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BOR - Papers in Press, published online ahead of print January 31, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056820
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BIOLOGY OF REPRODUCTION 76, 884–891 (2007)
DOI: 10.1095/biolreprod.106.056820
© 2007 by the Society for the Study of Reproduction, Inc.

Heat Shock Protein 1 and the Mitogen-Activated Protein Kinase 14 Pathway Are Important for Mouse Trophoblast Stem Cell Differentiation1

Quinton A. Winger 2, Jillian Guttormsen , Heather Gavin , and Frances Bhushan 

Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah 84322-5600

ABSTRACT

Differentiation of trophoblast cells is a critical process for the proper establishment of the placenta and is, therefore, necessary to maintain embryonic development. Trophoblast stem (TS) cells grown in culture can differentiate into different trophoblast subtypes in vitro mimicking normal trophoblast cell differentiation. Therefore, TS cells are a valuable model system that can be used to elucidate genetic factors that regulate trophoblast cell differentiation. Several transcription factors, when analyzed by targeted gene mutation in mice, have resulted in embryonic lethality due to placental defects and, more specifically, defects of the trophoblast lineages. These studies have helped improve our knowledge about trophoblast cell differentiation, but much is still unknown about the specific mechanisms involved. This study uses TS cell culture to detect proteins with differential expression in proliferating and differentiating TS cells in order to identify proteins with potential roles in the differentiation process. We identified four proteins with differential expression: dimethylarginine dimethylaminohydrolase1 (DDAH1), keratin 8, keratin 18, and HSPB1 (also known as heat shock protein 25, HSP25). Further investigation confirmed the presence of HSPB1 protein during in vitro TS cell differentiation. In addition, we confirmed that phosphorylation of HSPB1 and MAP kinase-activated protein kinase 2 (MAPKAPK2) increased in TS cells during differentiation. Inhibition of MAPK14 (also known as p38 MAPK) resulted in a reduction of HSPB1 phosphorylation and an increase in cell death during TS cell differentiation. These results suggest that HSPB1 and the MAPK14 pathway are important during TS cell differentiation.

early development, kinases, placenta, pregnancy, trophoblast

INTRODUCTION

Differentiation of trophoblast cells is essential during placental growth for the production of different trophoblast cell lineages necessary to perform specific functions. The formation of the blastocyst represents the first specification of cell type, resulting in the inner cell mass (ICM), which will become the embryo proper, and the trophectoderm, which is developmentally restricted to become the fetal portion of the placenta and the trophoblast giant cells [1, 2]. Mural trophectoderm cells are those not in contact with the ICM and, at implantation, these differentiate into primary trophoblast cells that will invade the uterus. Polar trophectoderm cells remain in direct contact with the ICM and receive mitogenic signals from the ICM and, later, the epiblast, to remain proliferative. These proliferative cells form a trophoblast stem cell population located in the extraembryonic ectoderm compartment. These stem cells populate the ectoplacental cone, and as they become removed from the epiblast, they become less proliferative and begin the differentiation pathway, developing into the secondary trophoblast giant cells [3]. Giant cells of the placenta are characterized by location, size, specific gene expression, and a large nucleus, the result of endoreduplication.

The Rcho-1 trophoblast stem cell line was derived from a rat choriocarcinoma [4] and has been used to study many aspects of TS cells, including differentiation [4–8] and cell-specific transcription [9, 10]. However, when using a cancer cell line like the Rcho-1 cells, caution must always be taken to ensure that any molecular mechanisms detected are the result of the characteristic TS cell type, and not associated with the transformed phenotype [8]. Therefore, it is advantageous to perform trophoblast differentiation studies using in vitro mouse TS cell models [3]. Several studies have been completed using mouse TS cell lines to demonstrate their potential as a research model [11–15]. TS cells have been established from Embryonic Day 3.5 mouse embryos, and have been maintained in culture by supplementing the culture media with FGF4, heparin, and embryonic fibroblast (EMFI)-conditioned media [3, 16]. TS cells can be maintained in culture in a proliferative state indefinitely, or can be induced to differentiate, mostly into giant cells, by the removal of FGF4, heparin, and conditioned media from the culture conditions [17]. Retinoic acid (RA) treatment of TS cells during culture has been shown to force differentiation of TS cells into giant cells even when the cells are cultured under proliferating conditions [13]. In addition, RA treatment of TS cells during differentiation preferentially produced giant cells and inhibited spongiotrophoblast cells usually seen during in vitro TS cell differentiation. This lack of spongiotrophoblast cells was confirmed by the inability to detect Tpbpa transcripts, a marker for spongiotrophoblast cells, in these cells. It is suggested that the RA acts on a population of cells that have begun differentiation, as indicated by the expression of Ascl2 (formerly known as Mash2) mRNA, and forces these cells into the terminally differentiated giant cell fate.

