# Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?

Céline Delestrain, Abdel Aissat, Stéphanie Simon, Agathe Tarze, Elodie Duprat, Elodie Nattes, Bruno Costes, Valérie Delattre, Stéphanie Finet, Pascale Fanen, et al.

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## To cite this version:

Céline Delestrain, Abdel Aissat, Stéphanie Simon, Agathe Tarze, Elodie Duprat, et al. Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?. British Journal of Clinical Pharmacology, 2021, 87 (5), pp.2361 - 2373. 10.1111/bcp.14645. inserm-04199312

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Submitted on 7 Sep 2023

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ORIGINAL ARTICLE

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BRITISH
PHARMACOLOGICAL
SOCIETY

# Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?

Céline Delestrain<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> | Abdel Aissat<fnref n="1" />,<fnref n="3" />,<fnref n="4" /> | Stéphanie Simon<fnref n="1" /> | Agathe Tarze<fnref n="1" /> |
Elodie Duprat<fnref n="5" /> | Elodie Nattes<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> | Bruno Costes<fnref n="1" />,<fnref n="3" />,<fnref n="4" /> | Valérie Delattre<fnref n="4" /> |
Stéphanie Finet<fnref n="5" /> | Pascale Fanen<fnref n="1" />,<fnref n="3" />,<fnref n="4" /> | Ralph Epaud<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> ID

Correspondence

Ralph Epaud, MD, Service de Pédiatrie, Centre Intercommunal de Créteil, 40 av de Verdun, 94000, Créteil, France

Email: ralph.epaud@chicreteil.fr

Funding information

Institut National de la Santé et de la Recherche Médicale

**Aims:** Mutations in the gene encoding surfactant protein C (SP-C) cause interstitial lung disease (ILD), and glucocorticosteroid (GC) treatment is the most recognized therapy in children. We aimed to decipher the mechanisms behind successful GC treatment in twins carrying a BRICHOS c.566G > A (p.Cys189Tyr) mutation in the SP-C gene (SFTPC).

**Methods:** The twins underwent bronchoscopy before and after GC treatment and immunoblotting analysis of SP-C proprotein (proSP-C) and SP-C mature in bronchoalveolar fluid (BALF). Total RNA was extracted and analysed using quantitative real-time PCR assays. In A549 cells, the processing of mutated protein C189Y was studied by immunofluorescence and immunoblotting after heterologous expression of eukaryotic vectors containing wild type or C189Y mutant cDNA.

**Results:** Before treatment, BALF analysis identified an alteration of the proSP-C maturation process. Functional study of C189Y mutation in alveolar A549 cells showed that pro-SP-C<sup>C189Y</sup> was retained within the endoplasmic reticulum together with ABCA3. After 5 months of GC treatment with clinical benefit, the BALF analysis showed an improvement of proSP-C processing. SFTPC mRNA analysis in twins revealed a decrease in the expression of total SFTPC mRNA and a change in its splicing, leading to the expression of a second shorter proSP-C isoform. In A549 cells, the processing and the stability of this shorter wild-type proSP-C isoform was similar to that of the longer isoform, but the half-life of the mutated shorter isoform was decreased.

**Conclusion:** These results suggest a direct effect of GC on proSP-C metabolism through reducing the SFTPC mRNA level and favouring the expression of a less stable protein isoform.

KEYWORDS

children, glucocorticoid, interstitial lung disease, surfactant, treatment

<footnote n="1">¹Université Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France</footnote>

<footnote n="2">²Centre Hospitalier Intercommunal de Créteil, Service de Pédiatrie Générale, Créteil, 94000, France</footnote>

<footnote n="3">³FHU SENEC, Créteil, France</footnote>

<footnote n="4">⁴AP-HP, Hôpital Henri Mondor, Pôle de Biologie-Pathologie, Département de Génétique, Créteil, 94000, France</footnote>

<footnote n="5">⁵Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, CNRS, Sorbonne Université, Muséum National d'Histoire Naturelle, Paris, France</footnote>
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# 1 | INTRODUCTION

Surfactant protein C (SP-C) is an essential proteolipid of pulmonary surfactant, which is needed to reduce alveolar surface tension and prevent end-expiratory atelectasis. SP-C is encoded by the SFTPC gene (SFTPC), which is expressed exclusively in alveolar type II cells.¹⁻³ The messenger RNA (mRNA) is translated into a proprotein (proSP-C), which is extensively post-translationally modified, yielding mature SP-C (3.7 kDa).⁴,⁵ Multiple human SP-C mRNAs, generated by alternative splicing, encode only two proSP-C isoforms distinguished by six amino acids (aa) in the BRICHOS domain: long isoform (197 aa) and short isoform (191 aa). In more detail, alternative splicing occurs at the beginning of exon 5 in the coding region which yields transcripts that differ by 18 nucleotides or at the end of exon 5 in the 3' untranslated region. Those two alternative splicings increase the number of different transcripts without affecting the size of the corresponding isoforms. Currently, we do not know whether both alternatively spliced transcripts are translated and whether any functional differences are carried by the 6-aa insertion.⁶ To our knowledge, all the in cellulo published data on SFTPC metabolism or functional studies on mutated SFTPC have been done on isoform 1.

