C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process

Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-β signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.


Introduction
Adult hepatic progenitor cells (HPCs), also known as oval cells in rodents, are bipotent cells that expand after chronic liver damage and can differentiate into hepatocytes or cholangiocytes to repair liver when hepatocytes are unable to do it [1,2]. Thus, during severe and chronic liver damage, HPCs expand, contributing to liver repair. Due to this function and its potentiality in therapy, full understanding of oval cell biology and how they are regulated is an important issue, considering that oval cells could have a potential implication in the generation of liver fibrogenesis and/or development of hepatocarcinoma (HCC) [2,3].
Different signals are involved in the regulation of oval cells. Among them, some signals generated by hepatic stellate cells (HSCs)/myofibroblasts, which are important components of oval cells niche, are key players, including HGF, TGF-β, TGF-α, EGF and b-FGF. Additional important signals are TWEAK, IL-6 or TNF-α, secreted by inflammatory cells [3]. It is worth highlighting the relevance of HGF, which has broad-ranging regulatory activities, inducing oval cell Ivyspring International Publisher survival, migration, invasion, and differentiation, critically counterbalancing TGF-β actions [4][5][6][7][8], being required for the repopulation capacity of oval cells in the injured liver [9]. Nevertheless, the regeneration process mediated by HPCs and its regulation has not been fully characterized yet.
C3G (Crk SH3-domain-binding guaninenucleotide-releasing factor) protein is a guaninenucleotide exchange factor (GEF) for Rap1 and other Ras proteins [10]. However, C3G can also act through mechanisms independent of its GEF activity [11,12], likely mediated by its interaction with other proteins through its proline-rich domain and/or its translocation to the nucleus [13]. C3G is a protein ubiquitously expressed, although some tissue-specific differences exist [10,14]. The main isoform is a protein with an apparent molecular weight of 140 kDa, which constitutes the isoform A. Other common isoforms are the B one, which differs in 21 aminoacids (aa) from the N-terminal region, and a mouse isoform with a deletion of 38aa in the N-terminal region [10]. A p87C3G isoform has also been described in chronic leukemia cells [15] and more recently, a new one of 175kDa was identified in the brain [16]. C3G is required for embryonic development [17] and regulates several cellular functions such as adhesion, migration, apoptosis, and differentiation [18][19][20][21].
C3G is expressed in mouse embryonic liver, where an enrichment in shorter isoforms, as compared to brain, was described [22]. We also found that C3G main isoform is expressed in neonatal and adult hepatocytes, as well as in oval cells, but at different levels [23,24]. Thus, C3G protein levels are high in both oval cells and neonatal hepatocytes, but low in adult hepatocytes. Moreover, C3G is upregulated in HCC cells, as compared to adult hepatocytes, promoting tumor growth [23]. Therefore, our data show that C3G levels are tightly regulated in the liver during development and their dysregulation has pathological implications, suggesting it might play a key role in oval cell function. In this work, we have evaluated the role of C3G in the regulation of oval cell biology using a gene silencing approach.

Wound healing and invasion assays
Wound healing was performed as described [21,25]. Invasion was assayed in Matrigel (333 µg/cm2; Corning#356234) coated transwells (BD#353097). 50,000 cells were seeded in the upper chamber in serum-free medium. 10% FBS-medium, placed in the lower chamber, acted as chemoattractant. After 24h, cells from the lower chamber were fixed with 4% paraformaldehyde (PFA), stained with crystal violet and counted using an Eclipse TE300 Nikon microscope. To evaluate HGF effect on invasion, cells in the upper chamber were treated with this growth factor (20 ng/mL) and no serum was added into the lower chamber.

Adhesion and clonogenicity assays
To measure adhesion, cells (50,000) were seeded in DMEM supplemented with 10% FBS. Adhered cells (15-30 min) were fixed, stained with crystal violet and counted using Eclipse TE300 Nikon microscope.

Analysis of F-actin organization
Cells seeded on 2% gelatin-coated glass coverslips were fixed with 4% PFA for 20 min. F-actin was stained with rhodamine-conjugated phalloidin (Sigma Aldrich#P1951) as described [26]. Images were analyzed by confocal microscopy.

Statistical analysis
Data are represented as the mean values ± S.E.M (n≥3) of independent experiments. Unpaired Student's t-test was used for comparison of two experimental groups and one-way or two-way ANOVA analyses to compare more than two groups with one or two variables using GraphPad Prism 7.0 software. Differences were considered significant when p value was p≤0.05.