In addition to Fgf4, several other genes have been identified that play a role in preventing differentiation or that act directly in the process of differentiation. Several gene mutations produce placental defects as a result of the disruption of trophoblast cell development. The deletion of the Ascl2 gene results in a phenotype characterized by a loss of spongiotrophoblast cells and an increase in giant cells [18, 19]. The opposite effect is observed in animals containing a Hand1 gene deletion, which results in a block of terminal differentiation of trophoblast cells into giant cells [6, 20]. As might be expected, targeted mutations for each of these genes result in embryonic lethality due to placental abnormalities. Interestingly, there are a number of targeted mutations, of other transcription factor genes, in addition to Ascl2 and Hand1 that lead to embryonic lethality due to placental defects [2] like Tcfap2c [21], Cdx2 [22], Eomes [23], Elf5 [12], and Gcm1 [24]. The retinoblastoma (Rb1) gene encodes a nuclear protein that acts as a cell-cycle control checkpoint and interacts with the transcription factor E2F3. Disruption of either Rb1 or the E2f3 gene results in abnormal cellular proliferation and produces placental defects [11].

The ability to maintain TS cells in culture and to control their differentiation in vitro has become a powerful tool for investigating gene regulation of trophoblast cell differentiation. Using this model system, it is possible to identify genes and proteins that are expressed during proliferative, differentiating, and differentiated stages of trophoblast cell growth. In an attempt to identify additional transcription factors or other nuclear regulatory proteins involved in the gene regulation of TS cell differentiation, the current study identified proteins present in the nucleus of differentiated TS cells that were not present in the proliferating cells.

MATERIALS AND METHODS

Trophoblast Stem Cell Culture

TS cells were derived from blastocyst-stage embryos at 3.5 days postcoitum (dpc) from naturally bred female mice. All animal experiments were performed in accordance with protocols approved by the Utah State University Institutional Animal Care and Use Committee. TS cells were derived and maintained according to previously described protocols [3, 16]. After initial establishment, TS cells were maintained in vitro without EMFI feeders in a proliferative state by culturing in conditions including 70% EMFI-conditioned medium, 30% TS cell medium containing, 20% ES cell-tested fetal bovine serum (Tissue Culture Biologicals, Tulare, CA), 25 ng/ml FGF4 (Sigma) and 1 µg/ml heparin (Sigma). EMFI-conditioned medium was produced by addition of 10 ml of TS cell media [3, 16] to 100-mm culture plates containing 2 x 106 mitomycin-C (Sigma)-treated EMFIs. Conditioned medium was collected following 3 days in culture and was filtered and stored at –20°C. Cells were used for up to three collections. TS cells were forced to differentiate into trophoblast giant cells by removing FGF4, heparin, and fibroblast-conditioned media from the culture medium. Maintenance of TS cell proliferation and confirmation of differentiation was performed by RT-PCR detection of the TS cell marker genes Esrrb and Cdx2 in proliferating TS cells, and the markers of TS cell differentiation, Tpbpa, placental lactogen I (Csh1), and placental lactogen II (Csh2), in differentiated cells. Primer sequences were: Esrrb-F 5'-CGC CAT CAA ATG CGA GTA CAT GC; Esrrb-R 5'-GAA TCA CCA TCC AGG CAC TCT G; Cdx2-F 5'-CGC CAC CAT GTA CGT GAG CT; Cdx2-R 5'-GTC ACT GGG TGA CAG TGG AG; Tpbpa-F 5'-TGA AGA GCT GAA CCA CTG GA; Tpbpa-R 5'-CAG GCA GTT CAT ATG TTG GG [25]; Csh1-F 5'-CTG CTG ACA TTA AGG GCA; Csh1-R 5'-AAC AAA GAC CAT GTG GGC [25]; Csh2-F 5'-TCC TTC TCT GGG GCA CTC CTG TT; Csh2-R 5'-CCA TGA AGG CTT TTG AAG CAA GAT CA [25]; β-actin-F 5'-GAT GAC GAT ATC GCT GCG CTG; and β-actin-R 5'-GTA CGA CCA GAG GCA TAC AGG [26]. PCR reactions were performed in the presence of 1.5 mM MgCl2 for 35 cycles with 30 sec at 94°C, 30 sec at 60°C, and 50 sec at 72°C, except for Cdx2, which was run for 40 cycles with 60 sec at 94°C, 60 sec at 68°C, and 60 sec at 72°C.

