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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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Modulation of chaperone levels in the cells expressing SP-CWT and SP-A116D by pharmacologic substances. Shown is the expression relative to β-actin of the chaperone proteins calnexin (A), calreticulin (B), Hsp70 (C) and Hsp90 (D) in MLE-12 cells expressing proSP-CWT or proSP-CA116D and either untreated (-) or exposed to cyclophosphamide (Cyc), azathioprine (Aza), methylprednisolone (Met), or hydroxychloroquine (Hyd). Only significant p-values are presented.
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Figure 4: Modulation of chaperone levels in the cells expressing SP-CWT and SP-A116D by pharmacologic substances. Shown is the expression relative to β-actin of the chaperone proteins calnexin (A), calreticulin (B), Hsp70 (C) and Hsp90 (D) in MLE-12 cells expressing proSP-CWT or proSP-CA116D and either untreated (-) or exposed to cyclophosphamide (Cyc), azathioprine (Aza), methylprednisolone (Met), or hydroxychloroquine (Hyd). Only significant p-values are presented.

Mentions: We determined the change in protein level of the two heat shock proteins Hsp90 and Hsp70, and the two ER-resident chaperones calreticulin and calnexin, in MLE-12 cells expressing SP-CWT and SP-CA116D, after exposure to pharmacological substances used in ILD therapy: cyclophosphamide, azathioprine, methylprednisolone or hydroxychloroquine (Figure 4A-D). While in the case of calnexin no significant alterations were seen, cells expressing SP-CA116D showed an increase in the expression of calreticulin, Hsp70 and Hsp90 at baseline and after treatment with pharmaceuticals. However, none of the applied drugs resulted in a significant change in expression level of any chaperone, neither in cells expressing SP-CWT nor in cells expressing SP-CA116D. However, we noted a slight increase in the expression levels of Hsp70 and Hsp90 in SP-CA116D cells treated with azathioprine (Figure 4C+D).


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)

Modulation of chaperone levels in the cells expressing SP-CWT and SP-A116D by pharmacologic substances. Shown is the expression relative to β-actin of the chaperone proteins calnexin (A), calreticulin (B), Hsp70 (C) and Hsp90 (D) in MLE-12 cells expressing proSP-CWT or proSP-CA116D and either untreated (-) or exposed to cyclophosphamide (Cyc), azathioprine (Aza), methylprednisolone (Met), or hydroxychloroquine (Hyd). Only significant p-values are presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Modulation of chaperone levels in the cells expressing SP-CWT and SP-A116D by pharmacologic substances. Shown is the expression relative to β-actin of the chaperone proteins calnexin (A), calreticulin (B), Hsp70 (C) and Hsp90 (D) in MLE-12 cells expressing proSP-CWT or proSP-CA116D and either untreated (-) or exposed to cyclophosphamide (Cyc), azathioprine (Aza), methylprednisolone (Met), or hydroxychloroquine (Hyd). Only significant p-values are presented.
Mentions: We determined the change in protein level of the two heat shock proteins Hsp90 and Hsp70, and the two ER-resident chaperones calreticulin and calnexin, in MLE-12 cells expressing SP-CWT and SP-CA116D, after exposure to pharmacological substances used in ILD therapy: cyclophosphamide, azathioprine, methylprednisolone or hydroxychloroquine (Figure 4A-D). While in the case of calnexin no significant alterations were seen, cells expressing SP-CA116D showed an increase in the expression of calreticulin, Hsp70 and Hsp90 at baseline and after treatment with pharmaceuticals. However, none of the applied drugs resulted in a significant change in expression level of any chaperone, neither in cells expressing SP-CWT nor in cells expressing SP-CA116D. However, we noted a slight increase in the expression levels of Hsp70 and Hsp90 in SP-CA116D cells treated with azathioprine (Figure 4C+D).

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