Mirdametinib

Discovering the role of VEGF signaling pathway in mesendodermal induction of human embryonic stem cells

Chenge Xin a, Chaonan Zhu a, Ying Jin a, b, c, *, Hui Li a, b, **
a Department of Histoembryology, Genetics and Developmental Biology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China
b Basic Clinical Research Center, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
c CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, CAS Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

A B S T R A C T

Human embryonic stem cells (hESCs) have the unique feature of unlimited self-renewal and differen- tiation into derivatives of all three germ layers in human body, providing a powerful in vitro model for studying cell differentiation. FGF2, BMP4 and TGF-b signaling have been shown to play crucial roles in mesendodermal differentiation of hESCs. However, their underlying molecular mechanisms and other signaling pathways potentially involved in mesendodermal differentiation of hESCs remain to be further investigated. In this study, we uncover that VEGF signaling pathway plays a critical role in the mesen- dodermal induction of hESCs. Treating hESCs with Lenvatinib, a pan-inhibitor of VEGF receptors (VEGFRs), impedes their mesendodermal induction. Conversely, overexpression of VEGFA165, a major human VEGF isoform, promotes the mesendodermal differentiation. Similar to the VEGFR inhibitor, MEK inhibitor PD0325901 hinders mesendodermal induction of hESCs. In contrast, overexpression of ERK2GOF, an intrinsically active ERK2 mutant, markedly reduces the inhibitory effect of the VEGFR inhibitor. Thus, the MEK-ERK cascade plays an important role for the function of VEGF signaling pathway in the mes- endodermal induction of hESCs. All together, this study identifies the critical role of VEGF signaling pathway as well as potential crosstalk of VEGF signaling pathway with other known signaling pathways in mesendodermal differentiation of hESCs.

Keywords:
Human embryonic stem cells Mesendoderm
VEGF signaling pathway Differentiation

1. Introduction

Human embryonic stem cells (hESCs) possess the ability to self- renew indefinitely and differentiate into any cell type in the body [1]. Because of these characteristics, hESCs become a powerful tool for developmental biology study, drug screening, disease modeling and cell replacement therapy. Signaling pathways and transcription factors play vital roles in regulating stem cell characteristics. On the one hand, ACTIVIN A/TGF-b, FGF2 and INSULIN/IGF signaling are the consensus players to modulate SMAD2/3, ERK1/2 and PI3K signaling, respectively, in the maintenance of hESCs at an undif- ferentiated state [2,3]. Core transcriptional factors (OCT4, SOX2 and NANOG) have been demonstrated to be the principal transcrip- tional regulators for hESC self-renewal [4e7]. On the other hand, BMP and WNT signaling pathways are known to play crucial roles for the exit from pluripotent state and lineage specification of hESCs, synthetically with FGF and TGF-b signaling pathways. Different combinations of these four pathways, together with their downstream targets, determine the main cell fate choice of hESCs [8]. These four pathways are reported to be involved in mesen- dodermal specification in hESCs [8e14]. We were interested in the question of whether there would be additional signaling pathways involved in the mesendodermal differentiation from hESCs, and if yes, what is the relationship between the newly identified pathway and the known ones. The answer to the question would be important for more efficient mesendodermal induction of hESCs.
The vascular endothelial growth factor (VEGF) signaling pathway is well known for its central roles in vasculogenesis, angiogenesis and lymphangiogenesis during embryonic develop- ment and in physiological homeostasis [15]. In human, five VEGF ligands (VEGFA, VEGFB, VEGFC, VEGFD and placental growth factor, PlGF) have been identified up to date. They display different binding affinities to the three receptor tyrosine kinases (VEGFR1-3) and the co-receptors in an overlapping pattern [16]. VEGFA, also known as VEGF, is the best characterized member of the VEGF family. VEGFA has nine isoforms [17,18], among which VEGFA165 is the most abundant one [19]. VEGFA binds to two receptors, VEGFR1 (FLT1) and VEGFR2 (KDR), in endothelial cells [20], and plays important physiological roles in vasculogenesis, angiogenesis as well as pathological roles in tumor angiogenesis [21e23]. In vitro, VEGF has been reported to be critical for the induction of meso- dermal endothelial cells from hESCs [24]. However, it remains un- known whether VEGF signaling pathway functions in the earlier cell fate decisions of hESCs such as mesendodermal induction.
Here, we employ loss- and gain-of function approaches to un-ravel the role of VEGF signaling in the process of the mesen- dodermal differentiation of hESCs. Our results show that inhibition of VEGF signaling activity by a pan-inhibitor of VEGFRs almost blocks mesendodermal differentiation of hESCs induced by FGF2, BMP4 and ACTIVIN A. Conversely, activation of the VEGF pathway by VEGFA165 overexpression facilitates the mesendodermal in- duction. Mechanically, the promotion of mesendodermal induction by VEGF signaling is mainly mediated through its downstream MEK-ERK cascade activity. Like the VEGFR inhibitor, MEK inhibitor PD0325901 significantly prevents the mesendodermal induction from hESCs. In contrast, overexpression of ERK2GOF, a constitutively active mutant isoform, markedly reduces the inhibitory effect of the VEGFR inhibitor. Therefore, this study discovers a critical role of the VEGF pathway in the regulatory signaling network that combines the essential extrinsic FGF2, BMP4 and TGF-b signaling pathways in mesendodermal induction from hESCs.