Protein Analysis by Two-Dimensional Gel Electrophoresis

Protein extraction was performed for 2-D gel analysis on proliferating TS cells (Day 0 [D0]) and differentiated cells (D6). Nuclear and cytoplasmic fractions were collected using the NucBuster Protein Extraction Kit (Novagen) according to the kit specifications. Nuclear and cytoplasmic fraction purity was confirmed by detection of a known nuclear-specific protein, activating protein (AP)-2{gamma}, by Western blot using a specific primary antibody against AP-2{gamma} (NovaCastra). Protein concentration was measured by the bicinchoninic acid protein assay (Pierce) using BSA as a standard, and samples were prepared for focusing using the 2-D Cleanup Kit (BioRad).

Isoelectric focusing (IEF) was performed on immobilized pH gradients (IPG; pH 4–7, 11 cm; BioRad) rehydrated in 200 µl of protein sample containing 300 µg of protein in ReadyPrep Rehydration/Sample buffer (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 50 mM dithiothreitol, and 0.2% [w/v] 3–10 ampholytes; BioRad). Strips were rehydrated for 12 h, followed by focusing on a Protean IEF Cell (BioRad) for 20 000–35 000 V-h at a setting of 8000 V at 20°C. The IPG strips were then equilibrated in Equilibration Buffer I and Buffer II (BioRad), and transferred to a second dimension of separation in an 8%–16% Tris-HCl gel (Critereon; BioRad). The gels were then fixed, and proteins were stained using Coomassie R-250.

Protein spots of interest were excised from the gel using an Ettan Spot Picker (Amersham Biosciences). Proteins were digested in trypsin in preparation for matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) performed by the Center for Integrated Biosystems, Utah State University. Mass fingerprinting database searching was performed using open access online databases.

Immunoblot Analysis

Quantification of heat shock protein 1 (HSPB1) and MAP kinase-activated protein kinase 2 (MAPKAPK2) protein was performed by Western blot analysis. Samples containing 50 µg of protein were separated by 12% SDS-PAGE, and proteins were transferred to polyvinylidene fluoride membranes (BioRad). Membranes were blocked in Super Block (Pierce) for 1 h at room temperature, the membrane was then incubated in primary antibody against HSPB1 (Calbiochem), phosphor-HSPB1 (Cell Signaling Technology), or phosphor-MAPKAPK2 (Cell Signaling Technology), followed by incubation with anti-rabbit secondary antibody (Rockland) and visualization using SuperSignal (Pierce) on a digital gel documentation system (UVP). To control for variation in loading, corrected values were calculated as the ratio relative to histone (nuclear, primary antibody; Chemicon) and β-actin (cytoplamic, primary antibody; Rockland).

Quantitative Real-Time RT-PCR

TS cells were lysed on the culture plates, and RNA was extracted using the RNeasy mini kit (Qiagen) according to the kit protocol. RNA quantification was performed on an Eppendorf Biosystem Photometer. Relative levels of gene expression were determined by quantitative real-time RT-PCR using the Taqman probes specific for Hspb1 (ABI). Complimentary DNA samples were prepared using 1 µg of RNA in each reaction, and 0.01 µg of cDNA was then added to each PCR reaction. β-Actin was used as an endogenous control for quantification. Reactions were performed in triplicate on a iCycler (BioRad) on three separate TS cell replicates. Relative levels of expression were compared by analyzing the cycle number (Ct) at which each reaction reached a threshold value of fluorescence. Relative level of expression was determined for the Ct from a standard curve for both Hspb1 and β-actin. Standard curves were generated using 10-fold serial dilutions of template cDNA. Relative levels for each sample were calculated using the Ct values and the respective standard curves. Values were normalized to endogenous β-actin levels and calculated as a percentage of maximum expression, with the maximum expression representing the level at D6. Differences in expression over days of differentiation were determined by a single-factor ANOVA with significance at P < 0.05. This analysis was followed by a pair-wise comparison between groups (Student t-test, P < 0.05) to evaluate differences between days.

