We used the global gene expression profile approach to identify context-dependent markers of the EMT obtained from the long-term TGFβ1 treatment of HUTEC in primary culture. Based on our previous data, we had speculated that the context-dependent EMT process we obtained was a dedifferentiating event. One of the aims of the present study was to further substantiate this hypothesis.
Various studies have shown that genes with a similar expression pattern frequently display common functions and form networks of interacting proteins [13
]. Assuming that the genes identified in our experiments belong to the TGFβ1-regulated pathways, we searched for interactions between the proteins encoded by the differentially expressed genes given in Additional file 1
. We reasoned that microarray analysis might identify only a part of the complex TGFβ1 network, due to other effects such as post-transcriptional regulation, so we used protein-protein interaction data to identify proteins interacting with those encoded by differentially expressed genes. We obtained a single connected component (interactome) consisting of 2630 proteins and containing 449 differentially expressed proteins that interact directly or with undifferentially expressed proteins. This analysis is extremely useful not only for detecting the network of interacting proteins that respond to TGFβ1, but also for identifying the network hubs, i.e. proteins with a high degree of connectivity that could have a crucial role in response to TGFβ1. We identified 27 hubs with more than 29 edges incident on them and encoded by genes found differentially expressed in our experiments. Of the three hubs identified as having more than 12 interactions with differentially expressed genes, SMAD3 was classified as a party hub while TGFBR1 and PXN were considered as date hubs. As shown by Han et al. [12
] in yeast, party and date hubs might have markedly different global properties in the interactome network. In accordance with this model, TGFBR1 and PXN, which interact with proteins that have a different cellular localization, might represent global or "high level" connectors between different biological modules and SMAD3, which functions within a module, works at a "lower level" in the organization of the proteome.
Among hubs, thrombospondin 1 (THBS1) – the principal activator of the TGFβ1 peptide [14
] – emerges because it links a wide range of matrix proteins mediating their interaction with cell-surface receptors. Its central role is confirmed by the 35 links connecting this protein to the other proteins in the interactome map. It could be considered as a date hub in the TGFβ1 interactome, according to the definition given by Han et al. [12
]. In Figure , the topological connections of THBS1 are magnified (A) and the differentially expressed genes interacting with it are indicated (B). Moreover, THBS1 is the gene that has the most significant correlation with TGFβ1 dosage (Table ).
Figure 5 Topological connections of the date hub THBS1. Thrombospondin 1 (THBS1) links directly 35 proteins in the TGFβ1 interactome: A) the topological connections are shown; B) the proteins encoded by differentially expressed genes are indicated and (more ...)
THBS1, PLS3 and ITGAV are known to have a pivotal role, both in cytoskeleton remodeling and in de novo extracellular matrix synthesis, two of the most relevant processes in EMT. The importance of these processes is highlighted by the considerable number of differentially expressed genes in the "extracellular matrix" and "cytoskeleton" GO categories (Figure ).
In the "extracellular matrix (EMC)" category, the different regulation of the various collagen types emphasizes the transition from an epithelial-like to a mesenchymal ECM. The up-regulation of TNC and FN1, one of the hubs of the TGFβ1 interactome, confirmed the switch to a mesenchymal ECM. The laminin α chain was found differentially down-regulated in our model, as in other EMT models [5
], while the β1 chain is up-regulated, as occurs in the precondensed mesenchyma [15
], suggesting that an embryonal mesenchymal ECM is being synthesized. This suggestion is reinforced by the types of integrins up-regulated during EMT. In fact, the "ECM receptor interaction" of the KEGG pathways (Additional file 2
) reveals not only the up-regulation of integrins generating the network for mesenchymal cell adhesion (ITGA5 and ITGAV) [16
], but also – and above all – the strong over-expression of ITGA11, which constitutes the receptor for the interstitial collagens involved in cell migration and collagen reorganization on mesenchymal cells during development [17
The "cytoskeleton" category is characterized by a large number of up-regulated genes, some of which have a crucial role in cytoskeleton remodeling. It is worth noting that four hubs of the TGFβ1 network, i.e. filamin A, alpha (FLNA), alpha 1 actin (ACTA1), paxillin (PXN) and beta actin (ACTB), belong to this category.
"Morphogenesis" is the GO category with the largest number of up-regulated genes, as shown clearly in Figure . The significance score assigned by the GOMiner software is not very high, however, because this category is one of the most represented in the microarray chip. It should nonetheless be noted that some of the genes belonging to this category are among the top ten up-regulated differentially expressed genes (Additional file 1
). Stimulated by TGFβ1, HUTEC seem to reactivate the developmental processes: this may point to a sort of stemness of tubular cells that enables them to dedifferentiate when stimulated (kidney repair and maintenance), but also to reawaken a silenced embryogenetic program. Sox 11, GADD45B, N-cadherin (CDH2), Activin A (INHBA), CTGF, FGF1/5, Angiopoietin (ANGPTL4), natriuretic peptide precursor B (NPPB), calcitonin receptor (CALCR) and caldesmon 1 (CALD1) are the most over-expressed genes belonging to the "morphogenesis" category. That tubular cells have the ability to reactivate an embryogenetic program has been demonstrated very recently by Kitamura et al. [18
] and Maeshima et al. [19
], who identified and isolated cells of the S2 segment with a potential tubulogenic ability and a capacity for integration in the developing kidney. Our results are in line with their findings.
