Regulation of TGF-β storage and activation in the human idiopathic pulmonary fibrosis lung

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-β (TGF-β) activation. Although TGF-β activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-β storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-β. Latent TGF-β-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-β signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-β signaling activity, respectively, suggesting that LTBP-1-mediated TGF-β activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-β-activating integrin β8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-β storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-β availability and activation in different pulmonary compartments in the fibrotic lung.     

Heavy resistance exercise training and skeletal muscle androgen receptor expression in younger and older men

Effects of heavy resistance exercise on serum testosterone and skeletal muscle androgen receptor (AR) concentrations were examined before and after a 21-week resistance training period. Seven healthy untrained young adult men (YT) and ten controls (YC) as well as ten older men (OT) and eight controls (OC) volunteered as subjects. Heavy resistance exercise bouts (5 × 10 RM leg presses) were performed before and after the training period. Muscle biopsies were obtained before and 1h and 48 h after the resistance exercise bouts from m.vastus lateralis (VL) to determine cross-sectional area of muscle fibers (fCSA) and AR mRNA expression and protein concentrations. No changes were observed in YC and OC while resistance training led to significant increases in maximal strength of leg extensors (1 RM), fCSA and lean body mass in YT and OT. Acute increases occurred in serum testosterone concentrations due to resistance exercises but basal testosterone remained unaltered. Mean AR mRNA expression and protein concentration remained unchanged after heavy resistance exercise bouts compared to pre-values. The individual pre- to post-training changes in resting (pre-exercise) AR protein concentration correlated with the changes in fCSA and lean body mass in the combined group of YT and OT. Similarly, it correlated with the changes in 1 RM in YT. Although mean AR expression did not changed due to the resistance exercise training, the present findings suggest that the individual changes of AR protein concentration in skeletal muscle following resistance training may have an impact on training-induced muscular adaptations in both younger and older men.     

Recovery after heavy resistance exercise and skeletal muscle androgen receptor and insulin-like growth factor-I isoform expression in strength trained men

The effects of heavy resistance exercise on skeletal muscle androgen receptor (AR) protein concentration and mRNAs of AR, insulin-like growth factor-I (IGF)-IEa, and mechano growth factor (MGF) expression were examined from biopsies of vastus lateralis (VL) muscle before and 48 hours after heavy resistance exercise (5 × 10 repetition maximum [RM] leg press and 4 × 10RM squats) in 8 adult strength trained men. The present exercise induced an acute decrease in maximal isometric force and increased serum total testosterone (T) and free testosterone (FT) concentrations. During 2 recovery days, maximal isometric force and subjective perception of physical fitness remained significantly lowered, whereas serum creatine kinase activity, subjective muscle soreness, and muscle swelling (i.e., thickness of VL by ultrasound) were significantly increased compared to pre-exercise values. Subjective perception of physical fitness was followed up to 7 days, and by 6 days postexercise, it was elevated above the pre-exercise level. Basal T and FT concentrations remained unaltered after the exercise. No statistically significant changes were observed in AR protein or mRNA expression, but IGF-IEa (p < 0.05) and MGF (p < 0.05) mRNA expression were increased compared to pre-exercise levels. These findings indicate that IGF-IEa and MGF responses may be related to acute regenerative processes in muscle because of exercise and may contribute to muscular adaptation to resistance exercise. Subjective perception of physical fitness suggests that recovery over a pre-exercise level of the present type of heavy resistance exercise can take approximately 6 days.     

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.Human gp340, also known as DMBT1 (deleted in malignant brain tumors 1), belongs to the innate immune protein family of scavenger receptor cysteine-rich (SRCR)² proteins, all of which contain one or more evolutionarily conserved SRCR domain linked to other conserved protein domains (1, 2). Many of these proteins serve as pattern recognition receptors for innate immunity. gp340 is expressed by epithelial cells and cells of the immune system, and its expression is up-regulated after inflammatory stimuli (3, 4). It inhibits bacterial invasion to epithelial cells and the secretion of proinflammatory cytokines (5–7). Thus, it appears to be an important mediator of host immune responses to various microbes and was recently linked to Crohn disease, a human inflammatory bowel disease (8). gp340 is also found in human secretions like tears and saliva, and the salivary form has long been known as salivary agglutinin, which is an important molecule in oral biofilm formation and is suggested to have a role in dental caries development (9–12). The mechanisms of gp340 action in these different biological contexts are not known.Common to all scavenger receptors, the ligand repertoire of gp340 is wide; it binds many different types of bacteria as well as viruses (10, 13, 14). The wide ligand recognition pattern of scavenger receptors is thought to be based on the recognition of common microbial structures, such as lipopolysaccharides and lipoteichoic acids, but in the case of gp340, specific bacterial surface proteins are reported to be involved in the interactions characterized (15–18). Because the importance of gp340/salivary agglutinin in the oral environment has been evident for a long time, most of our knowledge of gp340-microbial interactions is from studies with oral bacteria. For example, viridans streptococci, such as Streptococcus mutans and Streptococcus gordonii, interact with saliva gp340 via their surface proteins AgI/II and the sialic acid-binding Hsa or GspB adhesin. In these interactions, gp340 shows a peculiar fluid phase versus surface-adsorbed behavior, as evidenced by AgI/II polypeptides primarily mediating aggregation of bacteria by fluid phase gp340, whereas the Hsa adhesin primarily mediates adhesion of S. gordonii to surface-bound gp340 (18).gp340 binds also to many non-oral human Gram-negative and Gram-positive pathogens, such as Helicobacter pylori and S. pyogenes (10), but these interactions are less characterized. There are few studies suggesting that both carbohydrates and the protein core of the gp340 can be involved in these interactions. For example, a VEVLXXXXW peptide derived from the SRCR domain of gp340 is shown to bind different types of bacteria (19), whereas sialic acid residues may mediate binding to, for example, influenza virus (20). However, the molecular basis of the ability of gp340 or peptides derived thereof to bind a large ligand repertoire is not understood.The aim of the present study was to use the common human pathogen S. pyogenes as a model bacterium to identify novel bacterial proteins binding to gp340 and in this way shed light on the ligand recognition capability of gp340. We report a novel S. pyogenes-host interaction mediated by bacterial surface protein Spy0843 and gp340. gp340 binds to conserved leucine-rich repeat (Lrr) motifs in the Spy0843 and recognizes the same motif also in other bacterial proteins, from both Gram-negative and Gram-positive bacteria. Moreover, the inhibition of Lrr binding with SRCR-derived peptides differed from that previously reported, which suggests a novel mode for ligand recognition of human scavenger receptor gp340.     