2. Material and methods

2.1. Human embryonic stem cell culture

Human ESC line SHhES8 [25] was routinely cultured in mTeSR1 (STEMCELL Technologies, Canada) on Matrigel (STEMCELL Tech- nologies, Canada) coated plates. Cells were passaged every 4 days using Dispase (STEMCELL Technologies, Canada).

2.2. Mesendodermal differentiation of hESCs

For mesendodermal differentiation, a protocol described by Xie et al. [26]was used. Briefly, SHhES8 hESCs were individualized with ACCUTASE (STEMCELL Technologies, Canada) and then seeded onto a fresh Matrigel-coated 6-well plate at a density of around 2 105 cells per well and cultured in the mesendodermal induction medium [E8 medium supplemented with 25 ng/mL Activin A (STEMCELL Technologies, Canada) and 5 ng/mL BMP4 (PeproTech, America)] [12,26]. To increase cell survival, 10 mM of ROCK inhibitor Y-27632 (STEMCELL Technologies, Canada) was included in the medium during the whole process of experiments, except that the inhibitor was removed 24 h after plating for the culture of doxy- cycline (DOX)-inducible VEGFA165 overexpression (OE) cells. Me- dium was changed every day and cells were harvested after they were cultured for 48 h.

2.3. Small molecule inhibitors

The inhibitors used in this study were VEGFR inhibitor Lenva- tinib (1 mM, Selleck, America; 24-h treatment before cell harvest) and MEK inhibitor PD0325901 (0.1 mM, STEMCELL Technologies, Canada; 24-h treatment before cell harvest).

2.4. Plasmid construction

For overexpression of VEGFA165, its coding sequence was amplified by PCR from SHhES8 hESC complementary DNA (cDNA) using primers containing required restriction enzyme recognition sites (supplementary Table 1). The amplicon was subcloned into the pAAVS1-TRE3G-EGFP vector [27] to replace the original EGFP cod- ing sequence, generating the plasmid pAAVS1-TRE3G-VEGFA165. For overexpression of the ERK2 mutant, the HA-ERK2GOF fragment was amplified by PCR from pWZLblasti-HA-ERK2GOF [28] using specific primers (supplementary Table 1), and subcloned into the pAAVS1-TRE3G-EGFP vector to replace the original EGFP coding sequence, generating the pAAVS1-TRE3G-HA-ERK2GOF plasmid. The cloned sequences of both plasmids were verified by DNA sequencing.

