Abstract
BackgroundHeterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), however little is known about the cellular consequences of SP-CI73T expression.ResultsTo address this, we stably expressed SP-CI73T in cultured MLE-12 alveolar epithelial cells. This resulted in increased intracellular accumulation of proSP-C processing intermediates, which matched proSP-C species recovered in bronchial lavage fluid from patients with this mutation. Exposure of SP-CI73T cells to drugs currently used empirically in ILD therapy, cyclophosphamide, azathioprine, hydroxychloroquine or methylprednisolone, enhanced expression of the chaperones HSP90, HSP70, calreticulin and calnexin. SP-CI73T mutants had decreased intracellular phosphatidylcholine level (PC) and increased lyso-PC level without appreciable changes of other phospholipids. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CI73T cells secreted into the medium soluble factors that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine influence of SP-CI73T on neighboring cells in the alveolar space.ConclusionWe show that I73T mutation leads to impaired processing of proSP-C in alveolar type II cells, alters their stress tolerance and surfactant lipid composition, and activates cells of the immune system. In addition, we show that some of the mentioned cellular aspects behind the disease can be modulated by application of pharmaceutical drugs commonly applied in the ILD therapy.
Highlights
Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults
MLE-12 cells process proSP-CI73T differently from proSPCWT and accumulate proSP-CI73T processing intermediates SP-C is synthesized exclusively by AECII as a 21 kDa proSP-C which is processed to the 4.2 kDa mature protein through a sequence of C-terminal and N-terminal proteolytic cleavages [2,3]
To identify potential processing differences between proSP-CWT and proSP-CI73T, MLE-12 cells were transfected with plasmid vectors, allowing expression of fusion proteins of proSP-C with either C-terminal (N1) or N-terminal (C1) EGFP tag or N-terminal HA-tag
Summary
Heterozygous mutations of SFTPC, the gene encoding surfactant protein C (SP-C), cause sporadic and familial interstitial lung disease (ILD) in children and adults. The most frequent SFTPC mutation in ILD patients leads to a threonine for isoleucine substitution at position 73 (I73T) of the SP-C preprotein (proSP-C), little is known about the cellular consequences of SP-CI73T expression. Various pathologic mechanisms for these mutations causing chronic accumulation of misfolded proSP-C have been proposed, such as induction of endoplasmic reticulum (ER) stress, cytotoxicity, and caspase 3- and caspase 4-mediated apoptosis [14,15,16]. These factors might contribute to ILD through cell injury and death of AECII. Current knowledge on SP-CI73T lacks a precise understanding of the proSP-C processing abnormalities, concurrent cell stress response and cytotoxicity, as well as perturbations of the surfactant composition and secretion
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