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The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.

Zarbock R, Woischnik M, Sparr C, Thurm T, Kern S, Kaltenborn E, Hector A, Hartl D, Liebisch G, Schmitz G, Griese M - BMC Pulm Med (2012)

Bottom Line: Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy.Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

View Article: PubMed Central - HTML - PubMed

Affiliation: Childrens' Hospital of the Ludwig-Maximilians-University, Lindwurmstr, 4, 80337 Munich, Germany.

ABSTRACT

Background: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.

Methods: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.

Results: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.

Conclusions: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

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Processing features of proSP-CWT or proSP-CA116D. (A) Immunoblotting of total cell lysates with tag-specific antibodies. Cell lysates obtained from MLE-12 cells stably expressing fusion protein of proSP-C with an N-terminal HA-tag (left panel), transiently transfected cells expressing fusion protein of proSP-C with an N-terminal EGFP (EGFP-C1, middle panel) or a C-terminal EGFP (EGFP-N1, right panel), present with several bands corresponding to different proSP-C processing intermediates, in which the tag sequence is retained. (B) Based on the size of the bands, the projected corresponding intermediate species of the fusion constructs are depicted. The cleavage sites are only estimates due to the limited resolution of the technique. EGFP-C1 (band #5) and EGFP-N1 (band #9) are expressed as a full-length product of 48 kDa, HA-SP-C (band #1) of size 22 kDa.
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Figure 1: Processing features of proSP-CWT or proSP-CA116D. (A) Immunoblotting of total cell lysates with tag-specific antibodies. Cell lysates obtained from MLE-12 cells stably expressing fusion protein of proSP-C with an N-terminal HA-tag (left panel), transiently transfected cells expressing fusion protein of proSP-C with an N-terminal EGFP (EGFP-C1, middle panel) or a C-terminal EGFP (EGFP-N1, right panel), present with several bands corresponding to different proSP-C processing intermediates, in which the tag sequence is retained. (B) Based on the size of the bands, the projected corresponding intermediate species of the fusion constructs are depicted. The cleavage sites are only estimates due to the limited resolution of the technique. EGFP-C1 (band #5) and EGFP-N1 (band #9) are expressed as a full-length product of 48 kDa, HA-SP-C (band #1) of size 22 kDa.

Mentions: To identify the intracellular processing intermediates of proSP-C, MLE-12 cells were transfected with eukaryotic expression vectors, allowing expression of fusion proteins between proSP-C and either an EGFP- or a HA-Tag. Stable expression of HA-tagged proSP-CWT resulted in the appearance of a strong band at approximately 21 kDa and weaker bands at 22 kDa, 19 kDa, and 14 kDa (Figure 1A left). ProSP-CA116D yielded bands similar to the wild type at 21 kDa and 14 kDa. We also observed a much stronger band at 22 kDa and a band at 15 kDa that was not seen in the wild type, indicating accumulation of proSP-CA116D forms (Figure 1A, left). The postulated processing products are depicted in Figure 1B. Mature SP-C was never detectable because of the loss of the protein tag due to the final processing steps at the N-terminus.


The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.

Zarbock R, Woischnik M, Sparr C, Thurm T, Kern S, Kaltenborn E, Hector A, Hartl D, Liebisch G, Schmitz G, Griese M - BMC Pulm Med (2012)

Processing features of proSP-CWT or proSP-CA116D. (A) Immunoblotting of total cell lysates with tag-specific antibodies. Cell lysates obtained from MLE-12 cells stably expressing fusion protein of proSP-C with an N-terminal HA-tag (left panel), transiently transfected cells expressing fusion protein of proSP-C with an N-terminal EGFP (EGFP-C1, middle panel) or a C-terminal EGFP (EGFP-N1, right panel), present with several bands corresponding to different proSP-C processing intermediates, in which the tag sequence is retained. (B) Based on the size of the bands, the projected corresponding intermediate species of the fusion constructs are depicted. The cleavage sites are only estimates due to the limited resolution of the technique. EGFP-C1 (band #5) and EGFP-N1 (band #9) are expressed as a full-length product of 48 kDa, HA-SP-C (band #1) of size 22 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376036&req=5

Figure 1: Processing features of proSP-CWT or proSP-CA116D. (A) Immunoblotting of total cell lysates with tag-specific antibodies. Cell lysates obtained from MLE-12 cells stably expressing fusion protein of proSP-C with an N-terminal HA-tag (left panel), transiently transfected cells expressing fusion protein of proSP-C with an N-terminal EGFP (EGFP-C1, middle panel) or a C-terminal EGFP (EGFP-N1, right panel), present with several bands corresponding to different proSP-C processing intermediates, in which the tag sequence is retained. (B) Based on the size of the bands, the projected corresponding intermediate species of the fusion constructs are depicted. The cleavage sites are only estimates due to the limited resolution of the technique. EGFP-C1 (band #5) and EGFP-N1 (band #9) are expressed as a full-length product of 48 kDa, HA-SP-C (band #1) of size 22 kDa.
Mentions: To identify the intracellular processing intermediates of proSP-C, MLE-12 cells were transfected with eukaryotic expression vectors, allowing expression of fusion proteins between proSP-C and either an EGFP- or a HA-Tag. Stable expression of HA-tagged proSP-CWT resulted in the appearance of a strong band at approximately 21 kDa and weaker bands at 22 kDa, 19 kDa, and 14 kDa (Figure 1A left). ProSP-CA116D yielded bands similar to the wild type at 21 kDa and 14 kDa. We also observed a much stronger band at 22 kDa and a band at 15 kDa that was not seen in the wild type, indicating accumulation of proSP-CA116D forms (Figure 1A, left). The postulated processing products are depicted in Figure 1B. Mature SP-C was never detectable because of the loss of the protein tag due to the final processing steps at the N-terminus.

Bottom Line: Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy.Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

View Article: PubMed Central - HTML - PubMed

Affiliation: Childrens' Hospital of the Ludwig-Maximilians-University, Lindwurmstr, 4, 80337 Munich, Germany.

ABSTRACT

Background: Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.

Methods: SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.

Results: Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.

Conclusions: We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.

Show MeSH
Related in: MedlinePlus