and Orwig,

529

K.E. (1996) Differentiation of trophoblast endocrine cells. Placenta 17, 277-289

530

9. Soares, M.J., Chakraborty, D., Rumi, M.A.K., Konno, T., and Renaud, S.J. (2012)

531

Rat placentation: an experimental model for investigating the hemochorial

532

maternal-fetal interface. Placenta 33, 233-243

26

533 534 535 536 537 538 539 540 541 542 543 544 545 546 547

10. Simmons, D.G. and Cross, J.C. (2005) Determinants of trophoblast lineage and cell subtype specification in the mouse placenta. Dev. Biol. 284,12-24 11. Roberts, R.M. and Fisher, S.J. (2011) Trophoblast stem cells. Biol. Reprod. 84, 412-421 12. Pfeffer, P.L. and Pearton, D.J. (2012) Trophoblast development. Reproduction 143, 231-246 13. Latos, P.A. and Hemberger, M. (2014) Review: the transcriptional and signalling networks of mouse trophoblast stem cells. Placenta 35 (Suppl), S81-S85 14. Shaulian, E. and Karin, M. (2002) AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, E131-E136 15. Hess, J., Angel, P., and Schorpp-Kistner, M. (2004) AP-1 subunits: quarrel and harmony among siblings. J. Cell Sci. 117, 5965-5973 16. Eferl, R. and Wagner, E.F. (2003) AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer 3, 859-868 17. Bamberger, A.M., Bamberger, C.M., Aupers, S., Milde-Langosch, K., Löning, T.,

548

and Makrigiannakis, A. (2004) Expression pattern of the activating protein-1

549

family of transcription factors in the human placenta. Mol. Hum. Reprod. 10, 223-

550

228

551

18. Briese, J., Sudahl, S., Schulte, H.M., Löning, T., and Bamberger, A.M. (2005)

552

Expression pattern of the activating protein-1 family of transcription factors in

553

gestational trophoblastic lesions. Int. J. Gynecol. Pathol. 24, 265-270

554 555

19. Marzioni, D., Todros, T., Cardaropoli, S., Rolfo, A., Lorenzi, T., Ciarmela, P., Romagnoli, R., Paulesu, L., and Castellucci, M. (2010) Activating protein-1 family

27

556

of transcription factors in the human placenta complicated by preeclampsia with

557

and without fetal growth restriction. Placenta 31, 919-927

558

20. Kent, L. N., Rumi, M. A., Kubota, K., Lee, D. S., and Soares, M. J. (2011)

559

FOSL1 is integral to establishing the maternal-fetal interface. Mol. Cell. Biol. 31,

560

4801-4813

561

21. Renaud, S.J., Kubota, K., Rumi, M.A., and Soares, M.J. (2014) The FOS

562

transcription factor family differentially controls trophoblast migration and

563

invasion. J. Biol. Chem. 289, 5025-5039

564

22. Shida, M.M., Ng, Y.K., Soares, M.J., and Linzer, D.I. (1993) Trophoblast-specific

565

transcription from the mouse placental lactogen-I gene promoter. Mol.

566

Endocrinol. 7, 181-188

567

23. Sun, Y. and Duckworth, M.L. (1999) Identification of a placental-specific

568

enhancer in the rat placental lactogen II gene that contains binding sites for

569

members of the Ets and AP-1 (activator protein 1) families of transcription

570

factors. Mol. Endocrinol. 13, 385-399

571

24. Orwig, K.E. and Soares, M.J. (1999) Transcriptional activation of the

572

decidual/trophoblast prolactin-related protein gene. Endocrinology 140, 4032-

573

4039

574

25. Peters, T.J., Chapman, B.M., Wolfe, M.W., and Soares, M.J. (2000) Placental

575

lactogen-I gene activation in differentiating trophoblast cells: extrinsic and

576

intrinsic regulation involving mitogen-activated protein kinase signaling pathways.

577

J. Endocrinol. 165, 443-456

28

578 579

26. Bischof, P., Truong, K., and Campana, A. (2003) Regulation of trophoblastic gelatinases by proto-oncogenes. Placenta 24, 155-163

580

27. Abell, A.N., Granger, D.A., Johnson, N.L., Vincent-Jordan, N., Dibble, C.F., and

581

Johnson, G.L. (2009) Trophoblast stem cell maintenance by fibroblast growth

582

factor 4 requires MEKK4 activation of Jun N-terminal kinase. Mol. Cell. Biol. 29,

583

2748-2761

584

28. Kashif, M., Hellwig, A., Hashemolhosseini, S., Kumar, V., Bock, F., Wang, H.,

585

Shahzad, K., Ranjan, S., Wolter, J., Madhusudhan, T., Bierhaus, A., Nawroth, P.,

586

and Isermann, B. (2012) Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD

587

DNA-binding activity via acetylation: a novel mechanism regulating trophoblast

588

differentiation. J. Biol. Chem. 287, 5400-5411

589 590 591

29. Schorpp-Kistner, M., Wang, Z.Q., Angel, P., Wagner, E.F. (1999) JunB is essential for mammalian placentation. EMBO J. 18, 934-948 30. Schreiber, M., Wang, Z.Q., Jochum, W., Fetka, I., Elliott, C., and Wagner, E.F.