2.5. Generation of DOX-inducible VEGFA165 OE and HA-ERK2GOF OE hESC lines

pAAVS1-TRE3G-VEGFA165 and pAAVS1-TRE3G-HA-ERK2GOF plasmids were electroporated into SHhES8 hESCs, respectively. The pAAVS1-TRE3G empty vector was electroporated into SHhES8 hESCs to generate a negative control hESC line. Cells were selected by 0.5 mg/mL puromycin for 4 days.

2.6. Western blotting

Western blotting was performed as previously described [29]. Primary antibodies used were pERK1/2, ERK1/2, pSMAD1/5, SMAD5, pSMAD2/3, SMAD2/3 (Cell Signaling Technology, America), a-TUBULIN (Sigma, America) and T (R&D systems, America). The secondary antibodies were from Jackson Immuno Research, America. a-TUBULIN was used as an internal control.

2.7. Quantitative real-time PCR (qRT-PCR)

Cells were lysed by TRIzol (Thermo Fisher, America) to purify total RNA. RNA was reverse transcribed into cDNA. SYBR® (Takara, Japan), cDNA and primers were mixed to quantify gene expression using the ViiA 7 Real time PCR machine (Life Technologies, Amer- ica). Results were standardized by the GAPDH level and the primer sequences used are shown in supplementary Table 1.

