Published On: Wed, Mar 27th, 2019

The 1ALCTL and 1BLCTL isoforms of Arg/Abl2 induce fibroblast activation and extra cellular matrix remodelling differently [RESEARCH ARTICLE]

DISCUSSION

During the fibrosis process myofibroblasts, the activated form of fibroblasts continue to produce ECM resulting in abnormal scar tissue deposition. Myofibroblasts also occur in the context of growing tumour, and in some solid tumours they can generate a very abundant fibrotic stroma. The increase of the proliferation rate, the expression of several markers, the ECM production and remodelling are features of myofibroblasts (Yazdani et al., 2017) The knowledge of molecular players involved in fibroblast activation is important for the development of novel therapeutic strategies against fibrosis and growing tumours. Our data evidence the role of Arg in fibroblast activation. We previously showed that the 1ALCTL and 1BLCTL Arg isoforms determine some morphological and molecular changes in COS-7 cells (Bianchi et al., 2013).

Now, we have shown that Arg is necessary for fibroblast proliferation. Considering our two transfected isoforms, which maintained their tyrosine kinase activity (Fig. 1), the 1BLCTL isoform alone is sufficient to restore the proliferation rate of wt MEF (Fig. 2A,B). However, literature data show that Arg knockdown in MDA-MB-231, a breast tumour cell line, improves the proliferation of tumour epithelial cells (Gil-Henn et al., 2013), highlighting that the role of Arg in cellular proliferation could be cell specific. Numerous papers report the expression and localisation of α-sma as common markers of activated fibroblasts (O’Connell et al., 2011; Shi et al., 2016; Goffin et al., 2006). Our data showed that 1BLCTL isoform is able to restore both the α-sma expression and colocalization with stress fibres, negligible in Arg−/− MEF (Fig. 2C; Fig. S2A). This finding suggests that Arg is necessary for fibroblast activation and that the fibrotic or the primary tumour stroma could be characterised by 1BLCTL Arg upregulation. Also an intriguing finding, in Arg−/− MEF, showed us that TGFß1 expression was inversely related to Arg expression (Fig. 2D). We also described this interesting inverse relation between Arg and TGFß1 in renal tubular cells after Arg silencing (Torsello et al., 2016). It seems that the absence of Arg make the fibroblasts less responsive to the proliferative and activating effects of TGFß1 signals, suggesting that the TGFβ1 signalling is less efficient in absence of Arg, therefore, TGFβ1 production is upregulated. The TGFß1-mediated effects, carried out in wt MEF, were restored by 1BLCTL but not completely by 1ALCTL isoforms (Fig. 2A–C). A defined Arg isoform expression pattern may also be relevant for the movement ability of myofibroblasts in both tumour and fibrotic milieu. In accordance with this hypothesis, the wound recovery and the invasion of collagen matrices revealed that 1BLCTL isoform sustains this ability at the highest level in comparison with all the other MEF analysed. In particular, it seems that the Arg 1BLCTL protein level into the cell can modulate the migration. In fact, 1BLCTL, overexpressed either in Arg-endogenous-expressing or in Arg−/− fibroblasts, determines a decrement of migration ability (Peacock et al., 2007; Bianchi et al., 2013). However, when in Arg−/− cells the expression of recombinant Arg is only twofold over the endogenous Arg level the cells recover the migration abilty of wt cells (Peacock et al., 2007). In our transfected 1BLCTL MEF, in which the recombinant Arg is at the same level of wt MEF, their migration is higher than wt MEF. Instead, the 1ALCTL MEF were not able to reach the migration and invasion capacity shown by 1BLCTL MEF and an inhibitory activity of 1ALCTL cannot be excluded (Fig. 3A,B). The 4-h cultures evaluated in 2D or 3D environment of the different MEF could help to explain their behaviour. In fact, the highest spreading of wt MEF in 2D (Fig. S2B) may justify their adhesion capacity (Fig. 4B) (Nardone et al., 2017), despite the increased phosphorylation of Y118-paxillin. Instead, in 1ALCTL MEF the inhibition of paxillin phosphorylation, seems to account for the increment of cell adhesion. In 2D culture the decreased adhesion ability of 1BLCTL MEF (Fig. 4B) could contribute to a faster migration as it happens in Arg−/− MEF (Fig. 3A). In the 3D environment of collagen matrix, 1BLCTL MEF are present as single cells (Fig. 4D) and this 3D-organisation associated with the decreased adhesion ability (Fig. 4B) can justify their high invasion capacity. 1ALCTL and wt MEF in addition to higher adhesion were also grouped together and this conformation could have delayed their invasion ability (Wong et al., 2014). In 3D culture the different spatial organization of cells can be due even to different modulation of adherent junctions. However, preliminary data in our different MEF (not shown) did not evidence significant differences in N-Cadherin expression, a marker of cell–cell adhesion in fibroblasts (Labernadie et al., 2017) and other cell–cell adhesion molecules need to be evaluated.

The ECM-contraction ability, another feature of myofibroblasts (Calvo et al., 2013), demonstrated that Arg is essential for collagen I contraction and that both 1ALCTL and 1BLCTL Arg isoforms are able, after 72 h, to restore the wt MEF contraction ability (Fig. 4E). An efficient ECM-contraction is due to working stress fibres and focal adhesions as well as to α-sma expression and incorporation in intracellular stress fibres (Calvo et al., 2013; Shinde et al., 2017; Shuttleworth et al., 2018). Our 1ALCTL and 1BLCTL MEF grown for 4 h inside collagen showed no focal adhesions (Fig. 4D). Otherwise, no difference in plug contractions was observed after 24 h (not shown). However, after leaving the 3D culture for 72 h the focal adhesions could have taken place, giving rise to plug contraction. It has to be noted that the dynamics of focal adhesions seems to be different in a 2D culture compared to a 3D culture (Chiu et al., 2014). Moreover, the α-sma expression (Fig. 2C) with its incorporation in stress fibres (Fig. S2A) may counteract the lack of functional focal adhesion preserving cell contractility.

The MEF analysed in this study were able to secrete and organise fibronectin and collagen I differently (Fig. 5). It is of note that Arg−/− MEF failed to deposit the collagen I, which is currently deposited by myofibroblasts during fibrosis and tumour progression (Karsdal et al., 2017). This finding underlines the unique role of Arg in the production of collagen matrix. Otherwise, 1BLCTL MEF produced networks of collagen I and fibronectin that are similar to those produced by wt MEF and this condition correlated with the highest stiffness of ECM, measured after removal of MEF. The ECM stiffness is due to the amount of collagen and fibronectin fibres, their cross-link and ECM morphology (Di Stefano et al., 2016; Mierke et al., 2017). The specific role of Arg in producing fibronectin and collagen matrix turned out to be also significant in producing a specific matrix framework able to modulate the tumour cell morphology. In fact, the highest elliptical factor was induced in 786-O RCC cells when they grew in the high-stiffness-ECM produced by wt and 1BLCTL MEF (Fig. 5B,C). This finding is particularly relevant since it has been described that a fabricated matrix when forces the cells to assume an elongated morphology becomes able to select the cells with a more aggressive behaviour (Mazzini et al., 2015). In fact, the elongated morphology of tumour cells reveals a more invasive phenotype (Tanaka et al., 2016).

In conclusion, Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of fibroblasts and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activation, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features.

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