Mutations in the gene encoding SP-C cause familial and sporadic interstitial lung disease (ILD), which is associated with substantial morbidity and mortality. In affected individuals, all SFTPC mutations were found in the heterozygous state, which is consistent with the autosomal dominant pattern of inheritance observed in the familial case. Clinically, patients may develop acute neonatal respiratory distress or a chronic lung disease characterized by tachypnea and hypoxemia in childhood with significant morbidity and mortality. More than 50 mutations have now been identified in the SFTPC gene, and their classification is defined by their location within the proprotein. In particular, mutations in the BRICHOS domain alter the ability of proSP-C to fold correctly, thereby causing endoplasmic reticulum (ER) retention, aggresome formation and cellular dysfunction.⁷,⁸

Anti-inflammatory drugs, mostly corticosteroids (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7088), are the most common treatment for ILD, including surfactant disorders, in children.⁹ Apart from its anti-inflammatory effects, the specific mechanism of action of GCs in surfactant inherited disorders has not been elucidated. Short-term benefits have been reported, but they must be balanced with the well-known toxicity of steroids. The use of methylprednisolone pulses may reduce the side effects of steroids, but in the absence of studies the choice of azithromycin and/or hydroxychloroquine as first-line treatment remains highly dependent on the habits and experiences of different clinical centers.⁹⁻¹¹

Here, we report a positive outcome after methylprednisolone pulses treatment of ILD in dizygotic twins with an inherited heterozygous SFTPC mutation, g.7343G > A, previously described.¹² The authors hypothesized that this substitution of a cysteine by a tyrosine at codon 189 (C189Y) in the BRICHOS domain of proSP-C causes the retention of proSP-C in the ER, as with other BRICHOS mutations. The goal of the present study was to decipher the potential role of glucocorticosteroid (GC) treatment. We studied the profiles and levels

## What is the key question?

* Methylprednisolone pulse is the goal standard treatment of interstitial lung disease (ILD) in children but the mechanisms of its benefits remain unclear and must be balanced with the known toxicity of steroids. We aimed to decipher the mechanisms of methylprednisolone pulse with focus on surfactant protein C (SP-C) metabolism.

## Brief summary of the results

* We have demonstrated in twins with SFTPC mutation that methylprednisolone has a regulatory effect on SFTPC mRNA splicing leading to a modification of the different proSP-C isoforms which may, ultimately, account for the beneficial effects of these steroids.

## Potential significance of the results to human health and disease

* This report shows promise for improving our knowledge about methylprednisolone treatment mechanisms in patients with inherited surfactant disorders and ILD of unknown aetiology.

of SFTPC mRNA and proSP-C in bronchoalveolar lavage fluid (BALF) before and after treatment. We demonstrated that GC treatment impacts processing of proSP-C through an effect on expression and splicing of SFTPC mRNA. Interestingly, we demonstrated a new regulatory mechanism leading to a reduce expression of mutated proSP-C protein in the presence of GC treatment. More importantly, our study pinpoints the necessity to extend at both the short and the long proSP-C isoform the future functional studies.

# 2 | METHODS

## 2.1 | Study cases and genetics screening

We studied the clinical course of twins carrying a mutation in the SFTPC gene: c.566G > A (p.Cys189Tyr) previously identified after screening of 121 children with diffuse lung disease.¹² The genetic analysis was obtained in both children and their mother after informed consent according to French legislation.¹² Pathogenicity was evaluated using in silico tools (Alamut visual software v2.7, Interactive Biosoftware, Rouen, France) and by familial segregation. Molecular analysis of SFTPB (NM_00542.3), NKX2-1 (NM_001079668.2) and ABCA3 (NM_001089.2) was performed in the Genetics Department of Hôpital Henri Mondor (Creteil) by Sanger sequencing (primers available upon request). Before and after GC treatment, the evolution
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of lung lesions was evaluated by a high-resolution computed tomographic (HRCT) scan in both children. GC treatment consisted of methylprednisolone pulses at 300 mg/m² once daily for 3 days, every 3 weeks, with oral prednisolone between the pulse at an initial dose of 2 mg/kg/d progressively tapered. There was no other treatment administered, such as hydroxychloroquine or azithromycin, and the children received only GCs.

## 2.2 | Bronchoscopy and BALF analysis

A bronchoscopy with BALF was performed before and 5 months after GC treatment in both twins. With the informed consent of the parents, both patients underwent fiberoptic bronchoscopy after premedication with atropine and midazolam, local anaesthesia with 2% lidocaine and administration of premixed 50% N₂O and O₂ during the procedure.¹³ The bronchoscope was wedged in a segment or subsegment of the middle lobe or lingula, and BALF was obtained by using 5 mL/kg of 0.9% saline solution. Recovered BALF was pooled and used for cytological, virological and bacteriological examinations. The remaining BALF was centrifuged (500g, 5 min at 4 °C) to sediment cells. The supernatant and pellet were aliquoted and stored at -80 °C after parental consent for research purposes.

