Mutations in the SFTPC gene are a known cause of surfactant deficiency and very variable genetic ILD in children and adults. We investigated the consequences of the expression of SP-C with the mutation A116D for cell homeostasis and signaling in stably transfected MLE-12 cells. We further elucidated the ability of pharmaceutical drugs used in ILD therapy to modulate some of the cellular consequences of SP-C deficiency caused by the A116D mutation.
Stable transfection of MLE-12 cells with SP-CA116D
led to the intracellular accumulation of proSP-CA116D
processing intermediates which were not found in cells transfected with proSP-CWT
(Figure ). However, the observed species resembled those seen in another SP-C mutation, I73T, in vitro
as well as in BAL fluids from patients carrying this particular mutation [38
]. The accumulation we observed in the case of A116D may thus reflect alterations in folding, trafficking and/or processing of proSP-CA116D
. The first step in proSP-C processing is a cleavage at the C-terminal end [4
]. Using an EGFP tag fused to the C-terminus of proSP-C showed no difference in processing intermediates of proSP-CWT
(Figure , right). This suggests that the mutation does not interfere with the first cleavage step that takes place at the C-terminus. Furthermore, this finding implies that the mutation does not abrogate the export from the ER and Golgi completely, because this cleavage occurs after trafficking through these compartments [4
]. It is not known how the A116D mutation affects the folding of proSP-C, but subtle changes in conformation may be responsible for the appearance of a processing intermediate of approx. 17 kDa (Figure , band #3). A similar intermediate can be found in the BAL fluid of patients with the SP-CI73T
mutation, suggesting that this proSP-C form is being secreted from AT2 cells along with the mature SP-C that is produced by AT2 cells regardless of the presence of the I73T mutation [38
Immunofluorescence assay of stably transfected MLE-12 showed that proSP-CA116D
often colocalized with EEA1 positive vesicles (Figure ). Early endosomes generally contain material that is taken up by endocytosis and is either recycled or routed for degradation [39
]. Up to 80% of secreted lung surfactant is known to be reinternalized by AT2 cells from alveolar space [6
]. Colocalization of proSP-CA116D
with EEA1 would thus imply that mutant protein is secreted together with surfactant and subsequently taken up again. On the other hand, while proSP-CWT
colocalized with syntaxin 2, proSP-CA116D
rarely did so. Syntaxin 2 is a SNARE protein involved in the secretion of lung surfactant, found in the plasma membrane and lamellar bodies of AT2 cells. Surfactant secretion is dependent on the fusion of lamellar bodies with the plasma membrane, which requires the activity of SNARE proteins. The lack of colocalization with Syntaxin 2 implies that mutant SP-C mostly does not reach lamellar bodies. Our results thus suggest that while physiological proSP-C forms are secreted via lamellar body fusion with the plasma membrane, proSP-CA116D
might take a different route and eventually gets lysosomally degraded.
The expression of mutated proteins frequently results in elevated cell stress. This has been shown for the SP-C BRICHOS domain mutations L188Q and Δexon4 [14
]. We found that the constitutive expression of SP-CA116D
also significantly increased cell stress, compared to expression of SP-CWT
. Likewise, the treatment with azathioprine resulted in an elevated stress level in cells expressing SP-CWT
as well as in cells expressing the mutant SP-C form where it further exacerbated cell stress due to the mutated SP-C. No significant alteration of cell stress was observed when cells were treated with methylprednisolone, hydroxychloroquine or cyclophosphamide. It can thus be concluded that, similar to other BRICHOS domain mutations, SP-CA116D
leads to elevated cell stress that cannot be corrected by the ILD drugs currently applied. Quite the contrary, our data suggest that some substances used in ILD therapy, especially azathioprine, pose potent stress factors per se
After demonstrating that SP-CA116D
expression increases cell vulnerability to pharmacological stress stimuli, we further aimed to investigate the underlying intracellular mechanisms. Chaperone proteins assist in protein folding and are also involved in the folding of aberrantly processed proteins. Their synthesis is increased by cells as part of a cytoprotective mechanism to cope with increased intracellular stress and accumulation of misfolded proteins [27
]. Still, without pharmacological boost, such cytoprotective mechanisms may not always be sufficient to normalize the cell function and maintain production of bioactive surfactant with a physiological lipid/protein composition. We investigated the influence of ILD drugs on the expression of four important chaperones in WT and A116D cells. The expression of Hsp70, Hsp90 and calreticulin was markedly increased in cells expressing SP-CA116D
(Figure ). Only in the case of calnexin no significant increase was observed (Figure ). No drug did affect the expression of any chaperone in a significant fashion in WT cells. Furthermore, no drug was able to further increase the elevated chaperone levels seen in cells expressing A116D. However, azathioprine and methylprednisolone led to a slight, albeit not significant, increase in the expression of Hsp70 and Hsp90 by A116D cells. These findings imply that the drugs we tested did not possess the ability to significantly increase chaperone levels. It is thus questionable whether they exert their beneficial effects in ILD treatment by enhancing ER folding capacity.
