miRNA let-7e Modulates the Wnt Pathway and Early Nephrogenic Markers in Mouse Embryonic Stem Cell Differentiation

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.


Introduction
Mouse embryonic stem cells (mESCs) isolated from blastocysts are pluripotent cells with unlimited self-renewal capacity and the potential to generate all the cell types of the three germinal layers. These cells have been used to generate highly specialized cells and tissues in vitro such as pancreatic cells [1], motor neurons [2], hematopoietic cells [3] and renal cells [4].
Nowadays much research is trying to develop renal precursors that could integrate and regenerate damaged kidney. From our point of view it is important to study the possible mechanisms involved in ESCs differentiation because these cells could be a potential source of these precursors. mESCs in cell culture remain undifferentiated in the presence of leukemia inhibitory factor (LIF) [5]. Withdrawal of LIF, gives rise to embryoid bodies (EBs) formation that can be differentiated toward renal lineage using activin A, retinoic acid and BMP7 [6]. Retinoic acid and activin A stimulate expression of early intermediate mesoderm markers on the basis of pioneering work in Xenopus embryos [7,8] and in murine embryonic stem cells in a more recent study [9].
Stem cell differentiation towards renal lineage is associated with the sequential expression of different marker genes characteristic of early kidney development.
Pax2 is one of the earliest markers expressed in the intermediate mesoderm from where takes place the formation of the kidney. Pax2 and Wt1 are subsequently expressed in the metanephric mesenchyme and are two genes characteristic for initiation of nephrogenesis [9]. This gene expression is then followed by the secretion of many additional secreted factors, including Wnt4 and Wnt9b which are expressed in the condensing mesenchyme [10,11] and both are involved in the formation of epithelia [11]. Following that, the presence of Notch2 directs cells primarily to the proximal tubule fate [11].
Wnt/b-catenin signalling is essential during kidney development as well as in cell differentiation towards renal lineage [12,13]. Furthermore, Wnt is also believed to stimulate ESCs proliferation and maintain pluripotency [14], and its improper regulation is associated with cyst formation in the kidney [15]. Wnt/b-catenin activation should therefore be tightly regulated.
b-catenin production is dependent on Glycogen synthase kinase 3 beta (GSK3b) phosphorylation. GSK3b is a ubiquitously expressed, highly conserved serine/threonine protein kinase found in all eukaryotes and serves as a downstream regulatory switch for the Wnt signalling pathway [16]. Serine Phosphorylation of GSK3b is performed by protein kinase C beta (PKCb) [17,18]. microRNAs (miRNAs) are short noncoding RNAs of ,22 nt that post-transcriptionally regulate gene expression through the 39untranslated regions (39UTRs) of their target mRNAs. miRNAs are able to regulate the expression of numerous mRNAs, some of them belonging to critical pathways during differentiation such as the Wnt Pathway [19]. Some of these miRNAs, as is the case of the miRNA let-7 family, regulate cell proliferation and differentiation during development in different species [20]. Specifically, let-7e was detected in the adult kidney [21] and recent studies have started to investigate its role in renal cancer [a state of cell dedifferentiation], outlining that let-7e is downregulated and associated with metastasis and poor prognosis [22].
We hypothesized that miRNA let-7e was determinant in stem cell differentiation and expression of early nephrogenic markers.Therefore, EBs were differentiated using retinoic acid and activin A, a classical combination that promote the expression of genes characteristic of the intermediate mesoderm. miRNA let-7e silencing decreased the expression of these differentiation markers. In addition, since PKCb is an inductor of GSK3b phosphorylation (GSK3b P) , we hypothesized that miRNA let-7e could inhibit the formation of PKCb protein that in turn decreases serine phosphorylation and the negative regulation of GSK3b activity, destabilizing b-catenin during the differentiation process in mESCs.
Here we present our findings concerning the involvement of miRNA let-7e in stem cell differentiation via the modulation of GSK3b phosphorylation and b-catenin production.

Ethics Statement
This study has been approved by the bioethics committee of CSIC (Spanish National Research Council).

Induction of EBs Differentiation
EBs suspension was transferred to culture flasks coated with 0.1% gelatine and cultured for an additional 1 to 10 days with the described medium without mLIF and with presence or absence of: retinoic acid (ATRA) (Sigma-Aldrich) 0.1 mM and activin A Sigma-Aldrich)10 ng/ml.