2.8. Statistical analyses

Data are presented as mean ± standard deviation (SD) of three independent experiments. Statistical significance of differences between two groups was analyzed by the unpaired Student’s t-test and is shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. p < 0.05 was considered statistically significant. 3. Results 3.1. Inhibition of VEGF signaling pathway suppresses mesendodermal differentiation of hESCs To investigate the role of VEGF signaling pathway in mesen- dodermal differentiation of hESCs, we applied a previously pub- lished efficient mesendodermal induction medium, thereafter referred to as ME medium [26] for the induction of mesendodermal differentiation of hESCs. The role of the VEGF signaling pathway was first tested using Lenvatinib, a pan-VEGFR inhibitor. Cells were divided into three groups, including the self-renewal group (mTeSR1 DMSO), ME group (ME medium DMSO) and ME plus Lenvatinib group (ME medium Lenvatinib) (Fig. 1A). Cells of ME group displayed a flatter and differentiated colony morphology compared to cells in the self-renewal group (Fig. 1B). Our qRT-PCR results showed dramatic up-regulation of mesendodermal genes (GSC, EOMES, MIXL1, T, LHX1 and GATA4) in cells of ME groups (Fig. 1C). However, such massive changes in the expression level were not observed for ectoderm and trophoblast genes in the cells of ME group (Fig. 1D). Notably, the dramatic up-regulation of mesendodermal genes by the ME medium was significantly sup- pressed by Lenvatinib. Thus, the VEGFR inhibitor repressed hESC mesendodermal induction in terms of both colony morphology and mesendodermal gene expression pattern (Fig. 1B and C). Consis- tently, our Western blot analysis indicated the high expression of mesendodermal hall marker T in cells of ME group, suggesting the successful induction of hESCs to the mesendodermal lineage. However, the abundant expression of T in the ME group was abolished by Lenvatinib (Fig. 1E), verifying a suppressive effect of the VEGFR inhibitor on hESC mesendodermal differentiation. These findings indicate that endogenous VEGF signaling plays an indispensable role in hESC mesendodermal induction. As BMP4, ACTIVIN A/TGF-b and FGF2, being present in ME medium, were all reported to be closely involved in mesendodermal induction [30], we determined the effect of VEGFR inhibitor on the activities of these three pathways. As expected, Western blot analysis showed increased phosphorylation levels of pSMAD1/5, pSMAD2/3 and pERK1/2 in ME group cells compared to self-renewal group cells, indicating that BMP4, TGF-b and FGF2 signaling pathways were all activated (Fig. 1F). However, the activation of these three pathways, especially FGF2 and BMP4 pathways, was obviously repressed by the VEGFR inhibitor. These results imply the presence of crosstalk between endogenous VEGF signaling and BMP4, TGF-b and FGF2 signaling pathways. The crosstalk might play an important role for mesendodermal induction of hESCs. 3.2. Forced expression of VEGFA165 promotes mesendodermal induction Next, we asked whether forced expression of VEGF ligand would be able to enhance the mesendodermal differentiation of hESCs. To test the possibility, we established a doxycycline (DOX)-inducible VEGFA165 overexpressing (OE) hESC line, as VEGFA165 is the most abundant isoform of the VEGF family. A negative control hESC line was also established by transfection with an empty vector. As shown in Fig. 2A, the addition of DOX significantly induced expression of VEGFA165 at the mRNA level in DOX-inducible VEGFA165 OE cells. To investigated the effect of VEGFA165 OE on the mesendodermal induction, DOX-inducible VEGFA165 OE hESCs or negative control hESCs, were cultured in the ME medium in the presence or absence of DOX for 2 days after plating (Fig. 2B). DOX- inducible VEGFA165 OE cells in ME medium without DOX displayed a differentiated morphology and DOX treatment enhanced this phenomenon (Fig. 2C). qRT-PCR results revealed that culture in ME medium alone substantially up-regulated mesendodermal genes in DOX-inducible VEGFA165 OE cells in the absence of DOX. Notably, DOX treatment further elevated mesendodermal genes in these cells (Fig. 2D). However, DOX treatment did not cause such addi- tional enhancement of mesendodermal gene expression in nega- tive control cells, although ME medium also up-regulated mesendodermal genes in the absence of DOX in these cells (Fig. 2E). At the protein level, ME medium without DOX increased the steady-state level of T protein in inducible VEGFA165 OE cells. Treatment of these cells with DOX further elevated the T protein level (Fig. 2F). Furthermore, protein levels of pERK1/2, pSMAD1/5, (G) A representative Western blot result showing the protein levels of pERK1/2, ERK1/2, pSMAD1/5, SMAD5, pSMAD2/3 and SMAD2/3 in DOX-inducible VEGFA165 OE cells cultured under indicated conditions (left panel). The right panel shows the quantitative analysis of corresponding Western blot results shown in the left panel. Quantification was made with Tanon GIS Gel Imaging Processing System. The mean relative mRNA level in DOX-inducible VEGFA165 OE cells or negative control cells cultured in mTeSR1 in the absence of DOX was set at 1.0 (A, D and E). The mean relative phosphorylated protein level in DOX-inducible VEGFA165 OE cells cultured in mTeSR1 in the absence of DOX was set at 1.0 (G). pSMAD2/3 were all further enhanced by DOX treatment in induc- ible VEGFA165 OE cells (Fig. 2G). Therefore, forced expression of VEGFA165 could promote mesendodermal induction of hESCs. Combined with the effect of VEGFR inhibition on hESC mesen- dodermal induction, these results support the notion that the VEGF signaling pathway plays a critical role in the mesendodermal dif- ferentiation of hESCs. 