MAPK14 Inhibition Experiments

TS cells were grown in culture with the inhibitor, SB220025 (Calbiochem), a class of compounds that specifically inhibit both MAPK14{alpha} and -β, or the control compound, SB202474 (Calbiochem), at concentrations of 2 µM and 10 µM [27]. Cells were plated in media containing the inhibitor or the control compound under conditions supportive of TS cell proliferation. After 24 h in culture, protein and RNA, sample Day 0 was collected, and the remaining cultures were subjected to both differentiation and proliferation culture conditions until collection at 2, 4, and 6 days. Cultures were fed fresh media containing inhibitor every 24 h. Total protein was extracted using the MPER reagent (Pierce) containing the Halt Protease Inhibitor Cocktail (Pierce), following the kit protocol, and RNA was extracted as described in the previous section. Samples were analyzed by Western blot analysis or RT-PCR.

The effect of MAPK14 inhibition during TS cell differentiation was determined by uptake of neutral red stain—a measurement of cell viability. Cells were cultured for 2, 4, and 6 days in the presence of either SB220025 or SB202474 in both differentiation and proliferation conditions. A 0.034% neutral red solution was added to each well and incubated for 2 h at 37°C with 5% CO2. After incubation, the wells were rinsed, dried, and stored in the dark until dye extraction. The dye was extracted for 30 min at room temperature in the dark in absolute ethanol buffered with Sorenson citrate buffer. Buffer was transferred in duplicate to 96-well plates and was read at 540 nm on a Bio-Tek EL800 microplate reader (Bio-Tek Instruments, Inc.). Significance was determined by comparing optical density at 540 nm readings by a two-factor ANOVA for day of differentiation and effect of drug, with significance determined at P < 0.05.

RESULTS

Identification of Nuclear Proteins Expressed During TS Cell Differentiation

Nuclear proteins were extracted from proliferating and differentiated TS cells for 2-D analysis. TS cell differentiation was confirmed by cell morphology and by RT-PCR detection of mouse Esrrb and Cdx2 in proliferating TS cells, and Tpbpa, Csh1, and Csh2 in differentiated TS cells (Fig. 1A). The nuclear and cytoplasmic protein extractions were verified by Western blot detection of transcription factor AP-2{gamma} protein, a known nuclear protein localized to the nuclear compartment in trophoblast cells (Fig. 1B).


Figure 01
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  		FIG. 1. Verification of TS cell differentiation. A) Detection of TS stem cell markers Cdx2 and Esrrb mRNA transcripts in D0 confirmed the maintenance of TS cells in a proliferating stem cell state. Detection of spongiotrophoblast cell marker Tpbpa, primary giant cell marker Csh1, and secondary giant cell marker Csh2 confirms differentiation of TS cells following removal of conditioned media, FGF4, and heparin. B) Western blot detection of a nuclear protein, AP-2{gamma} verifies the isolation of nuclear and cytoplasmic protein fractions.

Two-dimensional experiments were repeated three times using different replicates of proliferating and differentiating TS cells. Similar protein spot patterns were obtained for the replicates. The overall pattern of proteins was similar in both the proliferating and the differentiated cells, indicating that the majority of nuclear TS cell proteins are expressed in both proliferating and differentiating cells (Fig. 2). To identify proteins potentially necessary for TS cell differentiation, we isolated six proteins expressed in the differentiated cells that were not detected in the proliferating cells. In addition, we isolated one protein from the proliferating cells that was not detected in the differentiated cells (Fig. 2). All seven spots were analyzed with MALDI-TOF. Proteins were identified by database searching for matching peptide fragment masses to known proteins. Protein spots 1, 2, and 3 were all determined by analysis of mass spectrometry peptide mass fingerprints to be type II keratin 8 (Krt8, formerly K8, or endoA), and spot 4 was determined to be type I keratin 18 (Krt 18, formerly K18, or endo B). Spots 5 and 6 were determined to be heat shock protein 1. The protein spot 7, expressed at greater levels in the proliferating cells, was identified as dimethylarginine dimethylaminohydrolase 1 (DDAH1).


Figure 02
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  		FIG. 2. Identification of proteins with increased expression during TS cell differentiation. Protein from proliferating and differentiated TS cells were separated by 2-D gel according to pH and molecular weight, and seven differentially expressed protein spots were collected for mass spectrometry identification.

