RESUMEN
Traditional thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. Accumulated data now suggests that the four major surfactant proteins (SPs) are a biochemically and functionally diverse group of mammalian peptides that have function beyond modification of alveolar surface tension. Alveolar SP-C (SP-C3.7, Mr 21,000) is 35 amino acid peptide isolated from lung surfactant that is synthesized and processed from a 191-197 amino acid precursor (proSP-C21). Although its solubility in organic solvents and avidity for lipid membranes impart properties important for its biophysical activity, SP-C represents a structurally and functionally challenging protein for the alveolar type II cell that must synthesize and traffic the peptide through the regulated secretory pathway. Despite technical and analytical difficulties imposed by its unique structure, our current understanding of SP-C biosynthesis has evolved over the past 10 years. Recent data now require us to consider proSP-C21 as a hybrid molecule incorporating structural and functional features both of bitopic integral membrane proteins as well us more classically recognized propeptide hormones. Our article highlights major developments related to characterization of molecular and cellular mechanisms underlying expression, post-translational processing, and targeting of proSP-C21 that result in production of secreted SP-C3.7.
Asunto(s)
Proteolípidos/biosíntesis , Proteolípidos/metabolismo , Surfactantes Pulmonares/biosíntesis , Surfactantes Pulmonares/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Biofisica , Humanos , Pulmón/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteolípidos/química , Proteolípidos/genética , Surfactantes Pulmonares/química , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/genética , Homología de Secuencia de Aminoácido , Tensión SuperficialRESUMEN
Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.
Asunto(s)
Proteolípidos/genética , Proteolípidos/metabolismo , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Acetatos/metabolismo , Acetatos/farmacología , Western Blotting , Cisteína/farmacocinética , Feto/metabolismo , Expresión Génica/fisiología , Genotipo , Humanos , Lactante , Recién Nacido , Metionina/farmacocinética , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/metabolismo , Proteolípidos/análisis , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/análisis , ARN Mensajero/análisis , Radioisótopos de Azufre , Transcripción Genética/fisiología , TritioRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.
Asunto(s)
Apoproteínas/genética , Células Epiteliales/fisiología , Pulmón/fisiología , Proteolípidos/genética , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Transcripción Genética , Factor de Crecimiento Transformador beta/farmacología , Apoproteínas/biosíntesis , Células Cultivadas , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Feto , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/ultraestructura , Proteolípidos/biosíntesis , Surfactantes Pulmonares/biosíntesis , ARN Mensajero/análisis , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.