## 2.3 | Surfactant protein and RNA analysis in BALF

The BALF supernatant was concentrated by lyophilisation, then pellets were resuspended in sample buffer (without Sodium dodecyl sulfate (SDS) as previously described.¹³ An equal amount of proteins (30 µg) from each sample, including a control (a 6-month-old girl with severe asthma) was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 16% Tris-Tricine gels (BioRad, Hercules, CA, USA). Proteins were further electrotransferred to a nitrocellulose membrane (Whatman, Dassel, Germany) and probed by immunoblotting with N-terminal proSP-C antibody (recombinant antihuman antibody from rabbit, dilution 1/5000; Seven Hills Bioreagents, Cincinnati, OH, USA) and then secondary antibody horseradish peroxidase-conjugated goat Ig-G antirabbit antibody from donkey (GE Healthcare, Buckinghamshire, UK). Bound antibodies were detected by using SuperSignal West Dura Extended Duration substrate (Pierce, Rockford, IL, USA) according to the manufacturer's instructions and exposed to a Bio-Rad Molecular Imager ChemiDoc XRS System. Images were analysed by using Quantity One (BioRad, Hercules, CA, USA).

Total RNA was extracted from BALF cells by using TRIzol Reagent (Life Technologies) according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using Maxima First Strand cDNA Synthesis kit with dsDNase (Thermo Scientific, St. Leon-rot, Germany). Amplified products were separated by capillary electrophoresis as previously described.¹⁴ Ratios of splicing isoforms were determined as the peak area of the

considered SFTPC isoform divided by the sum of peak areas for all isoforms for each variant. Unfortunately, only one of the two samples collected before corticosteroid treatment was available for our analysis. Quantitative real-time PCR assays were done using SFTPC, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (TATA-Box Binding Protein) (TBP) primers and probes (TaqMan FAM/NFQMGB probe format) designed by Applied Biosystems (Foster City, CA, USA). PCR reactions were carried out using TaqMan Gene Expression Assay Mix, TaqMan Universal PCR Master Mix, no AmpErase Uracil N-Glycosylase (UNG) and 1 µL of complementary deoxyribonucleic acid (cDNA) (or 1 µL of deoxyribonuclease/ribonuclease free water for the no template control) in a final volume of 20 µL. Samples were placed in 96-well plates and amplified in an ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Amplification conditions were 10 min at 95.0 °C, followed by 40 cycles of 15 s at 95.0 °C and 1 min at 60.0 °C. All reactions were run in triplicate and each sample was repeated in two independent quantitative PCR (qPCR) assays. To correct for variations in the amount of input RNA and efficiency of the reverse transcription, GAPDH and TBP levels were quantified and results were normalized to these values. Relative amounts of SFTPC mRNA were measured by the 2^ΔΔCT^ method with a control sample chosen as a calibrator (ie, the baseline value) for comparative results.¹⁵

## 2.4 | Immunofluorescence and immunoblotting analysis of human WT and mutated proSP-C in A549 cells

Human wild-type (WT) proSP-C cDNA (Origene, Herford, Germany) was subcloned into pcDNA3.1/V5-His-TOPO vector as described.¹⁶ The C189Y SP-C point mutations and the deletion to generate cDNA corresponding to the short isoform of proSP-C were introduced into SFTPC cDNA using a site-directed mutagenesis kit (Stratagene, Waghaeusel-Wiesental, Germany). In the A549 cells the maturation of proSP-C cannot be complete in the absence of ABCA3 disrupting lamellar body biogenesis.¹⁷ To fully and adequately evaluate the GC treatment on proSP-C processing, heterologous expression of ABCA3 is required. Therefore, we also used a plasmid containing the cDNA of the human WT ABCA3 subcloned into pEGFP-N1 plasmid.¹⁸ Human WT SFTPC or mutated cDNA was transiently transfected into alveolar A549 cells using lipofectamine 2000 (Life Technologies, Courtaboeuf, France) according to the manufacturer's instructions.

A549 cells seeded on coverslips were transfected for 24 hours, then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X100 at room temperature. Immunofluorescence was performed using the primary antibodies rabbit polyclonal anti-proSP-C-N-terminal (Abcam Inc, Toronto, ON, Canada) and mouse monoclonal anticalnexin (Abcam), with the secondary antibodies goat antirabbit Alexa Fluor 488 (green) or 555 (red) and goat antimouse Alexa Fluor 568 (red) (both Life Technologies). Nuclei were detected by To-Pro-3 Iodide staining (Life Technologies) according to the manufacturer's instructions. Cells were mounted with Vectashield medium
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(Vector Laboratories, Burlingame, CA, USA) without Di Aminido Phenyl
Indol (DAPI). Alexa Fluor and Green Fluorescent Protein (GFP) fluores-
cence were examined under a confocal Zeiss LSM 510 and quantified
by using Zeiss Zen 2009 software. At least 10 representative images
were taken by condition. Each experiment was repeated three times.