Quantitation of Hspb1 mRNA During TS Cell Differentiation

Hspb1 transcript expression was compared in TS cells during differentiation by quantitative real-time RT-PCR. A comparison of mRNA levels isolated from D0, D2, D4, and D6 of differentiation show a statistical increase in Hspb1 mRNA during differentiation (Fig. 3). Because the highest expression was detected in samples after 6 days, we have selected D6 to represent 100% expression. Hspb1 mRNA expression was 69% ± 8% (mean ± SEM) of maximal expression on D0 and increased to 85% ± 5% at D2. Hspb1 mRNA expression in proliferating TS cells at D0 was significantly lower than at D4 and D6 of differentiation.


Figure 03
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  		FIG. 3. Quantitation of Hspb1 mRNA. Taqman (ABI) probe-based real-time RT-PCR was used to compare Hspb1 mRNA levels. RT-PCR was run in triplicate on three different biological cell replicates. Expression of Hspb1 mRNA was significantly increased during TS cell differentiation, as determined by a single-factor ANOVA (P < 0.05). Difference in relative expression levels between days is represented by different subscripts, and was determined by a pair-wise comparison (Student t-test [P < 0.05; mean ± SEM; n = 3]).

Expression of HSPB1 Protein During TS Cell Differentiation

Protein from the nuclear and cytoplasm fractions at D0, D2, D4, and D6 of TS cell differentiation was extracted, and HSPB1 protein expression level was determined by Western blot. HSPB1 protein was detected in the cytoplasm and nuclear fractions in proliferating TS cells and during all days of differentiation (Fig. 4A). Quantitative analysis of HSPB1 protein in the nuclear fractions showed that levels did significantly increase from D0 to D6 of differentiation; however, the cytoplasmic HSPB1 levels were not significantly different at these stages (Fig. 4, B and C). Results represent the mean ± SEM of the ratio from three replicates in each group. Results from D0 and D6 were compared by pair-wise comparison (Student t-test) with P < 0.05 indicating significance.


Figure 04
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  		FIG. 4. Expression of HSPB1 protein during TS cell differentiation. A) Anti-HSPB1 antibody was used to detect levels of HSPB1 protein in both nuclear and cytoplasmic protein fractions. B and C) Quantitation of HSPB1 protein expression during TS cell differentiation. Expression of HSPB1 protein was significantly increased in the nuclear protein fractions by D6 of differentiation compared with D0 (B), but was not different in the cytoplasmic fractions (C). Student t-test (P < 0.05; mean ± SEM; n = 3).

Detection of Phosphorylated HSPB1 and MAPKAPK2 During TS Cell Differentiation

Phosphorylation of HSPB1 is regulated by the MAPK14 pathway. MAPK14 phosphorylates the intermediate protein MAPKAPK2, and this protein phosphorylates HSPB1. Since we detected an increase in HSPB1 protein during TS cell differentiation, we decided to investigate whether there was an increase in phosphorylated MAPKAPK2 and HSPB1 during differentiation. Total protein from D0, D2, D4, and D6 of TS cell differentiation was extracted and analyzed by Western blot to determine the levels of phosphorylated HSPB1 and phosphorylated MAPKAPK2 during TS cell differentiation. Levels of phosphorylated HSPB1 protein and phosphorylated MAPKAPK2 protein were low in the proliferating TS cell samples at D0, with phosphorylation increasing during TS cell differentiation (Fig. 5).


Figure 05
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  		FIG. 5. Detection of phosphorylated HSPB1 and MAPKAPK2 in TS cells during differentiation. Antibodies that detect the phosphorylated forms of HSPB1 and MAPKAPK2 were used to detect protein levels following Western blot. Expression of phosphorylated HSPB1 and MAPKAPK2 increases as TS cells differentiate.

