Reducing sarcolipin expression improves muscle metabolism in mdx mice.

Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mdx mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient mdx (mdx:sln+/-) and SLN-deficient mdx (mdx:sln-/-) mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of mdx:sln+/- and mdx:sln-/- mice. SR Ca2+ uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca2+ uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of mdx:sln+/- and mdx:sln-/- mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mdx mice from glucose intolerance.

NK-lysin from skin-secreted mucus of Atlantic salmon and its potential role in bacteriostatic activity.

NK-lysin, despite being a direct effector of cytotoxic T and natural killer cells, is an antimicrobial peptide (AMP) with known antibacterial function in vertebrates and so in fish. Its presence has been described in different tissues of teleost fish. One of the strongest antimicrobial barriers in fish is skin-secreted mucus; however, this mucus has been found to contain only a small number of AMPs. The present study describes for the first time the constitutive expression of NK-lysin in Atlantic salmon (Salmo salar) mucus produced by the skin, recording the AMP at a higher concentration than in serum with greater bacteriostatic activity. Hepcidin may be involved to a greater extent in systemic responses since it was expressed to a higher degree in serum which was more potent for alternative complement and peroxidase activities.

Endothermy in the smalleye opah (Lampris incognitus): A potential role for the uncoupling protein sarcolipin.

Sarcolipin (SLN) is a small transmembrane protein that in mice has been shown to uncouple the calcium ATPase pump of the sarcoplasmic reticulum, resulting in heat production. Mice up-regulate expression of SLN in response to cold challenge. This thermoregulatory mechanism is characterized as non-shivering muscle-based thermogenesis (NST). The current study was conducted to determine if the endothermic fish species, the smalleye opah (Lampris incognitus), has higher levels of sln transcription in tissues thought to be the main source of endothermic heat, namely the red aerobic pectoral fin musculature, which powers continuous swimming in this species. A search of the draft assembly of the opah genome reveals a single sln gene that is 95% identical to the zebrafish sln ortholog at the amino acid level. Quantitative PCR (qPCR) using opah-specific sln shows significantly higher sln transcript levels in the dark red pectoral fin muscle compared to both the light red pectoral muscle and white axial muscle tissues. The high ratio of sln transcripts to CaATPase (serca1) transcripts suggests that opah may utilize a futile calcium cycling NST mechanism in the dark red pectoral fin muscle to generate heat.

Both brown adipose tissue and skeletal muscle thermogenesis processes are activated during mild to severe cold adaptation in mice.

Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1-/- and SLN-/- mice. Interestingly, adaptation of SLN-/- mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1-/- mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes.

Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy.

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

Tissue transglutaminase activity is involved in the differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes during CNS remyelination.

During normal brain development, axons are myelinated by mature oligodendrocytes (OLGs). Under pathological, demyelinating conditions within the central nervous system (CNS), axonal remyelination is only partially successful because oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated state resulting in a failure to generate myelinating OLGs. Tissue Transglutaminase (TG2) is a multifunctional enzyme, which amongst other functions, is involved in cell differentiation. Therefore, we hypothesized that TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates remyelination. In vivo studies, using the cuprizone model for de- and remyelination in TG2(-/-) and wild-type mice, showed that during remyelination expression of proteolipid protein mRNA, as a marker for remyelination, in the corpus callosum lags behind in TG2(-/-) mice resulting in less myelin formation and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies showed that rat OPCs express TG2 protein and activity which reduces when the cells have matured into OLGs. Furthermore, when TG2 activity is pharmacologically inhibited, the differentiation of OPCs into myelin-forming OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in remyelination of the CNS, probably through stimulating OPC differentiation into myelin-forming OLGs. Therefore, manipulating TG2 activity may represent an interesting new target for remyelination in demyelinating diseases.

Decreased sarcolipin protein expression and enhanced sarco(endo)plasmic reticulum Ca2+ uptake in human atrial fibrillation.

Sarcolipin (SLN), a key regulator of cardiac sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase, is predominantly expressed in atria and mediates ß-adrenergic responses. Studies have shown that SLN mRNA expression is decreased in human chronic atrial fibrillation (AF) and in aortic banded mouse atria; however, SLN protein expression in human atrial pathology and its role in atrial SR Ca(2+) uptake are not yet elucidated. In the present study, we determined the expression of major SR Ca(2+) handling proteins in atria of human AF patients and in human and in a mouse model of heart failure (HF). We found that the expression of SR Ca(2+) uptake and Ca(2+) release channel proteins are significantly decreased in atria but not in the ventricles of pressure-overload induced HF in mice. In human AF and HF, the expression of SLN protein was significantly decreased; whereas the expressions of other major SR Ca(2+) handling proteins were not altered. Further, we found that the SR Ca(2+) uptake was significantly increased in human AF. The selective downregulation of SLN and enhanced SR Ca(2+) uptake in human AF suggest that SLN downregulation could play an important role in abnormal intracellular Ca(2+) cycling in atrial pathology.

