Cell surface-localised GRP78

The main role of GRP78 is to regulate the UPR in the ER, but it is also known to act as a binding receptor at the cell surface [189,248,464] where it plays a role in trophoblastic syncytialisation [93]. Several binding partners of sGRP78 have been identified; nevertheless, we focused our interest on α2M. The interaction of α2M with GRP78 was demonstrated in

146 prostate cancer cells to activate several signalling pathways leading to cell growth, survival and proliferation [238,241,250]. We studied the effects that α2M may have on trophoblastic syncytialisation and the activated mechanisms that could be involved in the promotion of cell fusion.

In the first part of the study, we observed increased α2M expression in trophoblastic cells, which corresponded to an increment in the gestational stage of the placenta. We also confirmed that BeWo cells did not express α2M, which allowed the treatment of these cells with exogenous α2M to observe its effects on trophoblastic fusion. We thus demonstrated a role for α2M in regulation of cell fusion that was mediated by sGRP78.

In the BeWo cell model, α2M treatment led to activation of ERK1/2, CREB and JNK. These proteins and their downstream signalling pathways were implicated in the expression of syncytin proteins and hCG secretion [465-467]. However, we did not observe the expected increase in expression of syncytin mRNA or hCG secretion in BeWo cells treated with α2M.

We speculated that activation of ERK1/2 and CREB may be involved in trophoblastic cell fusion by promoting certain intracellular pathways. It was previously reported that CREB can enhance activation of the UPR [468]. Additionally, ERK1/2 was demonstrated to activate CREB [469]. Indeed, we confirmed the involvement of ERK1/2 in CREB phosphorylation, which in turn is implicated in activation of the UPR in BeWo cells.

As we discussed in detail in the first part of this study, the UPR is activated during trophoblastic cell fusion and promotes cell survival through autophagy activation [320]. The α2M treatment in BeWo cells caused an increased expression of GRP78 and CHOP, the main markers of UPR activation. Nevertheless, in our previous study we demonstrated that the activation of the UPR in trophoblastic cells led to an increase in hGC secretion [320]. We

147 hypothesised that the CREB-dependent UPR activation that after α2M treatment may activate the IRE1α and PERK branches of the UPR, but not the ATF6 branch. It was already reported that CREB was involved in activation of IRE1α and PERK in breast cancer MDA-MB231 cells, while no effect was observed in ATF6 [468], supporting our hypothesis. Conveniently, we previously observed that the UPR-induced hCG secretion is mainly controlled by ATF6 (data not shown) in trophoblastic cells. This observation may explain why α2M-induced UPR activation does not lead to increased hCG secretion. To conclude, α2M induces BeWo cell fusion but not differentiation. Moreover, the absence of α2M expression in this cell line, which is able to fuse [470], suggests that this protein is not indispensable for BeWo cell fusion.

The results obtained with BeWo cells should be verified in primary cells that demonstrate high α2M expression. However, we failed to silence α2M expression in these cells (data not shown) and the addition of exogenous α2M had no effect since α2M is highly expressed in these cells.

One finding that we consider interesting when studying the potential of sGRP78 in trophoblastic cell fusion is the structural conformation of the α2M protein. α2M is a homotetrameric protein, which confers the ability of α2M to bind up to four different partners [265]. We suggest that α2M may bind to sGRP78 in two different cells, bringing them in close proximity and permitting an interaction between the fusogenic proteins that underpin fusion. The structural conformation of α2M in cell fusion may be easy to observe by labelling the cell membrane, sGRP78 and α2M prior to microscopy imaging. The confirmation of the bridge function of α2M through sGRP78 binding would confer an additional feature to GRP78 in trophoblastic cell fusion that may be essential to achieve complete fusion.

148 The binding promiscuity of α2M* [471-474], together with the possible bridge function of this protein, reinforces the importance of studying the role of monomeric α2M in cell fusion.

Other studies have already demonstrated the ability of monomeric α2M, most concretely a short version of the protein containing only the RBD, to induce intracellular protein activation in the same manner as tetrameric α2M* [475]. Additionally, treatment with the RBD mutant, K1370A (a mutated short version of α2M that eliminates the capacity of the RBD to bind LRP), also led to signalling pathway activation in prostate cancer cells [475].

