Karine Rousseau

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b−/− mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Proopiomelanocortin (POMC), the ACTH/ melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin‐1 receptor (MC‐1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melano‐cytes in culture. Much lower levels of α‐MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and α‐MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone conver‐tases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and α‐MSH, and this was enhanced in response to corti‐cotrophin‐releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably trans‐fected with the MC‐1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, α‐MSH, and β‐MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC‐1R highlight the importance of POMC processing as a key regulatory event in the skin.—Rousseau, K., Kauser, S., Pritchard, L. E., Warhurst, A., Oliver, R. L., Slominski, A., Wei, E. T., Thody, A. J., Tobin, D. J., White, A. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and mela‐nocytes and stimulates melanogenesis. FASEB J. 21, 1844–1856 (2007)

Expression of ErbB receptors and mucins in the airways of long term current smokers

Background: Airway epithelial goblet cell hyperplasia is known to occur in chronic smokers. Although the epidermal growth factor receptor has been implicated in this process, neither ErbB receptor expression nor the mucosecretory phenotype of the epithelium have been characterised in current smokers. Methods: Bronchial biopsies obtained from non-smokers (n = 10) and current smokers, with or without chronic obstructive pulmonary disease (n = 51), were examined immunohistochemically to measure the expression of epidermal growth factor receptor, ErbB2, ErbB3, ErbB4 and mucin subtypes (MUC2, MUC5AC and MUC5B) in the bronchial epithelium. The results were correlated with neutrophil counts measured in the airway wall and induced sputum. Results: Epidermal growth factor receptor, ErbB3 and MUC5AC expression, in addition to PAS staining, were significantly increased in all smokers compared with non-smokers, irrespective of the presence of chronic obstructive pulmonary disease. MUC5AC expression was significantly associated with both PAS staining and ErbB3 expression; no correlation was observed between either mucin or ErbB receptor expression and neutrophil counts. Conclusions: The results suggest that long term current smoking induces enhanced epidermal growth factor receptor, ErbB3, and MUC5AC expression in vivo; these increases are not associated with the presence of neutrophilic inflammation. ErbB receptors may contribute to epithelial responses to cigarette smoke.

Leptin and seasonal mammals.

Seasonal mammals commonly exhibit robust annual cycles of adiposity, food intake and energy metabolism. These cycles are driven by changes in the external daylength signal, which generates a diurnal melatonin profile and acts on neuroendocrine pathways. The white adipose tissue hormone leptin reflects overall adiposity in seasonal mammals, and consequently undergoes significant seasonal fluctuations in secretion. The seasonally breeding Siberian (Djungarian) hamster is a convenient laboratory model to study the effect of a seasonal time‐keeping clock on energy metabolism, appetite regulation and the control of adiposity. We have shown that administration of exogenous leptin at physiological doses induces significant loss of adipose tissue for short‐day housed winter‐like hamsters in which endogenous adipose tissue and leptin concentrations are already low. By contrast, long‐day housed hamsters with high adipose tissue reserves are refractory to the effects of leptin. This phenomenon of seasonal leptin resistance appears to be a general feature of other seasonally breeding mammals, and may reflect the operation of an annual timer controlling leptin uptake and/or action on central nervous system signal transduction pathways. The mobilization of fat by leptin in short‐day housed hamsters is not associated with changes in expression in either anorexic or anabolic peptides expressed in leptin‐receptor rich structures in the arcuate region of the hypothalamus, and suggests that leptin may target other structures. These data contrast with studies, which show that homeostatic mechanisms in response to feed‐restriction induce changes in hypothalamic peptides in a similar manner to nonphotoperiodic species. Thus, the long‐term seasonal regulation of body weight set point and leptin feedback may operate through separate pathways to those responsible for acute responses to food restriction.

Aberrant expression of MUC3 and MUC4 membrane-associated mucins and sialyl Le(x) antigen in pancreatic intraepithelial neoplasia.

Introduction Ductal adenocarcinoma of the pancreas has recently been suggested to arise from histologically identifiable ductal lesions known as pancreatic intraepithelial neoplasia (PanINs). Altered levels and patterns of mucin gene expression have been reported to occur in epithelial cancers. Aim To examine the pattern of expression of membrane-associated mucins, MUC3 and MUC4, and a mucin-associated carbohydrate tumor antigen, sialyl Lex, in these precursor lesions and ductal adenocarcinoma of the pancreas. Methodology A total of 144 PanIN lesions and 85 cases of ductal adenocarcinoma of the pancreas were examined by using immunohistochemistry and in situ hybridization methods. Results MUC3 showed a progressive increase in expression in PanINs of increasing dysplasia and was also highly expressed in ductal adenocarcinoma. In contrast, neoexpression of MUC4 and sialyl Lex antigen was observed, mainly in PanIN-3 and ductal adenocarcinoma. In addition, a decrease in the expression of MUC3 and MUC4 was correlated with the degree of de-differentiation of the tumor. Conclusion Aberrant expression of membrane mucins MUC3 and MUC4 and of a mucin-associated carbohydrate tumor antigen Sialyl Lex in PanINs and adenocarcinoma further supports the progression model for pancreatic adenocarcinoma.