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence.

BACKGROUND: Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients. METHODS: MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome. RESULTS: MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08-0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17-0.99, p = 0.048) had an independent prognostic value for disease-free survival. CONCLUSION: This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.

MAL promoter hypermethylation as a novel prognostic marker in gastric cancer.

T-lymphocyte maturation associated protein, MAL, has been described as a tumour-suppressor gene with diagnostic value in colorectal and oesophageal cancers, and can be inactivated by promoter hypermethylation. The aim of this study was to analyse the prevalence of MAL promoter hypermethylation and the association with mRNA expression in gastric cancers and to correlate methylation status to clinicopathological data. Bisulphite-treated DNA isolated from formalin-fixed and paraffin-embedded samples of 202 gastric adenocarcinomas and 22 normal gastric mucosae was subjected to real-time methylation-specific PCR (Q-MSP). Two regions within the MAL promoter (M1 and M2) were analysed. In addition, 17 frozen gastric carcinomas and two gastric cancer cell lines were analysed both by Q-MSP and real-time RT-PCR. Methylation of M1 and M2 occurred in 71 and 80% of the gastric cancers, respectively, but not in normal gastric mucosa tissue. Hypermethylation of M2, but not M1, correlated with significantly better disease-free survival in a univariate (P=0.03) and multivariate analysis (P=0.03) and with downregulation of expression (P=0.01). These results indicate that MAL has a putative tumour-suppressor gene function in gastric cancer, and detection of promoter hypermethylation may be useful as a prognostic marker.

Mammaglobin and lipophilin B expression in breast tumors and their lack of effect on breast cancer cell proliferation.

BACKGROUND: Mammaglobin (SCGB2A2) and lipophilin B (SCGB1D2) are members of the secretoglobin polypeptide family. Mammaglobin has been shown to be overexpressed in breast tumor tissue, indicating that mammaglobin might confer a growth advantage to mammaglobin-expressing tumor cells. MATERIALS AND METHODS: The mammaglobin and lipophilin B mRNA expression levels were investigated in seven breast tumors and matched nonneoplastic tissues from the same patients using quantitative real-time RT-PCR. The effect of mammaglobin and lipophilin B expression on breast cancer cell proliferation rates was investigated by analyzing retrovirally transduced Hs578T cell clones. Cell proliferation rates were determined during the exponential growth phase by analyzing the change in lactate dehydrogenase activity over time. RESULTS: All analyzed breast cancer tumors had lower expression levels of mammaglobin and lipophilin B than the respective mean level of the nonneoplastic breast tissues; no prominent overexpression was evident. There was high variability in the expression of mammaglobin and lipophilin B among the non-neoplastic samples, showing that caution should be taken when evaluating their over- and underexpression in tumors. The expression levels of mammaglobin and lipophilin B correlated with each other in the analyzed samples (p = 0.001). Ectopic overexpression of mammaglobin and lipophilin B did not affect the cell proliferation rate of Hs578T breast carcinoma cells in vitro. CONCLUSION: Our findings suggest that the overexpression of mammaglobin observed in certain breast tumors is an epiphenomenon not causally involved in breast carcinogenesis.

MiR-422a weakened breast cancer stem cells properties by targeting PLP2.

OBJECTIVE: This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were observed. METHODS: Flow cytometry and fluorescence-activated cell sorting was performed and CD24-/CD44+ cells were sorted from breast cancer cells and recognized as BCSCs. Microarray was applied to search for the differentially expressed miRNAs and mRNAs between MCF7 and BCSCs. The aberrant expression of miR-422a and PLP2 was further confirmed by RT-qPCR and the direct targeted relationship was verified by dual-luciferase reporter assay. After in vitro transfection, the expression of miR-422a and PLP2 were manipulated and biological functions of BMSCs were compared with CCK-8, colony formation and sphere formation assay. The tumorigenesis ability of transfected BMSCs was also investigated in NOD/SCID tumor mice models. RESULTS: BMSCs were successfully established from MCF7 cells and miR-422a expression was downregulated while PLP2 level decreased in BMSCs. MiR-422a directly targets the 3'UTR of PLP2 and suppressed its expression. Besides, the up-regulation of miR-422a contributed to weakened ability of proliferation and microsphere formation of BMSCs, while PLP2 overexpression facilitated those biological abilities. Tumorigenesis of BMSCs in mice models was impaired by either overexpression of miR-442a or silencing of PLP2. CONCLUSION: Up-regulation of miR-422a attenuated microsphere formation, proliferation and tumor formation of breast cancer stem cells via suppressing the PLP2 expression.

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-B Delta C). Structure and interaction with lipid interfaces.

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B(DeltaC), a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B(DeltaC) has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B(DeltaC) indicated that the protein is properly folded, showing around 32% alpha-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B(DeltaC) that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B(DeltaC) in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.

Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth.