Twenty-four hours after transfection, total cell proteins were
obtained by lysing cells in radioimmunoprecipitation assay buffer
(RIPA buffer) containing protease inhibitors (Roche, Basel,
Switzerland). Proteins were separated by electrophoresis on a 15%
polyacrylamide gel and transferred to PolyVinyliDene Fluoride
(PVDF) membrane. Immunoblotting of transferred proteins was
done using successive incubations with primary rabbit polyclonal
anti-proSP-C-N-terminal antibody (Abcam Inc, Toronto, ON,
Canada) and mouse monoclonal antibeta actine antibody (Abcam
Inc, Toronto, ON, Canada) overnight at 4°C and then with goat
antirabbit horseradish peroxidase-conjugated secondary antibody
(Life Technologies, Carlsbad, CA, USA) and goat antimouse horse-
radish peroxidase-conjugated secondary antibody (Life Technolo-
gies, Carlsbad, CA, USA) for 1 hour at room temperature. Signal
was detected by enhanced chemiluminescence using a commer-
cially available kit (GE Healthcare, Chicago, IL, USA).

## 2.5 | Half-life measurement of the two proSP-C isoforms

Human embryonic kidney (HEK) 293 cells, in six-well culture plates, were transiently transfected using human WT *SFTPC* or mutated cDNA. Fifteen hours later, cycloheximide (C7698, Sigma Aldrich, Saint-Louis, MO, USA) solubilized in dimethyl sulfoxide (Sigma Aldrich, Saint-Louis, MO, USA) was added to the culture medium for 30 minutes to 6 hours at a final concentration of 20 µg/mL. Cells were

lysed and proSP-C quantified by western-blot as previously described.
Each time course was repeated three times.

## 2.6 | Visualization of the 3D atomic coordinates of the human proSP-C<sup>WT</sup>

The 3D coordinates resolved using X-ray diffraction by Willander et al (PDB file 2YAD, chain A) were visualized using Pymol software (The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC, NY, USA). Whi1e residues 1-58 were absent in the recombinant protein expressed by Willander et al,¹⁹ residues 150-180 and 59-89 were disordered and consequently not visible in the electron density map.

# 3 | RESULTS

## 3.1 | Case report

Twins (a girl, Px, and a boy, Py) were born at term and presented
delayed but severe respiratory distress at 7 days and 1 day of
birth, respectively. Exogenous surfactant was administered twice,
and they were invasively ventilated for 11 and 19 days, respec-
tively. The twins were transferred in our paediatric department at
2 months of age. They were both hypoxic and exhibited
tachypnea, retractions, gastrointestinal reflux and failure to thrive.
HRCT scan of the chest revealed bilateral and diffuse ground glass
opacities in both children (Figure 1A). Before GC treatment, we
performed a BALF in both twins. Immunoblotting analysis of BALF
with proSP-C antibody revealed an isolated 24-kDa band in both
children with absence of bands corresponding to the proSP-C

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**FIGURE 1** Chest imaging and bronchoalveolar fluid (BALF) analysis of twins before treatment at 2 months. (A) High-resolution chest computed tomography before methylprednisolone treatment in twins (Px and Py). (B) and (C) Immunoblotting analysis with proSP-C and mature SP-C antibody of BALF samples before treatment: Px, girl; Py, boy; C, control; ProSP-C, surfactant proprotein C
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**FIGURE 2** ProSP-C trafficking and processing. Representative confocal images of A549 cells after 24 hours expression of proSP-C<sup>WT</sup> or proSP-C<sup>C189Y</sup>. (A) Immunostaining with rabbit polyclonal anti-proSP-C (green) and mouse monoclonal anticalnexin (ER marker; red) antibodies. (B) Immunostaining with rabbit polyclonal anti-proSP-C antibody after co-expression of proSP-C<sup>WT</sup> or proSP-C<sup>C189Y</sup> (red) and GFP-tagged wild-type ABCA3 (green). (C) Immunoblotting analysis with proSP-C antibody of the total proteins extracted from A549 cells 24 hours after transfection of WT or mutated plasmids

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processing intermediates of lower molecular weight observed in control patients (Figure 1B). Immunoblotting with mature SP-C antibody showed only a residual level of mature SP-C in both twins (Figure 1C). These results suggest an altered processing of proSP-C<sup>C189Y</sup> in vivo.

## 3.2 | Altered processing of proSP-C<sup>C189Y</sup> in A549 cells

To compare the intracellular localization of pro-SP-C<sup>C189Y</sup> with proSP-C<sup>WT</sup>, A549 cells were transiently transfected with WT or C189Y SFTPC cDNA vectors. The intracellular localization of proSP-C forms, monitored by immunofluorescence, differed between cells expressing proSP-C<sup>WT</sup> or proSP-C<sup>C189Y</sup>. Unlike proSP-C<sup>WT</sup>, proSP-C<sup>C189Y</sup> colocalized with calnexin, an ER marker, thereby suggesting a retention of this mutant within the ER (Figure 2A). In addition to documenting the impact of proSP-C<sup>C189Y</sup> on lamellar bodies, cells were co-transfected with WT or C189Y SFTPC cDNA and WT ABCA3 cDNA constructs. ProSP-C<sup>WT</sup> conferred a strong vesicular cytoplasmic signal that colocalized with ABCA3 while proSP-C<sup>C189Y</sup> trapped ABCA3 within the ER and disrupted lamellar body formation (Figure 2B).