The packaging and secretion of lung surfactant lipids is very closely linked to the expression of the hydrophobic surfactant proteins in AT2 cells [6
]. The lipid composition of stably transfected MLE-12 cells was similar to that previously described for human fetal AT2 cells, especially with regard to PC composition [41
]. In the cells expressing SP-CA116D
we found a pronounced drop of total cellular PC, while LPC was increased (Figure , Table ). It is known that PC is degraded to LPC by an intrinsic phospholipase A2-like activity, and that LPC is toxic to various cells [42
]. LPC is a known inhibitor of the lung surfactant activity and has the ability to penetrate directly into interfacial films to impair lowering of the alveolar surface tension during dynamic compression [43
]. Elevated LPC levels in the SP-CA116D
-expressing cells could also explain the heightened sensitivity towards exogenous stress described above. Generation of LPC cannot account for the decrease of PC mass in SP-CA116D
expressing cells, but additional factors, which directly interfere with the synthesis and packaging of PC, must also be responsible.
Among phospholipids secreted by cells expressing the A116D mutation into culture supernatant, PC was again decreased by 14% and LPC was increased by 184%, in accordance with a reduced surfactant function [41
]. Treatment with hydroxychloroquine ameliorated the increase in intracellular and secreted LPC and decrease in secreted PC, but did not completely correct it (Figure ). While methylprednisolone treatment resulted in a reduction of intracellular LPC, it did not have an effect on PC levels. The capacity of the treatment with methylprednisolone and hydroxychloroquine to correct the lipid disturbances caused by the A116D mutation likely represents one of the mechanisms by which these treatments are empirically helpful in some patients with A116D mutations [28
] (own unpublished results). However, their mode of action still remains a conundrum and needs to be investigated further.
Injury of lung epithelial cells resulting from endogenous or exogenous stress leads to accumulation of pulmonary leukocytes and finally to inflammation and tissue remodeling. Leukocytes are attracted to sites of inflammation along a chemokine gradient. This chemotaxis is mediated via chemokine receptors on the surface of leukocytes [45
]. In previous studies we demonstrated that, among a plethora of chemokine receptors involved in this network, specifically CCR2 on lymphocytes and CXCR1 on neutrophils modulate pulmonary immunity in human inflammatory lung diseases [37
]. We found that cells expressing SP-CA116D
released soluble factors into the medium that increase surface expression of CCR2 and CXCR1 on CD4+ lymphocytes and CXCR1 on neutrophils (Figure ). When activated, the high affinity IL-8 receptor CXCR1 mediates antibacterial killing capacity [46
]. Increases in surface expression levels of CCR2 and CXCR1, respectively, might have the potential to modulate the pulmonary immune response with regard to antibacterial (CXCR1) and profibrotic (CCR2) responses [46
]. However, the soluble factors involved in the induction of chemokine receptor expression as well as the functional consequences of this phenomenon remain to be addressed in future studies.
There are some limitations to our study. A potential limitation is that our system represents a homozygous mutation rather than a heterozygous SFTPC
mutation where one functional allele is still present. However, endogenous SP-C is expressed in the MLE-12 cells [29
]. Although expression of exogenous SP-C from the CMV promoter present on the plasmid vector is likely higher, both forms are present. Given that all known patients with SP-C mutations are heterozygous carriers, expressing one copy of the wild type gene, the experimental model closely reflects the in vivo
condition. A second important limitation to consider is that one has to be careful when extrapolating findings in MLE-12 cells in culture to the actual alveolar environment. In particular the compositional profile of lipids found in cell culture supernatant is not necessarily representative for surfactant lipids secreted by AT2 cells in vivo as cells in culture may secrete lipids by mechanisms other than via lamellar bodies. Finally, the A116D mutation seems to be rare and some of its features may be specific and not generalizable.