MAPK14 Inhibition

MAPK14 inhibition by SB220025 at 10 µM was verified by a decrease in detection of phosphorylated HSPB1 (Fig. 6A). The level of phosphorylated HSPB1 was lower at each day of differentiation when cultured in media containing the specific MAPK14 inhibitor SB220025 compared with the control SB202474 (Fig. 6B). In comparison an antibody that will detect both phosphorylated and nonphosphorylated HSPB1 showed that total protein levels were not affected by the inhibitor treatment (Fig. 6C). Significant differences were determined by a two-factor ANOVA for day of differentiation and inhibitor treatment (P < 0.05; n = 3). Following visual inspection of TS cells grown in differentiation media at this level of inhibition, it appears that cells were able to differentiate into giant cells. This differentiation is shown by the presence of cells resembling giant cells and the expression of Csh1 and Csh2 in D6 cultures (Fig. 7, A and B). However, visual inspection of the cells indicated a higher amount of cell lyses in the wells containing the MAPK14 inhibitor. Uptake of neutral red dye was measured to determine the viability of TS cells during differentiation in the presence of inhibitor. Significantly fewer viable cells were detected at each day of differentiation when cultured with inhibitor than with the control compound (Fig. 7C). Interestingly, neutral red uptake was not significantly different between SB220025 and SB202474 treatment groups when proliferation of cells was maintained by addition of FGF4, heparin, and conditioned media over the same culture period (Fig. 7D). This result suggests that the dramatic impact of MAPK14 inhibition on TS cell survival is specific to differentiating and not proliferating cells.


Figure 06
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  		FIG. 6. Inhibition of TS cells in SB220025 a specific inhibitor of MAPK14. Inhibition of phosphorylation of HSPB1 was verified for protein collected at D2, D4, and D6 of differentiation when grown in culture media containing 10 µM SB 220025, a specific inhibitor of MAPK14 compared to the control inactive analogue SB202474 (A). Incubation of TS cell in 10 µM of SB220025 resulted in a significant decrease in phosphorylated HSPB1 protein (mean ± SEM) during differentiation (B). The same inhibitor did not affect the levels of total HSPB1 protein expression (C). Significant differences were determined by a two-factor ANOVA for day of differentiation and inhibitor treatment with P < 0.05; n = 3.


Figure 07
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  		FIG. 7. Incubation of TS cells in 10 µM of SB220025 under differentiating conditions resulted in differentiation of TS cells, as shown by (A) the expected cell morphology changes associated with TS cell differentiation and (B) the expression of both Csh1 and Csh2 mRNA by RT-PCR in samples grown in SB202474 (–) and SB 220025 (+). However, incubation of TS cell in 10 µM of SB220025 under differentiating conditions (removal of FGF4, heparin, and conditioned media) resulted in a significant increase in cell death (mean ± SEM) compared with that with the inactive inhibitor SB202474 (C). Cell viability was not affected by inhibitor when cells were grown under proliferation conditions (D). Significance was determined by a two-factor ANOVA for day of differentiation and inhibitor treatment, with P < 0.05; n = 3.

DISCUSSION

The 2-D gel comparison of proteins expressed during proliferation and differentiation of TS cells identified three proteins with potential increase during differentiation: KRT8, KRT18, and HSPB1, and one protein, DDAH1, which was detected at higher levels in the proliferating TS cells. DDAH enzymes specifically hydrolyzes asymmetrically methylated arginine residues, which are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS), thereby increasing NOS activity [28]. Specific single-nucleotide polymorphisms in the Ddah1 gene have been implicated with increased pre-eclampsia susceptibility in humans [29]. DDAH2, a family member of DDAH1, has been studied in TS cells, and it was determined that mRNA expression is repressed in proliferating TS cells and is, therefore, expressed in higher amounts in differentiating cells [30]. This is the opposite expression pattern we found for DDAH1 protein by visual inspection of our 2-D gel results, in which DDAH1 appears higher in the proliferating cells and decreased in the differentiated cells.

KRT8 and KRT18 are the first intermediate filament proteins produced during mouse embryogenesis, and are detectable at the 4- to 8-cell stage [31, 32]. Both KRT8 and KRT18 are expressed in the trophoblast, where their role has been demonstrated to be necessary for embryo survival by studies utilizing targeted gene mutation [33–34]. Gene expression profiling by microarray of TS cells compared to embryonic stem cells has previously identified both Krt8 and Krt18 as genes with increased mRNA expression in TS cells compared with embryonic stem cells [15, 35]. We detected high levels of KRT8 and KRT18 protein expression in TS cell nucleus, and further suggest that an increase in protein levels occurs during differentiation, although this was not investigated in greater detail in this study. It is likely that the increase in intermediate filament proteins detected in the nucleus is a characteristic of giant cells being a larger cell and having a larger nucleus compared with proliferating TS cells. Therefore, it is possible that the increase in KRT8 and KRT18 is the result of the differentiation, but they do not play a regulatory role in the process. In addition, KRT8 and KRT18 have already been the focus of much research in the placenta and trophoblast cells [32–34, 36]; therefore, we decided to further investigate the differential expression of HSPB1.