Hemoglobin α and β are ubiquitous in the human lung,decline in idiopathic pulmonary fibrosis but not in COPD

Background

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are disorders of the lung parenchyma. They share the common denominators of a progressive nature and poor prognosis. The goal was to use non-biased proteomics to discover new markers for these diseases.

Methods

Proteomics of fibrotic vs. control lung tissue suggested decreased levels of several spots in the lung specimens of IPF patients, which were identified as Hemoglobin (Hb) α and β monomers and Hbα complexes. The Hbα and β monomers and complexes were investigated in more detail in normal lung and lung specimens of patients with IPF and COPD by immunohistochemistry, morphometry and mass spectrometry (MS).

Results

Both Hb monomers, in normal lung, were expressed especially in the alveolar epithelium. Levels of Hbα and β monomers and complexes were reduced/lost in IPF but not in the COPD lungs when compared to control lung. MS-analyses revealed Hbα modification at cysteine105 (Cysα105), preventing formation of the Hbα complexes in the IPF lungs. Hbα and Hbβ were expressed as complexes and monomers in the lung tissues, but were secreted into the bronchoalveolar lavage fluid and/or induced sputum supernatants as complexes corresponding to the molecular weight of the Hb tetramer.

Conclusions

The abundant expression of the oxygen carrier molecule Hb in the normal lung epithelium and its decline in IPF lung are new findings. The loss of Hb complex formation in IPF warrants further studies and may be considered as a disease-specific modification.     

Variability in the precursor proteins of collagen I and III in different stages of COPD

Background

Levels of precursor proteins of collagen I and III are increased in fibrotic pulmonary diseases. This study determined whether the expression of precursors of type I and III collagen proteins would be increased in small and large airways of COPD patients in various stages of the disease reflecting fibrogenesis.

Methods

The levels of precursor proteins of collagen I and III were studied by immunohistochemistry and quantified by image analysis in lung tissue of 16 non-smokers, 20 smokers with normal lung function, 20 smokers with stage I-II COPD and 8 ex-smokers with stage IV COPD.

Results

In large airways, the subepithelial layer which was positive for precursor proteins of collagen I and III was thicker in smokers and in stage I-II COPD compared to non-smokers. Large airways in stage IV COPD showed reduced expression of precursor protein of collagen I whereas precursor of collagen III was increased. The amount of precursor protein of collagen III was increased in small airways of smokers and stage I-II COPD but reduced in stage IV COPD.

Conclusions

Precursor proteins of collagen I and III revealed different expression profiles in large and small airways in various stages of COPD. Smoking enhanced expression of both precursors in large airways with a positive correlation with pack-years.     

Variable overoxidation of peroxiredoxins in human lung cells in severe oxidative stress

Peroxiredoxins (Prxs) are a group of thiol containing proteins that participate both in signal transduction and in the breakdown of hydrogen peroxide (H(2)O(2)) during oxidative stress. Six distinct Prxs have been characterized in human cells (Prxs I-VI). Prxs I-IV form dimers held together by disulfide bonds, Prx V forms intramolecular bond, but the mechanism of Prx VI, so-called 1-Cys Prx, is still unclear. Here we describe the regulation of all six Prxs in cultured human lung A549 and BEAS-2B cells. The cells were exposed to variable concentrations of H(2)O(2), menadione, tumor necrosis factor-alpha or transforming growth factor-beta. To evoke glutathione depletion, the cells were furthermore treated with buthionine sulfoximine. Only high concentrations (300 microM) of H(2)O(2) caused a minor increase (<28%, 4 h) in the expression of Prxs I, IV, and VI. Severe oxidant stress (250-500 microM H(2)O(2)) caused a significant increase in the proportion of the monomeric forms of Prxs I-IV; this was reversible at lower H(2)O(2) concentrations (< or =250 microM). This recovery of Prx overoxidation differed among the various Prxs; Prx I was recovered within 24 h, but recovery required 48 h for Prx III. Overall, Prxs are not significantly modulated by mild oxidant stress or cytokines, but there is variable, though reversible, overoxidation in these proteins during severe oxidant exposure.