592

(2000) Placental vascularisation requires the AP-1 component fra1. Development

593

127, 4937-4948

594

31. Kent, L.N., Konno, T., and Soares, M.J. (2010) Phosphatidylinositol 3 kinase

595

modulation of trophoblast cell differentiation. BMC Dev. Biol. 10, 97

596

32. Faria, T.N. and Soares, M.J. (1991) Trophoblast cell differentiation:

597

establishment, characterization, and modulation of a rat trophoblast cell line

598

expressing members of the placental prolactin family. Endocrinology 129, 2895-

599

2906

29

600

33. Sahgal, N., Canham, L.N., Canham, B., and Soares, M.J. (2006) Rcho-1

601

trophoblast stem cells: a model system for studying trophoblast cell

602

differentiation. Methods Mol. Med. 121, 159-178

603

34. Asanoma, K., Rumi, M.A.K., Kent, L.N., Chakraborty, D., Renaud, S.J., Wake,

604

N., Lee, D.-S., Kubota, K., and Soares, M.J. (2011) FGF4-dependent stem cells

605

derived from rat blastocysts differentiate along the trophoblast lineage. Dev. Biol.

606

351, 110-119

607

35. Lee, D. S., Rumi, M. A., Konno, T., and Soares, M. J. (2009) In vivo genetic

608

manipulation of the rat trophoblast cell lineage using lentiviral vector delivery.

609

Genesis 47, 433-439

610

36. Roby, K.F., Larsen, D., Deb, S., and Soares, M.J. (1991) Generation and

611

characterization of antipeptide antibodies to rat cytochrome P-450 side-chain

612

cleavage enzyme. Mol. Cell. Endocrinol. 79, 13-20

613

37. Hamlin, G.P., Lu, X.J., Roby, K.F., and Soares, M.J. (1994) Recapitulation of the

614

pathway for trophoblast giant cell differentiation in vitro: stage-specific expression

615

of members of the prolactin gene family. Endocrinology 134, 2390-2396

616

38. Loots, G.G. and Ovcharenko, I. (2004) rVISTA 2.0: evolutionary analysis of

617 618

transcription factor binding sites. Nucleic Acids Res. 32, W217-W221 39. Peters, T.J., Albieri, A., Bevilacqua, E., Chapman, B.M., Crane, L.H., Hamlin,

619

G.P., Seiki, M., and Soares, M.J. (1999) Differentiation-dependent expression of

620

gelatinase B/matrix metalloproteinase-9 in trophoblast cells. Cell Tissue Res.

621

295, 287-296

30

622

40. Terranova, P.F. and Garza, F. (1983) Relationship between the preovulatory

623

luteinizing hormone (LH) surge and androstenedione synthesis of preantral

624

follicles in the cyclic hamster: detection by in vitro responses to LH. Biol. Reprod.

625

29, 630-636

626

41. Rampon, C., Prandini, M., Bouillot, S., Pointu, H., Tillet, E., Frank, R., Vernet,

627

M., and Huber, P. (2005) Protocadherin 12 (VE-cadherin 2) is expressed in

628

endothelial, trophoblast, and mesangial cells. Exp. Cell Res. 302, 48-60

629

42. Moussian, B. and Uv, A.E. (2005) An ancient control of epithelial barrier

630 631

formation and wound healing. BioEssays 27, 987-990 43. Wang, S. and Samakovlis, C. (2012) Grainy head and its target genes in

632

epithelial morphogenesis and wound healing. Current Topics Dev. Biol. 98, 35-

633

63

634

44. Henderson, Y.C., Frederick, M.J., Jayakumar, A., Choi, Y., Wang, M.T., Kang,

635

Y., Evans, R., Spring, P.M., Uesugi, M., and Clayman, G.L. (2007) Human LBP-

636

32/MGR is a repressor of the P450scc in human choriocarcinoma cell line JEG-3.