In A549 cells expressing proSP-C<sup>WT</sup> or proSP-C<sup>C189Y</sup>, immunoblotting with anti-proSP-C antibody revealed a strong band with a molecular weight of about 21 kDa, corresponding to native proSP-C (black arrow) and three intermediates bands (white arrow): one with higher molecular weight and two with lower molecular weight (Figure 2C). For proSP-C<sup>C189Y</sup>, a single native protein without intermediates bands was visualized, suggesting the absence of proSP-C maturation in cellulo and the retention of proSP-C<sup>C189Y</sup> within the ER.

## 3.3 | Effect of methylprednisolone pulses on proSP-C processing

After 5 months of GC treatment, the twins were weaned off oxygen and gained 3.3 and 3.1 kg, respectively, without any cardiac, digestive or vascular effects. HRCT scan of the chest showed decreased ground-glass opacities but also lung cysts (Figure 3A). BALF investigation performed at this time revealed the persistence of the 24 kDa band (black arrow) but also the appearance of a 14 kDa band intermediate (white arrow) in both twins, suggesting better proSP-C protein processing along with clinical and radiological improvement (Figure 3B). Mature SP-C levels remained very low in both twins (data not shown).

## 3.4 | Effect of methylprednisolone pulses on mRNA expression level and splicing

To explain these results, our hypothesis was that GCs could increase the expression of the SFTPC gene. Thus, we performed a qPCR

analysis in BALF from Py before and after treatment. Surprisingly, the SFTPC mRNA level in BALF of this patient was decreased 8-fold after GC treatment (Figure 4A). This result could not explain how the GCs improve the maturation of proSP-C. Therefore, we then tested whether GCs could modify SFTPC mRNA expression qualitatively. In WT control, qualitative analysis of SFTPC mRNA by capillary electrophoresis revealed four transcript variants, 261 (base pairs) bp (variant 1, NM_003018.3), 253 bp (variant 2, NM_001172410.1), 243 bp (variant 3, NM_001317778.1) and 235 bp (variant 4, NM_001317780.1) (Figure 4B). Variants 1 and 2, which differ by eight nucleotides (nt) at the 3' untranslated region of exon 5, are translated as one protein, the longest isoform of 197 aa. The two smaller variants (3 and 4) resulted from an 18-nt deletion produced by alternative splicing at the 5' end of exon 5 of variants 1 and 2, respectively, and are translated as one shorter protein, the 191-aa isoform (Figure 4C). By measuring the peak area of the considered SFTPC isoform divided by the sum of peak areas for all isoforms for each variant, we evaluated the repartition between the different isoforms. Unexpectedly, variants 1 and 2 accounted for 44% of all peaks, whereas most of the mRNA corresponded to variants 3 and

<figure><img src="image_14.png" /><figcaption>**FIGURE 3** Chest imaging and BALF analysis of twins after GC treatment at 7 months. (A) High-resolution computed tomographic scan after treatment in twins (Px and Py). (B) Immunoblotting analysis with proSP-C antibody of BALF samples after treatment. Px, girl; Py, boy; C, control; pro-SP-C, surfactant proprotein C</figcaption></figure>
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<figure><img src="image_21.png" /><figcaption>**Transcripts**</figcaption></figure>

RefSeq ID
NM 003018.3

NM 001172410.1

NM 001317778.1

NM 001317780.1

<figure><img src="image_22.png" /><figcaption>**Pro-SP-C**</figcaption></figure>

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**FIGURE 4** RNA analysis in patients BALF. (A) Quantitative real-time PCR analysis of SFTPC mRNA in BALF from Py before and after GC treatment. (B) Qualitative capillary electrophoresis analysis of SFTPC mRNA extracted from BALF in control. (C) Schematic representation of SFTPC gene, splicing variants 1 to 4 and the short and long proSP-C isoforms (black box represents the mature SP-C domain). (D)-(F) Qualitative capillary electrophoresis analysis of SFTPC mRNA extracted from BALF in Py before treatment (D) and in both twins (Px and Py) after methylprednisolone pulse treatment (E and F)

4 (56%) in the WT control (Figure 4B). In Py, before GC treatment, the expressed transcripts did not differ in number, size and distribution to those observed in the WT control (Figure 4D). However, after GC treatment, analysis of SFTPC mRNA in both twins revealed only two peaks, at 243 and 235 bp, corresponding to variants 3 and 4, respectively (Figure 4E,F).

Altogether, these results indicate that distribution of the transcripts was modified by GC treatment, with a marked increase in variants 3 and 4 concomitantly with a complete disappearance of variants 1 and 2. To clarify if the effect of the GCs was specific to C189Y mutation, we analysed transcripts of four patients with ILD and absence of identified mutation in surfactant genes. Two of these
<figure><img src="image_26.png" /></figure>

patients did not receive GC treatment (Supporting Information Figure 1A,B) while the two others did (Supporting Information Figure 1C,D). Interestingly, four transcripts were identified in children before GC treatment whereas only two transcripts (variants 3 and 4) were identified in patients who received GC treatment, suggesting that the effect of GC treatment on SFTPC mRNA transcripts did not relate specifically to the C189Y proSP-C substitution.