Experimental Groups
EBs were cultured for 1 to 10 days under different conditions:

miRNA Microarray
A miRNA PCR Array System (SA Biosciences) that included specific primers for 88 different mouse miRNAs was performed according the manufacturer's instructions. RNA extracted from a pool of four control EBs (Control EBs d8) and a pool of 4 differentiated EBs (DIF EBs d8) were profiled. The resulting data was normalized to the average of the four small nuclear housekeeping RNAs (snoRNA251, snoRNA202, snoRNA142 and U6 snRNA). Results have been deposited in the Array Express database, accession number (E-MTAB-1468). In order to validate the expression levels obtained in the microarray experiments, we used quantitative real-time RT-PCR, and Northern Blot analysis.

Northern Blot
miRNA Northern Blot Assay Kit (Signosis) was performed using 5 mg total RNA according to the manufacturer's protocol using a complementary probe to miRNA mmu-let-7e.

Reverse Transcription and RT-PCR microRNA
Total RNA was extracted as described previously. Specific reverse transcription for miRNA mmu-let-7e and U6 small nuclear RNA (U6 snRNA) was performed using TaqManH microRNA Assay (Applied Biosystems). The RT-PCR was performed using the TaqManH Universal PCR Master Mix (Applied Biosystems) and results were normalized to U6 snRNA.

Statistical Analysis
Data are expressed as means 6 SE. Means of different groups were compared using one way ANOVA. The Student-Newman-Keuls test was used for the evaluation of significant differences between groups. The existence of significant differences was already assumed when P,0.05.

ATRA and Activin A Treatment Differentiates EBs and
Promote the Expression of Early Nephrogenic Markers and miRNA let-7e  [metanephric mesenchyme] increased significantly from day 5 to day 10 in differentiated EBs compared with non treated EBs (Fig. 1A). Protein expression of Pax2 and Wt1 also increased in differentiated EBs compared with non treated EBs (Fig. 1B).
A microRNA microarray was performed on EBs cultured for 8 days under differentiation conditions (he 8th day was selected based on the high expression of markers found that day). As shown in table 1, a total of 88 miRNAs were profiled, and a set of 26 miRNA were found upregulated. The expression levels of the miRNA let-7 family members were significantly upregulated; [.1.5] fold difference for the eight members (let-7e,let-7i,let-7a,let-7d,let-7g,let-7c,let-7b,let-7f) in treated EBs with respect the non treated EBs (Fig. 1C, Table 1). The most upregulated miRNA with a [.9] fold difference was the miRNA let-7e.
To confirm the results of the microRNA microarray, we examined expression levels of let-7e miRNA in treated and non treated EBs by northern blot analysis and additionally by real-time PCR. As presented in Fig. 1D, miRNA let-7e expression in treated EBs was dramatically upregulated on day 8 relative to non treated EBs.

miRNA let-7e Overexpression is Involved in EBs Expression of Early Nephrogenic Markers
In order to evaluate the role of miRNA let-7e during the EBs differentiation, let-7e was silenced. Efficient silencing of miRNA let-7e was observed since expression levels of miRNA let-7e decreased significantly after silencing ( Fig. 2A).
When miRNA let-7e was silenced, mRNA expression of the differentiation markers WT1, Pax2, Wnt4 were significantly decreased on day 10 (Fig. 2B), thus indicating the role of miRNA let-7e in EBs differentiation. Similar results were observed on day 6 (data not shown). In addition miRNA let-7e is an upstream regulator of PKCb since Western blot analysis of PKCb, revealed a decrease in the expression in differentiated EBs and an increase when miRNA let-7e was silenced (Fig. 2C) indicating the inhibitory influence of miRNA let-7e on PKCb levels.

miRNA let-7e Inhibitory Effect on PKCb Prevents Phosphorylation of GSK3b and b-catenin Formation
The differentiation treatment applied to embryoid bodies (EBs) provokes a decrease in GSK3b serine phosphorylation (GSK3b P ])and b-catenin expression compared to EBs control. Conversely, miRNA let-7e silencing induces an increase in the expression of GSK3b P and b-catenin, indicating the relation between miRNA let-7e and Wnt pathway (Fig. 3).
Addition of a PKCb inhibitor to the miRNA let-7e silenced group reversed the increase in GSK3b P and b-catenin, thus indicating the direct relationship between PKCb, GSK3b P and bcatenin levels.
Addition of a PKCb inhibitor to the miRNA let-7e silenced group reversed the decrease in differentiation markers provoked previously by let-7e silencing (Fig. 4). The addition of the Wnt pathway activator BIO to differentiated EBs, decreased the expression of differentiation markers.