C3G knock-down enhances the migratory capacity of oval cells while reducing adhesion
We have previously described that oval cells express higher levels of C3G than adult hepatocytes [23]. To evaluate the functional relevance of C3G in these cells we generated oval cells with permanent C3G knock-down using different shRNAs. A reduction in C3G protein levels upon shRNAmediated knock-down was confirmed by western-blot and immunofluorescence analysis ( Fig. 1A and 1B, and Supplementary Fig. 1A).
Considering that migration is necessary for the expansion of oval cells to repair the chronically damaged liver, and that C3G regulates cell adhesion, migration and invasion [21,23,26], we evaluated the effect of C3G down-regulation on these cellular processes. The analysis of cell migration using wound healing assays (Fig. 1C) revealed that wound closure in C3G-silenced cells was significantly higher at all analyzed time points (6, 8 and 24h). In agreement with the enhanced motility of C3G-silenced oval cells, invasion through Matrigel using serum as chemoattractant was also increased (Fig. 1D). As a reference, we used oval cells chronically treated with TGF-β, a well-characterized model of epithelial to mesenchymal transition (EMT) [8]. The increase in invasion elicited by C3G down-regulation was similar to that induced by TFG-β in non-silenced cells ( Supplementary Fig. 1B), but TGF-β further enhanced the invasive capacity of C3G-silenced cells ( Supplementary Fig. 1B). Moreover, C3G knock-down oval cells showed decreased adhesion (Fig. 1E).
Therefore, oval cells with low levels of C3G show a higher migratory and invasive capacity and a lower adhesion, all of which could favor oval cell expansion.

C3G down-regulation in oval cells promotes an EMT associated process with increased stemness markers
Previous data from the literature have demonstrated the relevance of TGF-β-induced EMT [8] for oval cell pro-regenerative capacity upon liver damage. C3G down-regulation is known to promote the acquisition of a more mesenchymal phenotype in HCC and glioblastoma cells [23,26]. Hence, we analyzed the potential induction of an EMT process in oval cells by C3G knock-down as a mechanism to enhance migration and invasion. Figure 2A shows increased mRNA levels of EMT-inducing transcript-ion factors, Snail1, Zeb1 and Zeb2, in C3G-silenced oval cells, when maintained both in the presence or absence of serum. Interestingly, this increase was not observed in Twist1, which showed a tendency to decrease. The increased expression of these transcription factors elicited by C3G down-regulation was similar to that induced by TGF-β treatment [8]. Consistent with the increase in EMT-inducing transcription factors, the levels of the mesenchymal proteins, N-cadherin and Vimentin, were also higher in C3G-knock-down oval cells (Fig. 2B and 2C). This was corroborated upon C3G silencing with additional shRNAs (Supplementary Fig. 1A). Moreover, immunofluorescence analysis by confocal microscopy showed that Vimentin was differently distributed within C3G-silenced oval cells, being mainly present in cell extensions ( Fig. 2C and Supplementary Fig.  1C).
Confocal analysis also revealed the internalization and disorganized distribution of the epithelial marker, E-cadherin, in C3G knock-down cells, both when they were maintained in the presence or absence of serum ( Fig. 2D and Supplementary Fig.  1D, respectively). The subcellular localization of the tight junction protein zona occludens-1 (ZO-1) also changed from a cell-to-cell membrane contact site distribution to a diffuse pattern within the membrane in C3G-silenced cells (Fig. 2E and Supplementary Fig.  1E). Lastly, F-actin staining revealed its accumulation at focal adhesions and cell extensions in C3G-knock-down oval cells, consistent with features of migratory cells, especially when maintained in the absence of serum ( Fig. 2F and Supplementary Fig. 1F). All these data support the induction of an EMT process in oval cells by C3G silencing.
The EMT process is often associated with the acquisition of stemness properties and markers [27,28]. To analyze whether C3G down-regulation could have an impact on the expression of hepatic stem/progenitor cell markers, Cd133 and Cd44 mRNAs levels were quantified by RT-qPCR (Fig. 3A). Cd133 mRNA expression was significantly increased in C3G-knock-down cells, both in the presence and absence of serum. Likewise, Cd44 mRNA levels were higher in C3G-silenced cells, although differences did not reach statistical significance. Moreover, flow cytometry analysis revealed that C3G down-regulation increased the percentage of CD44 positive cells and CD44 levels (Fig. 3B), while decreasing the percentage of cells expressing the epithelial marker EpCAM (Fig. 3C). These data indicate that the EMT process promoted in oval cells by C3G down-regulation is associated with enhanced expression of stemness-related markers. Based on the need of a high clonal growth capacity for liver repair by oval cells, this was analyzed under adhesion conditions. As shown in Figure 3D, shC3G cells generated a significant higher number of colonies, reflecting a clonal growth advantage. However, the number of cells per colony was markedly reduced in shC3G cells. This could be explained by the enhanced pro-migratory capacity of these cells, which would facilitate cell escape from the original colony, leading to secondary colonies. This is supported by the morphological appearance of shC3G cells colonies, showing a pattern of scattered cells with less cell-to-cell contacts (Fig. 3E). This was already detected at early stages (day 4), when the number of scattered colonies was significantly increased in shC3G cells (Fig. 3F), and continued up to day 8, when cells were in closer proximity, but rarely in tight contact. However, it is important to highlight that C3G down-regulation did not promote anchorage-independent growth of oval cells (data not shown) and therefore, it did not confer tumorigenic capacity.  In addition, in agreement with the higher stemness of shC3G cells, changes in the expression of lineage markers were found, specifically, decreased mRNA levels of Albumin and CK19 ( Supplementary  Fig. 2), a hepatocyte and cholangiocyte marker, respectively, widely used as oval cell markers; and reduced mRNA levels of the transcription factor HNF4α, a known driver of hepatocyte differentiation (Fig. 4A). No changes were found on the expression of Afp, an early hepatocyte differentiation marker ( Supplementary Fig.2). Altogether, these data support that C3G down-regulation helps to maintain a non-differentiated phenotype in oval cells. Nevertheless, when cells were forced to differentiate into hepatocytes in vitro by treatment with a growth factor/hormone cocktail, a similar increase in the protein levels of E-cadherin and a decrease in CK19, were found in both parental and C3G-silenced cells (Fig. 4B). HNF4α mRNA expression increased in both control and shC3G oval cells, although it remained lower in shC3G cells at all the time points (Fig. 4A). All this suggests that either C3G is not essential for hepatocyte lineage commitment in oval cells or that low levels of C3G are sufficient for it.
In summary, low levels of C3G enhances pro-migratory, stemness and clonogenic properties without impairing differentiation into hepatocytes.