HSPB1 is a member of the family of small heat shock proteins that, in addition to responding to heat shock and other environmental stresses, also function as molecular chaperones and in signal transduction pathways [37]. Hspb1 mRNA has been detected in all stages of mouse preimplantation development, where it is involved in MAPK14 signaling [27, 38]. Inhibition of MAPK14 resulted in halted embryonic development at the 8- to 16-cell stage, related to a lack of promotion of filamentous actin beginning at the 8-cell stage. In this pathway, MAPK14 activates MAPKAPK2 or MAPKAPK5, which phosphorylate HSPB1 [39]. Inhibiting MAPK14 in the mouse completely blocked phosphorylation of HSPB1 [27, 38]. Targeted inactivation of Mapk14{alpha} in the mouse resulted in early embryonic lethality due to placental defects around Embryonic Day 10.5 [40, 41]. In the rat, HSPB1 was strongly expressed in trophoblast giant cells beginning at Day 11 of gestation [42]. In human pregnancies, HSP27 (HSPB1 in mouse) protein has been detected in the decidua and in the placenta in the intermediate trophoblast and syncytiotrophoblast cells during the first two trimesters [43], and has been suggested to play a role in trophoblast differentiation [44]. HSP27 is more commonly found in placenta from patients with severe preeclampsia compared with that from normal term gestations [45].

Other heat shock proteins have been shown to be necessary for placental development. Targeted gene mutation of the 90-kDa heat shock protein resulted in embryonic lethality due to placental defects as a result of a failure to form a placental labyrinth layer [46]. HSF1 is the major heat shock transcriptional factor controlling rapid heat shock protein induction. Mice mutant for Hsf1 had a common phenotype that produced a thinning of the spongiotrophoblast layer and resulted in embryonic lethality [47].

Nuclear levels of HSPB1 protein increased as TS cells differentiated. This suggests that HSPB1 and the MAPK14 signaling pathway may be involved in TS cell differentiation. HSPB1 mRNA expression increases by the second day of TS cell differentiation, and is significantly greater at Days 4 and 6 of differentiation. Levels of phosphorylated HSPB1 and MAPKAPK2 protein increases during differentiation, and this increase can be inhibited when cells are differentiated in inhibitor specific for MAPK14. This indicates a role for the MAPK14 pathway, ultimately resulting in phosphorylation of HSPB1 during TS cell differentiation. TS cells appear to differentiate in the presence of MAPK14 inhibitor; however, the cell viability is greatly compromised. This result indicates that the removal of FGF4 from the culture is forcing the TS cells to differentiate; however, with MAPK14 inhibition, the differentiated cells lose survivability. Interestingly, the inhibition of MAPK14 does not affect cell viability in proliferating cells, suggesting that the MAPK14 pathway is only critical during TS cell differentiation. Further studies are necessary to determine how the inhibition of MAPK14 results in molecular, structural, or other defects that affect the viability of TS cells during differentiation.

During the inhibition experiments, some phosphorylated HSPB1 protein was detected, indicating an incomplete inhibition, or perhaps that other MAPK pathways are capable of phosphorylating HSPB1. Other MAPK pathways are known to be involved in trophectoderm differentiation, as is shown by the disruption of Mapk1, which leads to embryonic lethality with mutant embryos failing to form the ectoplacental cone and extraembryonic ectoderm [48].

The objective of this research was to identify proteins involved in TS cell differentiation. We have identified several proteins with differential expression, and have further identified HSPB1 and the MAPK14 pathway as playing a role in TS cell differentiation.

ACKNOWLEDGMENTS

The authors thank Andy Watson and David Natale for critically reading the manuscript, Anne Howlett, Erin Young, Kie-Hoon Jung, and Brian Gowen for their assistance, and Shelby Sorenson and Dr. Dominick Roche for their assistance with the 2-D gels.

FOOTNOTES

1Supported by a Utah State University College of Agriculture Assistantship to J.G., Utah Agriculture Experiment Station Project Number UTA00493 (Q.W.), and is published as UAES paper number 7842. Back

Correspondence: 2Quinton Winger, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-5600. FAX: 435 797 3959; e-mail: qwinger{at}cc.usu.edu

Received: 28 August 2006.

First decision: 12 September 2006.

Accepted: 18 January 2007.

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