Discussion
In this study we differentiate EBs towards cells that express markers of intermediate mesoderm and metanephric mesenchyme using retinoic acid and activin A [9]. After this treatment, we detected increases in the expression of some differentiation markers such as Pax2 and WT1, characteristics of the metanephric mesenchyme and related to the initiation of nephrogenesis [9].
Wnt9b and Wnt4, expressed in the condensing mesenchyme [10,11], and Notch2, a gen involved in the proximal tubule fate determination [11] increased significantly during the differentiation protocol. Similar to our results, other authors, using an identical protocol, differentiated ESCs towards intermediate mesoderm-like cells. Furthermore, they obtained terminally differentiated renal cells with the subsequent addition of conditioned medium [9].
Our work indicates that activin A and retinoic acid provokes the differentiation towards cells that express markers of intermediate mesoderm and metanephric mesenchyme.
A microarray analysis revealed the overexpression of different miRNAs in the differentiated EBs compared to control EBs. Among the miRNAs overexpressed, we found let-7e, let-7i, let-7a, let-7d, let-7g, let-7c, let-7b and let-7f. miRNA let-7 family was observed to regulate proliferation and differentiation of neural stem cells [23]. Here we show that let-7e is involved in the expression of early nephrogenic markers in mESCs, since let-7e miRNA silencing reduced the expression of Pax2, WT-1 and Wnt4, thus indicating the role of let-7e in differentiation.
PKCb is a multifunctional serine/threonine protein kinase that plays important roles in the regulation of cell cycle, differentiation and proliferation [24]. Recently its inhibition has also been associated with T cell differentiation [25], osteogenic differentiation [26] or cardiomyocyte differentiation [27]. Moreover, the inhibition of PKCb could block GSK3b serine phosphorylation, since it has been described that GSK3b phosphorylation by cholinergic stimulation is probably mediated by a mechanism involving protein kinase C [PKC], as it was blocked by a PKC inhibitor [17]. A close relationship between PKCb protein expression and miRNA let-7e has been detected in our work, thus miRNA let-7e is an upstream regulator of the PKCb protein expression. The fact that PKCb has been listed in the micro-RNA.org database as one potential target gene of the miRNA let-7e reinforce our findings.
GSK3b constitutively phosphorylates the b-catenin protein and promotes its degradation. However, serine phosphorylation of GSK3b provokes the opposite effect and b-catenin degradation is avoided [28]. Our results show an increased expression of GSK3b serine phosphorylation and b-catenin, concomitant with a decrease in the expression of differentiation markers when let-7e is silenced. Therefore indicates that let-7e acts in differentiation as an inhibitor of b-catenin through the reduction of the inactive form of GSK3b.
The use of the PKCb inhibitor Gö 6983 that acts avoiding phosphorylation of several PKC isoforms, including PKCb [29], provoked a decrease in the Wnt pathway components, GSK3b serine phosphorylation and b-catenin, concomitant with an increase in the expression of the differentiation markers, indicating the relationship between PKCb and Wnt pathway in this process.
Our results indicate that Wnt/b-catenin signalling is reduced in differentiated EBs. Moreover BIO, an activator of Wnt signaling pathway [30], reduced the expression of differentiation markers. In line with our work, it has been described that Wnt signalling activators such as BIO induced the maintenance of pluripotency in human and mouse embryonic stem cells [14,28] and stimulates proliferation [31].
In the other hand, Wnt pathway is essential during kidney development and in cell differentiation toward renal lineage since it has been shown that Wnt9b 2/2 mice die within 24 h of birth due to agenesis of the kidneys [12] and also targeted disruption of Wnt4 results in kidney agenesis and impairs mesenchymal-to-epithelial transition [32]. Thus it can be argued that activation of the Wnt pathway should be present in embryonic stem cell differentiation but in a way in which the end result of b-catenin production is well balanced in order to avoid pluripotency [31]. This work could well indicate that one potential interfering factor favoring the correct balance between Wnt signaling and b-catenin production is the miRNA let-7e.
In summary, our results indicate that miRNA let-7e is critically involved in the expression of early nephrogenic markers during EBs differentiation and the concomitant reduction of b-catenin production. The inhibitory effect of miRNA let-7e on GSK3b serine phosphorylation through PKCb could explain these effects.