Treatment of BeWo cells with the RBD and RBD mutated K1370A may serve to confirm the promotion of trophoblastic cell fusion by the interaction of α2M with sGRP78. Furthermore, the results would also serve to decipher the potential bridge function of tetrameric α2M*, since the cell fusion rate could easily be compared to that observed with RBD and RBD mutated K1370A, confirming our results and solving our hypothesis. We could conclude that an interaction between sGRP78 and α2M can promote trophoblastic cell fusion by activating ERK1/2, CREB and JNK, triggering the UPR. The exact mechanisms involved in the cell fusion are still unknown but may be deciphered in the near future following the previously mentioned hypothesis.

The promising results obtained with the sGRP78–α2M interaction in trophoblastic cell fusion lead us to expect similar observations with members of the same family of proteins such as PZP. PZP is macroglobulin protein specifically found during pregnancy that shares a 71%

amino acid sequence correlation with α2M [476,477]. In a similar manner to α2M, PZP is transcribed as a monomer of 180 kDa, but unlike α2M, the active form of PZP is a homodimer of 360 kDa [478]. PZP is a trace plasma protein (<0.01 mg/ml). However, the female reproductive hormones modify its expression, altering the plasma levels up to 100–200-fold

149 above pre-conception levels at term [479,480]. Additionally, PZP has been detected in STB [481]. PZP function during pregnancy is not completely understood but it is believed to play a role in immune regulation [482]. Neither its mechanisms nor its functions have been completely deciphered.

PZP is known to bind a wide variety of proteins such as vascular endothelial growth factor, placental growth factor and placental protein-14 [483]. The capacity of PZP to bind different proteins, together with its similarity to α2M, lead us to hypothesise a possible interaction between this protein and sGRP78, and subsequent signalling pathway activation in trophoblastic cells. The impact of this probable interaction and signalling pathway activation in cell fusion would be interesting to analyse. Finally, the dimeric structure of PZP could also be involved in cell fusion through the bridge function, increasing interest to study the possible PZP–sGRP78 interaction.

It is clear that multiple proteins are implicated in the syncytialisation process and several more might be involved even if their contribution is unknown at present. The path to a total understanding of the process is still long; nevertheless, the results we have obtained during this study help to pave the way, facilitating future research and bringing to light certain essential aspects.

4.2 P

The different processes that we have discovered as active during physiological syncytialisation may also be implicated in the development of certain placental disorders such as PE. It has already been reported that in vCTBs of patients with PE, the mRNA levels of GRP78, CHOP and ATF4 (UPR-associated genes) are increased when compared with

150 those of healthy patients [181,484]. In a similar way, it has been demonstrated that autophagy was higher in vCTBs from patients suffering from PE [485-489]. Some of these studies observed an increase in the expression of Beclin-1 and LC3bII protein, which corresponded to the results we obtained in our laboratory at the mRNA level [489,490]. Moreover, comparisons of apoptotic activation in patients with PE and in healthy patients highlighted a greater incidence of apoptotic nuclei in the complicated pregnancies [491,492], which is in agreement with the abnormal mRNA levels of the apoptotic genes, p53, Bak, Bcl2 and Bcl-Xl, that we observed [491,493-495]. Together, we hypothesise that an upregulation of the UPR may generate an imbalance in autophagy and apoptosis of vCTBs.

Overactivated autophagy may be detrimental for the vCTBs, generating excessive self-degradation and activating autosisa non-apoptotic cell death mechanism [445]. At the same time, the increased activation of apoptosis also promotes cell death and nuclei degradation [491,492]. We could assume that increased autophagy and apoptosis intensify the number of degrading nuclei that need to be eliminated from the STB in form of syncytial knots [170,171]. Syncytial knots are aggregates of apoptotic nuclei packed into protrusions that are released in the maternal blood [95]. The importance of the syncytial knot increment resides in the inability of the maternal clearance system to cope with the abnormally high levels of apoptotic fragments in the blood, promoting secondary necrosis within the blood and leading to the clinical symptoms of PE [155]. The findings we made link the UPR with autophagy control and cell fate regulation in trophoblastic cells and demonstrate the role the UPR and GRP78 may have in preventing and treating PE.