MUC5B Is the Major Mucin in the Gel Phase of Sputum in Chronic Obstructive Pulmonary Disease

RATIONALE Overproduction of mucus is a contributory factor in the progression of chronic obstructive pulmonary disease (COPD). The polymeric mucins are major macromolecules in the secretion. Therefore, we hypothesized that the polymeric mucin composition or properties may be different in the sputum from individuals with COPD and smokers without airflow obstruction. OBJECTIVES To determine the major polymeric mucins in COPD sputum and whether these are different in the sputum from individuals with COPD compared with that from smokers without airflow obstruction. METHODS The polymeric mucin composition of sputum from patients with COPD and smokers without airflow obstruction was analyzed by Western blotting analysis. The tissue localization of the mucins was determined by immunohistochemistry, and their size distribution was analyzed by rate-zonal centrifugation. MEASUREMENTS AND MAIN RESULTS MUC5AC and MUC5B were the major mucins. MUC5AC was the predominant mucin in the smoker group, whereas MUC5B was more abundant from the patients with COPD, with a significant difference in the ratio of MUC5B to MUC5AC (P = 0.004); this ratio was correlated with FEV(1) in the COPD group (r = 0.63; P = 0.01). The lower-charged glycosylated form of MUC5B was more predominant in COPD (P = 0.012). No significant associations were observed with respect to sex, age, or pack-year history. In both groups, MUC5AC was produced by surface epithelial cells and MUC5B by submucosal gland cells. Finally, there was a shift toward smaller mucins in the COPD group. CONCLUSIONS Our data indicate that there are differences in mucin amounts and properties between smokers with and without COPD. Further studies are needed to examine how this may impact disease progression.

Differential mucin expression in colon carcinoma HT‐29 clones with variable resistance to 5‐fluorouracil and methotrexate

Abstract A current challenge is to define the biological characteristics of colon tumor cells resistant to chemotherapy. Distinct sub‐populations of mucus‐secreting cells were previously obtained from the colon cancer cell line HT‐29 after long‐term treatment with the anti‐cancer drugs, 5‐fluorouracil (5‐FU) and methotrexate (MTX). Since mucins are increasingly implicated as playing a role in carcinogenesis, we studied the pattern of mucin expression in two HT‐29 clones of mucus‐secreting and two clones of enterocyte‐like phenotype which differ in their capacity to resist to 5‐FU and/or MTX. The expression of both transmembrane (MUC1, MUC3, MUC4) and secreted gel‐forming (MUC2, MUC5AC, MUC5B, MUC6) mucins in clones was studied by northern and/or western blotting. The four HT‐29 clones showed three cellular phenotypes: (1) The mucus‐secreting clone HT29‐5F12 consists of unpolarized cells with mucus secretions that have anti‐colonic mucin immunoreactivity, and mainly expresses MUC2 and is resistant to 5‐FU and sensitive to MTX; (2) The mucus‐secreting clone HT29‐5M21 forms a monolayer of polarized cells with strong anti‐gastric mucin immunoreactivity and mainly expresses MUC5AC and MUC5B and is resistant to MTX and sensitive to 5‐FU; (3) The two enterocyte‐like clones, HT29‐5F7 and HT29‐5M12 are resistant to both MTX and 5‐FU and express mainly MUC1 and MUC5B, respectively. These clones which originate from a same colorectal tumour and display different patterns of mucin expression as well as differing resistance to MTX and 5‐FU will make useful in vitro models for studying the potential role of mucins or other biological markers in drug resistance pathways.

Ex Vivo Sputum Analysis Reveals Impairment of Protease-dependent Mucus Degradation by Plasma Proteins in Acute Asthma

RATIONALE Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies. OBJECTIVES To investigate mechanisms of airway mucus clearance in health and in acute severe asthma. METHODS We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus. MEASUREMENTS AND MAIN RESULTS Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation. CONCLUSIONS Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.

Cocaine and Amphetamine-Regulated Transcript mRNA Regulation in the Hypothalamus in Lean and Obese Rodents

Cocaine and amphetamine‐regulated transcript (CART) mRNA and immunoreactivity are expressed abundantly in the hypothalamus. Central administration of various fragments of this neuropeptide decreases food intake in rodents. To find out whether CART might play a role in the physiological regulation of energy balance, we used in situ hybridization to investigate whether CART mRNA abundance changed in two chronic obese/fat versus lean states and after acute dietary restriction. In the first study, mice were treated with goldthioglucose to destroy glucose‐responsive neurones in the ventromedial hypothalamus. This produced hyperphagia and obesity: 7 weeks after treatment, those receiving goldthioglucose weighed 70% more than the controls. CART mRNA abundance in the arcuate nucleus of goldthioglucose‐treated mice was decreased by 71% compared to levels in the control mice, but CART expression was unaffected in the dorsolateral hypothalamus. In the second study, male Siberian hamsters were exposed to short days to induce a physiological winter response in which body weight decreases as fat reserves are catabolized, and food intake correspondingly declines. After 8 weeks in short days, body weight had declined by 18% relative to controls maintained in long days in a summer fat state. CART mRNA levels did not differ significantly between the two groups in any hypothalamic areas. In the third study, male Siberian hamsters, either in long days or after 12 weeks exposure to short days to induce weight loss, were subject to a 48‐h period of fasting. Although photoperiod per se did not affect CART expression, fasting produced a significant decrease in CART mRNA in the arcuate nucleus of hamsters in both the long‐ and short‐day state. We conclude that CART‐producing cells are involved in energy homeostasis: the marked decrease in CART expression in the arcuate nucleus in goldthioglucose‐lesioned mice may contribute to the development of obesity, and the decrease following acute dietary restriction in hamsters may reflect a compensatory mechanism to reduce caloric expenditure, but our results do not indicate that CART is involved in long‐term seasonal regulation of body weight.

New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7.

The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.