### 3.4.1 | Consequence of partial expression of variants 3 and 4

We have demonstrated that GC treatment reduces the expression of total SFTPC mRNA and modifies its splicing by promoting the

expression of variants 3 and 4 encoding a shorter isoform of proSP-C. Figure 5 illustrates the difference in primary structure of the two proSP-C isoforms (Figure 5A) and the environment of the C189 residue involved in a disulphide bridge in the three-dimensional structure of the BRICHOS domain (WT proSP-C, long isoform; Figure 5B). In the shorter isoform, the 6 aa length deleted fragment encompasses the C148 residue involved in a second internal disulphide bridge. Currently, the impact of the 6 aa deletion on proSP-C properties and processing in humans is not documented. Moreover, the C189Y mutation might have different consequences on short or long isoforms. Thus, we decided to assess the in cellulo localization and processing of WT and C189Y proSP-C with 6 aa deletion (short isoform). The WT short isoform had a vesicular cytoplasmic signal whereas the C189Y short isoform was mostly in a location suggesting its retention in the ER (Figure 6A).

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>**FIGURE 5** Primary, secondary and tertiary structure of the human ProSP-C<sup>WT</sup>. The amino acid positions of the two disulphide bridges are depicted: C120-C148 (*) and C121-C189 (**). (A) Amino acid sequence (a.a.) of the long isoform of the human ProSP-C<sup>WT</sup> (Swiss-Prot accession P11686). Cysteines are underlined. The amino acid fragment deleted in the short isoform (P11686-2) is shown in purple. Residues 90-197 correspond to the BRICHOS domain. The secondary structure elements (s.s.) are indicated as assigned by Willander et al<sup>19</sup> from the three-dimensional coordinate file (PDB code 2YAD, chain a) encompassing the residues described by bold characters. E and H represent residues involved in a five-stranded β-sheet and two α-helices, respectively. (B) Three-dimensional structure of the BRICHOS domain of the human ProSP-C<sup>WT</sup> long isoform (PDB code 2YAD, chain a)</figcaption></figure>
<figure><img src="image_29.png" /></figure>

Additionally, the WT short isoform strongly colocalized in ABCA3 vesicles while the C189Y short isoform was still mainly as trapped in the ER and not in ABCA3 vesicles (Figure 6B). In the immunoblotting experiment, we observed a native band of about 20 kDa (black arrow)

and three intermediates bands (white arrows) after expression of short isoform proSP-C<sup>WT</sup>. The expression of the short proSP-C<sup>C189Y</sup> led to the synthesis of the single native bands (black arrow) without intermediates of maturation (Figure 6C). These results were similar to those

<figure><img src="image_30.png" /><figcaption>**FIGURE 6** Trafficking and processing of the ProSP-C short isoform. Representative confocal images of A549 cells after 24 hours expression of the WT and C189Y short isoform.</figcaption></figure>

(A) Immunostaining with rabbit polyclonal anti-proSP-C (green) and mouse monoclonal anti-calnexin (ER marker; red) antibodies.

(B) Immunostaining with rabbit polyclonal anti-proSP-C antibody after co-expression of proSP-C<sup>WT</sup> or proSP-C<sup>C189Y</sup> (red) and GFP-tagged wild-type ABCA3 (green). (C) Immunoblotting analysis with proSP-C antibody of the total proteins extracted from A549 cells 24 hours after transfection of plasmids containing WT or C189Y short isoform
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /><figcaption>**FIGURE 7** Results of CHX-chase experiment on transfected HEK 293 cells. Half-time measurement of the two isoforms proSP-C, wild-type (A) and mutated (C189Y) after CHX treatment (B). The half-time measurement of wild-type short proSP-C compared to mutated short proSP-C after CHX treatment (C)</figcaption></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

observed with the longer isoform of proSP-C described above (Figure 2C). Regarding the fact that the clinical status of the twins was improved by GC treatment which promoted the short proSP-C isoform expression, we hypothesized that the presence or absence of 6 aa in the BRICHOS domain could modify the stability of the isoforms. To test this hypothesis, we performed cycloheximide chase experiments on transfected HEK 293 cells. Our results revealed that the 6 aa deletion did not change the half-life of WT proSP-C isoforms (Figure 7A). In contrast, the C189Y short isoform half-life was significantly reduced compared to that of the C189Y long isoform (Figure 7B). Moreover, the comparison between WT and C189Y short isoforms revealed a significant decrease of C189Y short isoform half-life in line with the effect of GC treatment in this case (Figure 7C).

4 | DISCUSSION

This study reports the successful use of GC treatment in twins with ILD related to SP-C mutation. The twins, their mother and grandfather showed a cysteine-to-tyrosine substitution at amino acid 189 (C189Y). This mutation was also identified by Turcu et al in a 5-year-old child with neonatal respiratory distress syndrome evolving to ILD, who received GC treatment with a favourable outcome, although the patient remained corticosteroid-dependent.²⁰

The consequences of this mutation have not been described before but may induce a retention of proSP-C in the ER, as most of the mutations are located in the BRICHOS domain.⁷,⁸,²¹,²² In accordance with this hypothesis, we showed that heterologous expression of pro-SP-C<sup>C189Y</sup> in alveolar A549 cells led to the retention of proSP-C within the ER and consequently the absence of its maturation. This ER accumulation is likely responsible for the cytotoxicity and suggests a conformational disease due to an abnormal folded protein. This mutation may disrupt the disulphide bridge between cysteines C189 and C121, both strictly conserved in the BRICHOS domain superfamily,²³ which is critical for correct proSP-C folding.²²,²⁴ According to the available three-dimensional structure of the BRICHOS domain of the human pro-SP-C, this internal covalent bond links the beta-sheet and an alpha-helix.¹⁹ Altogether, this indicates that the disulphide bridge is crucial for domain stability.