3.3. The MEK-ERK cascade is implicated in the function of VEGF signaling pathway in mesendodermal differentiation As VEGF signaling has been shown to activate MEK-ERK signaling cascade [31], the activity of which is critical for mesen- dodermal differentiation [10,12], we hypothesized that MEK-ERK cascade might contribute to the function of VEGF signaling in mesendodermal differentiation of hESCs. To test this hypothesis, we first determined the effect of MEK inhibitor PD0325901 on mesendodermal induction in our mesendodermal differentiation system described above (Fig. 3A). Results of qRT-PCR analyses showed that cells cultured in the ME medium expressed dramati- cally higher levels of mesendodermal markers as compared to cells cultured in mTeSR1. However, PD0325901 markedly repressed the up-regulation of mesendodermal markers induced by the ME me- dium, in a way similar to the VEGFR inhibitor (Fig. 3B). Moreover, like the VEGFR inhibitor, PD0325901 also remarkably reduced ME medium-induced elevation of protein levels of pERK1/2, pSMAD1/5 and pSMAD2/3 as well as T proteins (Fig. 3C). These results indicate that ERK signaling inactivation and VEGF signaling inhibition have similar effects on hESC mesendodermal induction. Given that the VEGFR inhibitor reduced pERK1/2 level, while VEGFA165 OE enhanced the pERK1/2 level in ME medium-induced mesendodermal differentiation of hESCs, and that the inhibition of MEK-ERK signaling cascade hindered hESC mesendodermal induction, we anticipated that MEK-ERK signaling cascade could be critical for the function of VEGF signaling pathway in mesen- dodermal induction. To test this, we established a DOX-inducible HA-ERK2GOF mutant OE hESC line. ERK2GOF is an intrinsically active mutant of ERK2 [28]. DOX was added to the ME medium to induce HA-ERK2GOF mutant OE at the start of differentiation. Twenty-four hours later, the VEGFR inhibitor was added (Fig. 4A). Western blot analysis verified the induction of HA-ERK2GOF expression by DOX in cells of the ME DOX group (Fig. 4B). As expected, the ME medium-induced up-regulation of mesen- dodermal genes was repressed by VEGFR inhibitor Lenvatinib. Importantly, the repressive effect of Lenvatinib on mesendodermal gene expression was significantly attenuated by DOX treatment (Fig. 4C), suggesting that the MEK-ERK cascade could, at least, partially mediate the function of endogenous VEGF signaling pathway in mesendodermal induction of hESCs. 4. Discussion Cell differentiation is controlled by sophisticated networks including signaling pathways, transcription factors and other reg- ulatory elements. BMP, TGF-b and FGF signaling pathways play crucial roles in mesendodermal differentiation of hESCs. However, interactions in the integrated network remain to be revealed. Here, using both loss- and gain-of function approaches, we show that endogenous VEGF signaling pathway is required for normal mes- endodermal differentiation of hESCs. VEGF signaling is well known for its function in vasculogenesis, which involves the differentiation of vascular endothelial precursor cells emerging de novo from the mesoderm [32]. In this study, we uncover a novel role of VEGF signaling pathway for promotion of mesendodermal induction from human pluripotent stem cells, which occurs at an earlier developmental stage than vasculogenesis from the mesoderm. Mechanically, MEK-ERK cascade may play an important role downstream of VEGF signaling pathway in mesendodermal in- duction of hESCs. Inhibition of VEGF signaling by VEGFR inhibitor abrogated the enhancement of pERK1/2 protein levels induced by mesendodermal induction (Fig. 1F). In addition, MEK inhibitor PD0325901 inhibited mesendodermal induction in a way similar to the VEGFR inhibitor (Fig. 3B and C). Most importantly, ectopic expression of HA-ERK2GOF significantly compromised the inhibi- tory effects of VEGFR inhibitor on mesendodermal induction (Fig. 4C). Together, these results indicated that MEK-ERK signaling cascade plays a key role for the function of VEGF signaling pathway in mesendodermal induction. Nevertheless, the intrinsically active ERK2 mutant did not completely block the inhibitory effect of the VEGFR inhibitor on mesendodermal induction (Fig. 4C), suggesting that other factors involved in the function of VEGF signaling in mesendodermal induction remain to be discovered. In addition to MEK-ERK signaling cascade, we found that VEGF signaling pathway had effects on the activity of BMP and TGF-b signaling pathways in mesendodermal induction (Figs. 1F and 2G). It remains unclear whether these effects are direct or indirect. Moreover, WNT signaling is reported to participate in mesendodermal differentia- tion [33]. It is interesting to investigate whether VEGF signaling affects the activity of WNT signaling pathway in mesendodermal differentiation. Further studies are needed to determine the role and precise mechanisms responsible for the crosstalk of these signaling pathways. 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