C3G is required for HGF/MET signaling and functionality in oval cells and to maintain a proper TGF-β pathway activation
As mentioned previously, HGF/MET axis plays a key role in oval cells, enhancing survival and migration [6,7], contributing to counterbalance TGF-β-induced EMT, and to maintain and/or promote their epithelial properties [8]. On the other hand, we previously demonstrated that C3G is required for a full activation of MET signaling in HCC cells [23]. Therefore, we analyzed whether C3G downregulation had any effect on MET signaling activation and HGF/MET-induced invasion in oval cells. Figure 5A shows that HGF-induced MET phosphorylation decreased in shC3G oval cells. The levels of P-Akt and P-p38 MAPK were also highly reduced in these cells and ERKs activation was delayed and diminished. Contrarily, phosphorylation of SHP2 phosphatase was much higher both in unstimulated and HGF-stimulated shC3G cells up to 10 min, although after 15 min P-SHP2 levels were higher in parental cells. These results indicate that HGF/MET signaling is defective in oval cells with low levels of C3G. Therefore, we evaluated its functional consequences. We found that shC3G oval cells presented a lower invasive capacity in response to HGF (Fig. 5B). Furthermore, lack of a functional MET receptor mimicked the low adhesion shown by C3G-silenced oval cells (Supplementary Fig. 3).
To determine the mechanism responsible for this reduced HGF/MET signaling in oval cells with C3G down-regulation, we search for a potential alteration in MET membrane localization and/or recycling as C3G facilitates recycling and membrane localization of EGFR in glioblastoma (GBM) cells [26]. However, we did not find significant changes in the levels of MET in the surface of shC3G oval cells (Fig. 5C). There was a tendency to decrease MET surface levels in non-silenced cells in response to HGF, which was not so well appreciated in shC3G cells. However, this effect was not significant. Alteration in HGF/MET signaling upon C3G knock-down could lead to an imbalance in the signals regulating oval cells, facilitating TGF-β effects, as HGF/MET counteract its action in oval cells [8]. Indeed, P-Smad2 and P-ERKs levels were increased in shC3G cells in response to TGF-β (Fig. 5D), suggesting that canonical and non-canonical TGF-β signaling is enhanced in oval cells with C3G down-regulation, favoring TGF-β actions.