Intraamniotic infection is associated with increased IL-1 activity in amniotic fluid, increased incidence of preterm labor, and with decreased incidence of respiratory distress syndrome in infants born prematurely. We hypothesized that an elevated IL-1 in amniotic fluid promotes fetal lung maturation. On day 23 or 25 of gestation (term 31 d), either IL-1alpha (150 or 1,500 ng per fetus) or its antagonist IL-1 receptor antagonist (IL-1ra, 20 microg) was injected to the amniotic fluid sacs in one uterine horn, whereas the contralateral amniotic sacs were injected with vehicle. Within 40 h, IL-1alpha caused a dose-dependent increase in surfactant protein-A (SP-A) and SP-B mRNAs (maximally, fivefold), without affecting lung growth or increasing inflammatory cells in the lung. Both genders, and upper and lower lung lobes were similarly affected. IL-1ra did not modify SP-A, -B, or -C mRNA. IL-1 increased the intensity of staining of alveolar type II cells for SP-B, and the concentrations of SP-B, -A, and disaturated phosphatidylcholine in bronchoalveolar lavage. The dynamic lung compliance and the postventilatory expansion of lungs were increased two- to fourfold after IL-1alpha treatment. In fetal lung explants, IL-1alpha increased the expression of SP-A mRNA. IL-1 in amniotic fluid in preterm labor may promote lung maturation and thus be part of a host-defense mechanism that prepares the fetus for extrauterine life.

Post-transcriptional downregulation of sarcolipin mRNA by triiodothyronine in the atrial myocardium.

Thyroid hormone-mediated positive cardiotropic effects are differently regulated between the atria and ventricles. This regulation is, at least in part, dependent on sarcoplasmic reticulum (SR) proteins. Sarcolipin, a homologue of phospholamban, has been recently identified as an atrium-specific SR protein. The expression of sarcolipin mRNA was significantly decreased in the atria of mice with hyperthyroidism and in 3,5,3'-triiodo-l-thyronine-treated neonatal rat atrial myocytes. Promoter activity and mRNA stability analyses revealed that thyroid hormone post-transcriptionally down regulated the expression of sarcolipin mRNA. The atrium-specific effect of thyroid hormone may occur in part through the regulation of atrial sarcolipin gene expression.

Expression of tumor protein D52-like genes in childhood leukemia at diagnosis: clinical and sample considerations.

The tumor protein D52 gene or protein is frequently overexpressed in several carcinomas, and has been identified as a B cell differentiation marker. D52-like genes are also differentially expressed in particular haematological malignancies, where transcript or protein levels may reflect cellular proliferative or differentiative status. We used RT-PCR to analyse the expression of three D52-like genes in bone marrow at the time of ALL or AML diagnosis in children. Whereas D53 transcripts were undetectable in all samples, D52 and D54 transcripts were frequently detected in ALL and AML, where they were frequently co-expressed. While D52 and D54 transcripts were detected in T-ALL and pre-B ALL at comparable frequencies, D52 was less frequently detected in ALL bone marrow with hyperdiploid karyotypes, compared with samples with normal karyotypes. We also found that total RNA yields significantly differed according to D52 and D54 expression status, and that bone marrow freezer storage time (up to 945 days) differed significantly according to D52 expression status. These results indicate that D52-like genes are not ubiquitously expressed in leukemic bone marrow in children, and that RNA sample parameters may influence measures of gene expression more than commonly appreciated.

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species.

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

MAL gene expression in esophageal cancer suppresses motility, invasion and tumorigenicity and enhances apoptosis through the Fas pathway.

We isolated the MAL (T-lymphocyte maturation associated protein) gene from differentially expressed products of esophageal epithelium relative to esophageal carcinoma tissues. The Mal protein has been demonstrated as being a component of the protein machinery for apical transport in epithelial polarized cells. In this study, we describe the reduced expression of MAL in all 39 cases of esophageal carcinoma tested and 60 other human carcinomas. MAL gene transcription was induced in three out of 13 esophageal carcinoma cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), and in nine additional cell lines by simultaneous treatment with trichostatin A, an inhibitor of deacetylation, and DAC. We established a stable MAL gene transfectant whose expression was regulated by subcutaneous doxycycline injection in nude mice. Tumor growth was suppressed in cells expressing TE3-MAL compared with TE3 parent cells or cells not expressing TE3-MAL with doxycycline injection (20 microg/body) (P<0.01). Additionally, the TE3-MAL transfectant cells exhibited decreased cellular motility, a G1/S transition block and increased levels of apoptosis, concomitant with increased expression of Fas receptor in vitro. The apoptotic staining in MAL-expressing tumors was confirmed by TUNEL assay. Therefore, we conclude that expression of MAL was frequently decreased or diminished in gastrointestinal tract cancers, and that Mal expression confers reduced tumorigenicity in vivo to tumor TE3 cells through the induction of apoptosis via the Fas signaling pathway.

Members of the C/EBP transcription factor family stimulate expression of the human and rat surfactant protein A (SP-A) genes.

Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBP delta expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 microM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.

Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence.

Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.