Additionally, we showed that the proSP-C<sup>C189Y</sup> had a dominant-negative effect in cellulo on wild-type ABCA3, which is mainly retained within the ER. This observation is in line with previous studies which reported potential effects of ABCA3 variants in modifying the phenotype of subjects with SFTPC mutations.²⁵,²⁶ This effect may induce ER stress and thereafter disrupt lamellar body formation. It has been reported that ER stress induced by SFTPC BRICHOS mutations promote proinflammatory signalling, a main target of GCs.²²,²⁷
<figure><img src="image_36.png" /></figure>

After the successful GC treatment of the patients, we demonstrated here that GCs could directly influence the metabolism of SP-C. After GC treatment, proSP-C processing was improved, as revealed by the appearance of proSP-C intermediates on immunoblotting of protein extracts from patient's BALF. Our results are consistent with a previous ex vivo study showing that dexamethasone promotes proSP-C post-translational processing.²⁸ The authors suggested that dexamethasone treatment results in an acceleration of the alveolar type II cells secretory pathway, which in turn is able to process an increased amount of translated proSP-C.²⁹ The administration of exogenous GCs to animal models accelerated the transition from the pseudo-glandular stage during pulmonary development and early secretion of surfactant.³⁰,³¹ Indeed, glucocorticoid receptor (GR) is highly expressed in the developing lung.³² The effect of GCs on lung terminal maturation in vivo are reflected in vitro and in cellulo in both developing lung explants and cultured cell models. With respect to the foetal period, the mechanism by which GCs regulate the post-translational processing of proSP-C is not yet elucidated.

In this study, we extracted SFTPC mRNA from cells in BALF. This technique is also used to determine whether variants of uncertain significance that potentially affect mRNA splicing are deleterious or not. Surprisingly, we found that GC treatment in twins with SFTPC mutations induced an 8-fold decrease in the expression of SFTPC mRNA in patient BALF cells. Expression of surfactant genes is known to be regulated by numerous hormones or factors, including insulin, GCs or cAMP (for review see Mendelson³⁰). In human lung explants, it has been shown that GCs directly increase the expression of SFTPC mRNA in alveolar type II cells or indirectly by inducing NKX2-1 expression, the transcription factor activating in turn SFTPC expression.³³⁻³⁶ However, GCs have a distinct effect on SFTPC and SFTPB transcription depending on the cell culture mode. While dexamethasone increases the transcription rate of both SFTPB and SFTPC mRNA, the impact on SFTPC mRNA stability differs from significant in vitro³⁷ to low ex vivo.³⁸,³⁹ Boggaram and Marzana pointed to a discrepancy between the temporal induction of SFTPC gene transcription and mRNA accumulation which seems to be different during prenatal lung development and adult lung.³⁷ Interestingly, different time courses induction by dexamethasone were observed for SFTPC mRNA level, with a decrease in explants cultured without hormones,³⁴ while SFTPB transcription constantly increased after 5 days of hormone deprivation.³⁹ Like the results we observed, this decrease in SFTPC transcription between GCs pulses may involve a complex and indirect mechanism involving a labile transcription factor or protein, as illustrated by protein synthesis inhibition experiments which blocked GC-induced SFTPC transcription.³⁹ The inducible effect of GCs on NKX2-1 may be a potential mechanism by which GR signalling pathways stimulate SP-C expression at the cellular level during alveolar epithelium differentiation. However, this hormonal regulation may occur during lung development and differ after birth, which could explain the difference between our results and those of previous studies.³³⁻³⁵ This indirect regulation is also corroborated by the fact that the SFTPC gene contains fewer glucocorticoid response elements

(GRE), unlike SFTPB.⁴⁰ Further experiments studying SFTPC mRNA transcription levels and stability from human foetal to adult lung development in response to GC treatments would be of interest.

Altogether, this decrease in global SFTPC mRNA level may improve the conformational disease induced by the proSP-C¹⁸⁹Y by reducing accumulation of proSP-C in alveolar type II cells. Therefore, the dominant negative effect of mutated proteins on wild-type proteins could also be reduced, which accordingly may explain the appearance of proSP-C intermediates on immunoblotting in patients after GC treatment.

