Phorbol ester‐induced podosomes in normal human bronchial epithelial cells

Abstract

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z-stack scanning confocal microscopy showed that PDBu-induced podosome-like structures contain actin-rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration.

The Protein Kinase C Cascade Regulates Recruitment of Matrix Metalloprotease 9 to Podosomes and Its Release and Activation

“…Ventral podosome-like structures and dorsal circular ruffles were identified with Z-stack scanning confocal microscopy, together with immunostaining of multiple marker proteins (55). In the present study, PDBu-induced podosomes in primary human bronchial epithelial cells identified with Zstack scanning confocal microscopy with cortactin as a marker of podosomes are shown as an example.…”

“…Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin (BSA), and stained with the designated antibodies Oregon Green 488 phalloidin (Molecular Probe) and Hoechst dye 33342. After gentle washing, the coverslips were mounted on glass slides with Dako fluorescence mounting medium (Dako, Mississauga, Canada) (51,55).…”

“…Fluorescent images were processed with Olympus FV10-ASW version 01.07.02 imaging software. Cells with at least five small dots (Ͼ1 m), rosettes, or belts in cytoplasm and with actin-rich circular ruffles at the periphery were counted as podosome-positive cells and cells with clear, long stress fibers and weaker peripheral actin as podosome negative (55). The quantitative assessment of fluorophore colocalization in the confocal section images was done by Olympus FV10-ASW version 01.07.02 imaging software for Pearson's correlation coefficient, which is calculated according to the equation…”

“…BEAS2B cells were transfected with plasmids expressing pEGFP alone, pEGFP-PKC, or pEGFP-PKC␣, with or without mCherry-Tks5 (56). After 48 h, cells were subjected to live cell imaging (55). The time lapse series of photographs were taken using the confocal microscope mentioned above, equipped with a computer-driven camera and an autofocus system, in a humidified chamber with 5% CO 2 at 37°C.…”

“…Cells were plated on coated coverslips in 6-well plates and incubated at 37°C for 8 h. Foci of degraded matrix were observed as dark holes (Ն3 m) on the green fluorescent gelatin matrix. Cells with black digested areas under the cell or near the cell edge were counted as positive to degrade matrix in a blinded fashion (55).…”