Discussion
Adult HPCs/oval cells can proliferate and differentiate into hepatocytes and bile duct cells in response to a chronic and severe liver damage. However, they could also be potentially implicated in the generation of fibrosis and/or HCC [3,29,30]. Therefore, a tight control of oval cell activation and expansion is essential. Moreover, due to its regenerative potential, a deep knowledge of how oval cell fate and biology is regulated constitutes a relevant issue.
C3G is highly expressed in oval cells and neonatal hepatocytes [23], pointing to its relevance in the liver and, particularly, in oval cells. However, it remains unknown the function of C3G in the liver [31]. In this work, we have uncovered C3G as a protein that controls oval cell fate and physiology. C3G down-regulation by stable shRNA-mediated knock-down enhanced oval cell migratory and invasive properties to a similar extent to chronic treatment with TGF-β through inducing an EMT process. This effect of C3G down-regulation resembles that observed in colon carcinoma, HCC and GBM cells [21,23,26] and agrees with the inhibition of invasion promoted by C3G in breast cancer cells [32]. Moreover, this EMT was accompanied by a greater expression of the stemness markers, CD133 and CD44, in shC3G oval cells, which may facilitate the maintenance and expansion of HPCs/oval cells, in agreement with its enhanced clonogenic capacity. Interestingly, this differs from the TGF-β-induced EMT in oval cells [8], which is not associated to increased stemness, suggesting different mechanisms to promote EMT in oval cells. This is further supported by the additional increase in invasion induced by chronic TGF-β treatment in C3G-silenced oval cells.
It is important to mention that the EMT induced by C3G down-regulation is a partial EMT. Hence, although the epithelial marker E-cadherin is partially internalized, it still coexists with mesenchymal markers (e.g. Vimentin and N-cadherin). This coexpression of epithelial and mesenchymal markers has been previously observed in HPCs in different contexts [33], including after TGF-β-induced EMT [8]. Moreover, this is in line with the mesenchymalepithelial transitional phenotype of HPCs from human fetal liver [34] and it could represent a mechanism to enhance the plasticity of oval cells, contributing to liver repair. In addition, we could hypothesize that reduced levels of C3G in HPCs/oval cells might prevent HCC development based on our previous published data showing that C3G is upregulated in these tumors promoting their growth [23]. However, this deserves further investigation.
On the other hand, it should be noticed that a reduced expression of C3G did not impair in vitro oval cell differentiation into hepatocytes in response to HGF, dexamethasone and oncostatin M in the presence of serum, even though HGF/MET signaling was defective. These results could partially contradict previous data showing that C3G promotes the differentiation of muscle cells [35] and megakaryocytes [20], and with the fact that C3G knock-out in mouse embryonic stem cells (ESCs) impairs lineage commitment, while enhancing self-renewal and clonogenicity [36]. This potential discrepancy might be due to differences in the levels of C3G expression. Thus, low levels of C3G in oval cells could still allow hepatocyte differentiation, while a total absence would not. On the other hand, the enhanced stemness of C3G knock-down hepatic progenitor/oval cells would agree with the above mentioned increased self-renewal found in C3G knock-out ESCs [36].
Considering the above comments, in the in vivo liver context, where several growth factors and cytokines are produced in the HPCs niche such as HGF, TGF-β, EGF, TWEAK, IL-6 or TNF-α [3], it is likely that C3G expression could be finely tuned in oval cells to promote a successful liver repair. Thus, reduced expression of C3G could facilitate oval cell expansion, particularly after the proliferation peak, to enhance migration. In agreement with this idea, a time-dependent decrease in C3G protein levels occurred in livers from DDC-treated mice to induce oval cell expansion ( Supplementary Fig. 4A). In this line, in response to other types of liver damage, the expression of its mRNA (RapGEF1) was also reduced in the liver. Hence, the analysis of data from public databases indicate that RapGEF1 mRNA expression decreased in liver samples from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) patients compared to healthy livers ( Supplementary Fig. 4B). However, there are some discrepancies in other studies that could be due to the complexity of the liver, where several cell types are present, in addition to oval cells, which can express RapGEF1 mRNA and protein. On the other hand, since HPCs gradually lose EpCAM expression along with its maturation into hepatocytes [37,38] and EpCAM inhibits hepatocytic differentiation in human liver progenitors, the decreased EpCAM levels in C3G knock-down oval cells might facilitate its differentiation into hepatocytes [39], also preventing the development of liver fibrosis [40].
It should be also mentioned that the defective HGF/MET signaling and function found in C3G knock-down oval cells is not due to a failure in MET membrane localization, but rather a consequence of an altered formation of signaling complexes, as described in HCC cells [23].
In conclusion, according to our work, C3G plays a key role regulating oval cell phenotype and fate, facilitating a balance in the signaling induced by different signals. Reduced C3G levels enhance the migratory and stemness capacity of oval cells, which might facilitate liver repair in response to chronic injury. Future studies will aim to further understand and characterize the mechanisms of C3G actions in oval cells and how C3G levels are regulated in the context of liver regeneration upon chronic damage.