More importantly, our study revealed that GCs induced an effect on the regulation of alternative splicing of SFTPC mRNA in patient BALF. In control subjects and in patients before treatment, we confirmed the presence of four SFTPC alternative transcripts expressed in BALF cells, as previously described by Warr et al⁵ with human lung cDNA libraries, and we documented their relatively balanced distribution among them. These four splice variants encode the two proSP-C isoforms (197 and 191 aa). Remarkably, GC treatment abolished almost completely the expression of the longest isoform by reducing drastically the amount of splice variants 1 and 2. We showed this effect in children with SFTPC mutation in the BRICHOS domain (C189Y) but also in two children with an idiopathic ILD and without SFTPC mutation. These results are concordant with the presence of four transcript variants in a cDNA library carrying the c.566G > A substitution (p.C189Y) in a previous study by Glasser et al,⁴ which corroborates the fact that the GC effect on splicing is not due to the mutation itself but could be the result of a more global and less specific mechanism. This effect has been previously shown on murine calcium-activated potassium channels, where GCs induced new alternative transcripts and isoforms with different functions depending on the cell type.⁴¹

Taking these results together, we observed both a lower level of SFTPC mRNA in infant BALF and a change in alternative splice variant diversity after GC treatment. This suggests that the impacts of GCs on both transcription rate/stability and alternative splicing are closely related, one can be the consequence of the other or concomitants. The composition of regulatory elements within the SFTPC promoter itself in reaction to GC-induced transcription factors could influence RNA-processing decisions.⁴² Here, GC treatment induced the choice of alternative transcripts harbouring a more distal alternative acceptor site within SFTPC exon 5. Several studies have demonstrated that hormones could modulate the amount and exon composition of their target-gene transcripts (for review see Auboeuf et al⁴³) and GCs could similarly modulate coregulators that might affect splicing decisions. A long-term treatment with GCs may modify expression and post-translational modifications of several activators/silencers splicing factors,⁴⁴ whereas acute GCs treatments influence splicing through promoter or transcriptional coregulators.⁴⁵ Additionally, several transcriptional coregulators modulated by GCs could affect splicing decisions by modulating the dynamics of RNA polymerase II elongation, causing both RNA stability modification and alternative splice site selection.⁴⁶ Further high-throughput RNA experiments studying both global alternative transcript distribution and mRNA abundance in response to
<figure><img src="image_37.png" /></figure>

GCs would give clues to understanding the fine regulation of surfac-
tant genes expression along lung development.

This treatment promotes the expression of variants coding short proSP-C isoforms. Currently little is known about the ontogeny of proSP-C isoforms. Examination of SP-C mRNAs in other species would suggest that it is the shorter isoform which is the ancestral one and hence might want to refer to the longer one as having an insertion rather than the shorter one having a deletion. The processing of the short proSP-C isoform has not been previously described in cellulo. In our study, we showed that the processing of the short isoform seems similar to that of the long one (WT vs WT or C189Y vs C189Y). The CHX-chase experiment revealed that the short isoform proSP-C<sup>C189Y</sup> has a drastic decreased half-life compare to the long proSP-C<sup>C189Y</sup> or the two isoforms of proSP-C<sup>WT</sup>. By promoting the synthesis of the short isoform in twins, GCs lead to the synthesis of less stable mutated isoforms and probably contribute to limiting the accumulation of unfolding protein in the cell.

This study had several limitations. First, we examined only two patients with SFTPC mutations. However, only a few children have BALF before and after GC treatment and no other drugs, limiting the access to relevant samples. Second, we did not study the effect of GCs on SFTPC mRNA expression in cellular or murine models. Currently, no animal model is available that has both proSP-C isoforms to allow studies of the effect of GCs on expression and splicing of SFTPC mRNA. Furthermore, mice have only one proSP-C isoform, corresponding to the shortest isoform found in humans, and therefore the metabolism of SP-C could be different to that of humans. Most of the in cellulo studies regarding proSP-C processing after transfection experiments use cDNA corresponding to variants 1 or 2, which encode the long isoform. Caution should be therefore taken for further researches in considering the presence of variants 3 and 4 which encode the shorter isoform. Third, the GC used in most studies is dexamethasone, a sustained-delivery GC, unlike methylprednisolone in our study, and comparison studies between these two GCs and their effects on the expression of surfactant genes would be relevant.

In conclusion, we have identified and documented a beneficial effect of GC treatment in twins with SFTPC BRICHOS mutation through inducing the synthesis of a less stable proSP-C isoform that results in limitation of proSP-C precursor accumulation with the ER and subsequently decreases its deleterious effects on cellular homeostasis.

ACKNOWLEDGEMENTS

We thank Natacha Martin for her help during transcript analyses and Xavier Decrouy for help in confocal microscopy. This work was supported by public grants from the Institut National de la Santé et de la Recherche Médicale.

COMPETING INTERESTS

There are no competing interests to declare.

CONTRIBUTORS

C.D., A.A., P.F., S.S. and R.E. designed the study. R.E. and P.F. coordinated the study. C.D., A.A., A.T., S.S., P.F. and

R.E. conceived the experiments and drafted the manuscript. C.D., A.A., V.D., S.S., P.F., B.C. and E.N. performed experiments and analysed data. All authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT

Data generated or analysed during this study are included in this pub-
lished article.

ORCID

Ralph Epaud https://orcid.org/0000-0003-3830-1039

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## SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Delestrain C, Aissat A, Simon S, et al. Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story? *Br J Clin Pharmacol.* 2021;87:2361-2373. https://doi.org/10.1111/bcp.14645