In the context of placental disorders, a surprising observation was made by a pathologist in PE tissue, which led us to speculate an additional involvement of the UPR in PE development

151 and its role in controlling cell fusion. The thickness of the STB layer in PE placenta and in healthy placenta showed no significant differences [496,497]. The increased autophagy and apoptosis may increase the number of apoptotic nuclei that are eliminated from the STB as syncytial knots [170,171], causing a reduced cellular material in STB. Consequently, the thickness of the STB layer was expected to be smaller and less nucleated. The equality in STB thickness reported between PE and healthy patients suggested an enhancement on vCTB cell fusion to balance the material loss [498].

Conversely, syncytin expression at the mRNA and protein levels in patients with PE showed that fusogenic protein expression was reduced [499-501]. A reduced expression of syncytins is generally accompanied by decreased trophoblastic cell fusion [67,502], which disagrees with our hypothesis. A possible explanation for these results is a nomenclature confusion derived from the different subdivisions of PE. As previously mentioned, PE can be subdivided into early-onset PE and late-onset PE [155,164], displaying differences between them, and it can also be accompanied by IUGR (PE + IUGR) [155]. The volume and surface area of the STB layer in PE + IUGR patients are reduced [496,497,503-505], together with a decreased number of STB nuclei [496,497,503-505]. In this specific case of PE, we would expect reduced cell fusion and a decreased syncytin expression. Therefore, we propose that some studies may have collected the three different types of PE placenta, without separating them by subdivision, in order to analyse syncytin expression. Thus, the conclusions from these studies may not be correct for early-onset PE.

We suggest that in early-onset PE there is an increased cell fusion that aids to accomplish an STB layer of normal thickness in these patients. A study separating the different PE placenta by subdivision may help to discriminate the expression of syncytins depending on the specific

152 type of disorder. Interestingly, preliminary results obtained by some colleagues in early-onset PE placenta suggest an increased expression of syncytins at the mRNA and protein level, together with an increased fusion index (data not shown). Confirmation of these results may be essential to better understand early-onset PE and could reinforce the differences between the subdivisions of PE.

Additionally, despite activation of the UPR and overexpression of GRP78 in PE vCTBs, the relocalisation of GRP78 to the cell surface of PE vCTBs is reduced in severe cases [93]. We hypothesised that in PE there is an accelerated trophoblastic cell fusion, accompanied by increased nuclear apoptosis and clearance from the STB. The decreased relocalisation of GRP78 to the cell surface should lead to a decrease in the fusion capacities of trophoblastic cells, which in principle do not fit with our hypothesis. In the PE model that we propose, there is an accelerated cellular turnover that may be essential for pathology progression. In this specific PE model, we could hypothesise the relocation blockage as a cellular protection mechanism. PE is a severe placental disorder that despite its gravity, allows foetal development [155], demonstrating the partial functionality of the STB layer. An even greater cell fusion rate during STB formation in PE may lead to an unsustainable state where the newly formed cell is completely disorganised and unable to perform its functions such as maternal–foetal exchange and hormone secretion. Additionally, in contexts where the GRP78 in PE would normally relocate to the cell surface, increased activation of the UPR may be observed and may lead to uncontrolled apoptosis and definitive STB collapse. The deficient GRP78 relocation to the cell surface may be an evolutionary mechanism present in PE that secures the STB functionality during pregnancy disorders and foetal development.

153 In conclusion, we hypothesise that in PE there is an accelerated cellular turnover. The increased activation of the UPR [181,484] may cause overactivation of autophagy [485-490]

and apoptosis [491-494], which may lead to an increase in syncytial knot formation and nuclei elimination [170,171]. At the same time, the UPR may be involved in promoting additional vCTB fusion to the STB, permitting formation of a layer displaying normal thickness [496,497]. Finally, the defective GRP78 relocation to the cell surface of trophoblastic cells may protect the STB, allowing it to remain functional and ensure foetal development.