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. 2015 Mar;143(3):277-87.
doi: 10.1007/s00418-014-1279-x. Epub 2014 Sep 12.

Murine CLCA5 Is Uniquely Expressed in Distinct Niches of Airway Epithelial Cells

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Free PMC article

Murine CLCA5 Is Uniquely Expressed in Distinct Niches of Airway Epithelial Cells

Kristina Dietert et al. Histochem Cell Biol. .
Free PMC article

Abstract

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.

Figures

Fig. 1
Fig. 1
mCLCA5 is expressed in lungs of naive mice in a highly selective pattern. a Whole lungs of naive mice were embedded in paraffin, and multiple sections of defined layers were prepared to ensure that the entire bronchial stem and its branching points are available for systematic investigation. mCLCA5 protein is expressed in bronchial epithelial cells at the transition from the extrapulmonary main bronchi to the intrapulmonary bronchi (black lines) only. HE staining. b This region was characterized by CC10-positive club cells, PAS-positive mucus cells as well as mCLCA5- and mCLCA3-expressing cells. Bar (a) 2 mm, bar (b) 20 µm
Fig. 2
Fig. 2
mCLCA5 is predominantly located in club cells, to lesser extent in mucus cells and ciliated cells. a For co-localization studies of mCLCA5 and club cell protein CC10, immunofluorescence and spectral confocal laser scanning microscopy was performed. (mCLCA5: left, green; CC10: center, red; merged image: right). Blue DAPI (4,6-diaminidino-2-phenylindole) staining of the DNA in the nuclei. b, c Double staining of mCLCA5 either with PAS reaction, identifying mucus cells, or with mCLCA3 by immunohistochemistry was conducted. mCLCA5 is primarily located in club cells (a arrow), followed by fewer ciliated cells (a arrowhead) and mucus cells (b arrowhead). In mucus cells, mCLCA5 was occasionally co-localized with mCLCA3 (c arrowhead). d Club cells and mucus cells showed an either fine or coarse, diffuse, granular, cytoplasmatic, subcellular labeling pattern of mCLCA5, in contrast to ciliated cells, which displayed a clumpy and perinuclear labeling pattern. However, only a limited number of the investigated cells were positive for mCLCA5 protein expression. Bar (a) 5 µm, bar (b, c) 10 µm
Fig. 3
Fig. 3
mCLCA5 mRNA and protein are strongly decreased in challenged lungs. ac 24 h after mice were treated with PBS or infected with S. aureus, lung mRNA expression levels of Muc5ac, Muc5b, mClca3 and mClca5 were determined by RT-qPCR in comparison with naive mice. Only mClca5 mRNA was significantly decreased in both challenge models (c). Dotted lines indicate fold changes of 0.5 and 2, respectively, as limits for valid statement of lowered and elevated parameters. Values are given as mean ± SEM (n = 8 each group). Ct cycle threshold. *p < 0.05 versus the naive control group. d, e Numbers of CC10-, PAS-, mCLCA3- and mCLCA5-positive cells per mm basement membrane were quantified by immunohistochemistry or PAS reaction. mCLCA5-positive cells were significantly reduced in both challenge models at indicated time points, without any further changes in number or composition of the bronchial epithelial cells. Values are given as mean ± SEM (n = 4 each group). *p < 0.05 versus the naive control group
Fig. 4
Fig. 4
mCLCA5 protein expression disappeared in various challenge models. Lungs from naive (n = 4) and PBS-treated (n = 4) mice as well as from mice infected with S. aureus (n = 4), S. pneumoniae (n = 2) and influenza virus (n = 2) were examined at the extrapulmonary to intrapulmonary junction to characterize the presence and the course of mCLCA5 protein expression in this specific location at various time points. a, b Comparison of naive lungs to lungs from PBS-treated or S. aureus-infected mice revealed a significant reduction in mCLCA5 protein expression 24 h after infection, with a slight tendency toward a return after 48 h. c, d After infection with S. pneumoniae and influenza virus, the immunosignal of mCLCA5 disappeared over time. Bar 20 µm
Fig. 5
Fig. 5
Species-specific differences in expression patterns of mCLCA5 and its human and porcine orthologs. Murine (n = 4), human (n = 2) and porcine (n = 3) lung tissues were investigated by immunohistochemistry. ac A species-specific distribution pattern in the submucosal glands (blue lines) was observed, and all species investigated had mCLCA5-, hCLCA2- or pCLCA2-positive cells, respectively, in the epithelial cells of these SMGs (d1, e1, f1, left picture). However, only the mouse had mCLCA5-positive cells in this specific location within the bronchial epithelium (d2, e2, f2, right picture). Bar 40 µm

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