637

Placenta 28, 152-160

638

45. Henderson, Y.C., Frederick, M.J., Wang, M.T., Hollier, L.M., and Clayman, G.L.

639

(2008) LBP-1b, LBP-9, and LBP-32/MGR detected in syncytiotrophoblasts from

640

first-trimester human placental tissue and their transcriptional regulation. DNA

641

Cell Biol. 27, 71-79

642 643

46. Knöfler, M. (2010) Critical growth factors and signalling pathways controlling human trophoblast invasion. Int. J. Dev. Biol. 54, 269-280

31

644

47. Spencer, T.E. and Bazer, F.W. (2002) Biology of progesterone action during

645

pregnancy recognition and maintenance of pregnancy. Front. Biosci. 7, 1879-

646

1898

647

48. Librach, C.L., Werb, Z., Fitzgerald, M.L., Chiu, K., Corwin, N.M., Esteves, R.A.,

648

Grobelny, D., Galardy, R., Damsky, C.H., and Fisher, S.J. (1991) 92-kD type IV

649

collagenase mediates invasion of human cytotrophoblasts. J. Cell. Biol. 113, 437-

650

449

651

49. Plaks, V., Rinkenberger, J., Dai, J., Flannery, M., Sund, M., Kanasaki, K., Ni, W.,

652

Kalluri, R., and Werb, Z. (2013) Matrix metalloproteinase-9 deficiency

653

phenocopies features of preeclampsia and intrauterine growth restriction. Proc.

654

Natl. Acad. Sci. USA 110, 11109-11114

655 656 657

50. Miller, W.L. and Auchus, R.J. (2011) The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr. Rev. 32, 81-151 51. Escalante-Alcalde, D., Hernandez, L., Le Stunff, H., Maeda, R., Lee, H.S., Jr-

658

Gang-Cheng, Sciorra, V.A., Daar, I., Spiegel, S., Morris, A.J., and Stewart, C.L.

659

(2003) The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis

660

and axis patterning. Development 130, 4623-4637

661 662 663

52. Hilberg, F., Aguzzi, A., Howells, N., and Wagner, E.F. (1993) c-Jun is essential for normal mouse development and hepatogenesis. Nature 365, 179-181 53. Kashif, M., Hellwig, A., Hashemolhosseini, S., Kumar, V., Bock, F., Wang, H.,

664

Shahzad, K., Ranjan, S., Wolter, J., Madhusudhan, T., Bierhaus, A., Nawroth, P.,

665

and Isermann, B. (2012) Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD

32

666

DNA-binding activity via acetylation: a novel mechanism regulating trophoblast

667

differentiation. J. Biol. Chem. 287, 5400-5411

668

54. Anson-Cartwright, L., Dawson, K., Holmyard, D., Fisher, S.J., Lazzarini, R.A.,

669

and Cross, J.C. (2000) The glial cells missing-1 protein is essential for branching

670

morphogenesis in the chorioallantoic placenta. Nat. Genet. 25, 311-314

671

55. Lu, J., Zhang, S., Nakano, H., Simmons, D.G., Wang, S., Kong, S., Wang, Q.,

672

Shen, L., Tu, Z., Wang, W., Wang, B., Wang, H., Wang, Y., van Es, J.H.,

673

Clevers, H., Leone, G., Cross, J.C., and Wang, H. (2013) A positive feedback

674

loop involving Gcm1 and Fzd5 directs chorionic branching morphogenesis in the

675

placenta. PLoS Biol. 11, e1001536

676

56. Reinke, A.W., Baek, J., Ashenberg, O., and Keating, A.E. (2013) Networks of

677

bZIP protein-protein interactions diversified over a billion years of evolution.

678

Science 340, 730-734

679 680 681

33

682

FIGURE LEGENDS

683

Fig. 1 FOSL1 regulation of the trophoblast cell phenotype. A) qRT-PCR analysis of

684

Fosl1, Prl3d1, Tpbpa, Prl5a1, Gcm1, and Pcdh12 expression in stem (Stem) and

685

differentiated (Diff) Rcho-1 TS cells. B) Western blot analysis of FOSL1 and GAPDH

686

expression in stem (Stem) and differentiated (Diff) Rcho-1 TS cells. FOSL knockdown

687

efficiency was validated by qRT-PCR (C) and western blot (D) analyses in differentiated

688

Rcho-1 TS cells expressing control (Ctrl) or Fosl1 shRNAs. GAPDH was used as an

689

internal control for the western blot analysis. E) FOSL1 localization and knockdown

690

efficiency were assessed by immunocytochemistry in stem and differentiated Rcho-1 TS

691

cells expressing Ctrl or Fosl1 shRNAs. FOSL1 was localized to nuclei (red). Cells were

692

counterstained for cytokeratin (CK, green) and/or DAPI (blue). Scale Bar = 100 μm. F)

693

DNA content was estimated by propidium iodine staining followed by flow cytometry. G)

694

qRT-PCR analysis of Prl3d1, Tpbpa, Prl5a1, Gcm1, and Pcdh12 transcripts in

695

differentiated Rcho-1 TS cells expressing Ctrl or Fosl1 shRNAs. H) Scatter plot of

696

microarray analysis shown as the natural logarithm of raw value signals for

697

differentiated Rcho-1 TS cells expressing Ctrl (x axis) or Fosl1 (y axis) shRNAs. The

698

magnitudes of fold changes (FC) are depicted by different colored dots. Bars from

699

histograms in panels A, C, and F represent the mean ± SEM. Values with an asterisk or

700

different characters indicates significantly difference (P