Real-time PCR was performed in an I-Cycler (Bio-Rad, Munich, Germany) using a QuantiTec SYBR Green PCR kit (Qiagen)

Real-time PCR was performed in an I-Cycler (Bio-Rad, Munich, Germany) using a QuantiTec SYBR Green PCR kit (Qiagen). respectively, of cav-1 and cav-2 in airway epithelium. Real-time quantitative RT-PCR analysis of abraded tracheal epithelium revealed a higher expression of cav-2 than of cav-1. Immunoreactivities for cav-1 and for cav-2 were co-localized in the cell membrane of the basal cells and basolaterally in the ciliated epithelial cells of large airways of rat and human. However, no labeling for cav-1 or cav-2 was observed in the epithelial cells of small bronchi. Using conventional double-labeling indirect immunofluorescence combined with CLSM-FRET analysis, we detected an association of cav-1 and -2 in epithelial cells. The presence of caveolae was confirmed by electron microscopy. In contrast to human and rat, cav-1-immunoreactivity and caveolae were confined to basal cells in mice. Epithelial caveolae were absent in cav-1-deficient mice, implicating a requirement of this caveolar protein in epithelial caveolae formation. Conclusion These results show that caveolae and caveolins are integral membrane components in basal and ciliated epithelial cells, indicating a crucial role in these cell types. In addition to their physiological role, they may be involved in airway infection. Background Caveolae are omega-shaped invaginations of the plasma membrane measuring 50 to 100 nm in diameter. They are found in numerous cell types such as type I pneumocytes, endothelial cells, adipocytes, fibroblasts, smooth muscle cells, cardiac and striated muscle cells [1]. Caveolar formation is dependent on the expression of caveolins. Three caveolins (cav) are known. Cav-1 and cav-2 are widely expressed, whereas cav-3 is thought to be restricted to muscle cells [2]. Cav-1 is expressed in two isoforms, cav-1 and cav-1, exhibiting a cell type-specific distribution (endothelial vs. alveolar type-1 cells) in the alveolar region [3]. Caveolae are involved in diverse cellular functions such as organizing signal Rabbit polyclonal to NF-kappaB p65.NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA, or RELB (MIM 604758) to form the NFKB complex. transduction mechanisms, endocytosis and intracellular transport [2]. Several pathogenic microorganisms selectively use caveolae to enter cells [4]. After accumulation in the caveolae, they are delivered to the endoplasmatic reticulum bypassing the classical endosome-lysosome trafficking and thereby preventing inactivation [5,6]. It has been shown that the infectivity of C-type human adenovirus can Prohydrojasmon racemate be greatly reduced by the expression of a dominant negative cav-1 mutant in plasmocytic cells [7], indicating that caveolae are involved in this process. In addition, it was recently shown for Chlamydia pneumoniae that it co-localizes intracellularly with cav-1 and cav-2 after infection, and a role of these proteins for the developmental cycle of Chlamydiae is discussed [8]. Also, the human coronavirus 229E that is known to induce respiratory tract infections enters cells via a caveolae dependent mechanism [9,10]. Although the airway epithelium serves as entry site for microbes, fulfils functions that are associated with caveolae such as endo- and transcytosis, and harbors receptors that are associated with caveolae [1], the expression of caveolins, their interaction, and the presence of caveolae in tracheal and bronchial epithelial cells have not yet been determined. Interestingly, the presence of “vesicles that sometimes are connected with the membrane” has earlier been described at the electron-microscopic level in mouse basal cells [11]. Moreover cav-1 and cav-2 were detected in cell lines derived from bronchial epithelium [12], pointing to the presence of caveolae in the airway epithelium. Both cav-1 and cav-2 exhibit a similar expression, but seem to have different functions. Cav-1 is sufficient to drive caveolar formation [13]. In general, it is thought that cav-2 only is not adequate for caveolae formation, and the absence of caveolae in cav-1-deficient mice Prohydrojasmon racemate is definitely associated with designated reduction in cav-2 levels [14]. In contrast, although Prohydrojasmon racemate caveolae are still present in cav-2 deficient mice, these mice display the.