CD133+ cells have been found to have stem cell potential in the adult kidney [20
]. Hypothesizing that CD133 up-regulation should occur if a stemness property is activated by TGFβ1, we looked specifically for prominin 1 (CD133) mRNA activation in our EMT model. Surprisingly, we found CD133 down-regulated, indicating that CD133 is expressed in control conditions. This finding supports the view taken by Florek et al. [21
], who showed that prominin 1 transcript and the alphahE2 epitope immunoreactivity of CD133 (obtained using a novel antibody instead of AC133) occur in several adult tissues and in the proximal tubular cells of the adult kidney in particular.
Signaling members of the Wnt (WNT5B) and FGF (1/5) families and transcription factors such as Sox 11, known to have a crucial role in nephrogenesis and cell fate determination during kidney development [22
], were found up-regulated. The involvement of the Wnt pathway was also confirmed by KEGG analysis.
The non-canonical Wnt signaling pathway centered on WNT5/Ca2+
] seems to be activated through Nemo-like kinase (NLK) in our EMT model and to antagonize the canonical beta-catenin Wnt signaling (Additional file 2
). In fact, we observed the down-regulation of both wingless-type MMTV integration site family, member 2B (WNT2B) and catenin, beta 1 (CTNNB1) and the up-regulation of both WNT5B and the calcium signaling pathway (see KEGG pathways). This effect seems to be reinforced by the up-regulation of dapper, antagonist of beta-catenin, homolog 1 (DACT1), which is a known beta-catenin Wnt signal inhibitor. It has been reported that inhibiting the beta-catenin system strongly inhibited TGFβ1-induced αSMA expression in tubular cells [26
]. Our data confirm this finding, since no αSMA expression was triggered by TGFβ1 in our EMT model [6
The non-canonical Wnt pathway, which includes planar cell polarity – an important process in embryonal axis development involving cytoskeletal polarity, as well as in the calcium pathway regulating cell adhesion [24
]-, was thus found up-regulated in our EMT model, reinforcing the idea that an embryological program is awakened. Very recently, Osafune et al. [25
] reported that the capacity of renal progenitor cells of the metanephric mesenchyme to form colonies in vitro and undergo mesenchymal-epithelial transition is positively regulated by planar cell polarity pathways downstream from Wnt.
Although the canonical Wnt signaling was repressed, the final effector of the pathway – and one of the most important – Cyclin D1 (CCND1) was up-regulated, whereas Cyclin B2 (CCNB2) – which is assumed to bind to TGFβ R2 and thus play a key part in TGFβ-mediated cell cycle control – was down-regulated.
Apoptosis and EMT are two distinct and opposite signal modules for TGFβ1-downstream effects. There is growing evidence that SMAD3 is an important signaling anchor for the apoptotic network for TGFβ1 too. In particular, the loss of SMAD3 function due to a decrease in its expression might be a requirement for epithelial cells to survive in the presence of prolonged TGFβ1 stimulation [27
]. This was also confirmed in our EMT model: visual inspection of the TGFβ-SMAD KEGG pathway (Additional file 2
) reveals what we demonstrated previously using RT/PCR analysis [6
], i.e. that Smad signaling was down-regulated. Despite the number of up-regulated inducers (TGFβ1, INHBA), the key transducers are all down-regulated, as are the Id genes (the effectors of cell differentiation). The ID2B gene was specifically down-regulated. Mad expression and ID2 down-regulation are important events in the TGFβ1 cytostatic program in epithelial cells and ID2 suppression by TGFβ1 is essential for EMT to occur [28
The central role of SMAD3 is also demonstrated by its position in the TGFβ1 network: it is one of the hubs, most likely a date hub since it works within a single module (Figure ), at a low level of network organization. This might explain why apoptosis seems to be induced in our model, despite SMAD2 and SMAD3 down-regulation. In fact, a visual inspection of the KEGG apoptosis pathway clearly shows the up-regulation of caspase 3 (CASP3), a known inducer of cell death, and the under-expression of BCL2 and BIRC3 (IAP gene in the KEGG map), which counteract this action.
Figure 6 Topological connections of the party hub SMAD3. SMAD3 links directly 119 proteins in the TGFβ1 interactome: A) the topological connections are shown; B) the proteins encoded by differentially expressed genes are indicated and colored respectively (more ...)
On the other hand, the up-regulation of several genes implicated in the cell cycle pathway (Additional file 2
), such as CCND1, GADD45, YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), indicates that cells are entering the cell cycle. In this pathway, however, the up-regulation of wee1 tyrosine kinase (WEE1), one of the genes strictly regulated by TGFβ1, seems to indicate a sort of actual control of cell proliferation, so both apoptosis and cell cycle entry seem to be strictly controlled in the EMT process. Indeed, neither apoptosis nor proliferation were seen in our model by immunocytochemistry [6
]. It is tempting to speculate that a concerted and strictly controlled action between signals for cell death and proliferation might be taking place in cells after long-term TGFβ1 exposure, miming the developmental process in which morphogens such as TGFβ1 act not as positive regulators of cell differentiation but as key regulators of cell survival [30
]. It is worth noting that key proteins of the Wnt signaling and apoptosis/cell cycle control pathways (i.e. CTNNB1, CCND1, CASP3, BCL2) were found to constitute some of the hub proteins of the TGFβ1 network.
Finally, KEGG analysis of microarray data highlighted that RAS/MAPK signaling was the principal downstream effector of chronic TGFβ1 stimulation in our EMT model, confirming the suggestions advanced by other authors [5
], i.e. that both Smad-dependent and Smad-independent signaling cascades are activated by TGFβ1 and that they regulate mesenchymal transition in a context- and cell-dependent manner. The MAPK signaling pathway has an important role in connecting the signal triggered by TGFβ1 to important downstream processes such as apoptosis/proliferation and the Wnt cascade [5
]. Our results confirm reports from other authors on the role of this signaling in other EMT processes [31