PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression

Koji Kubouchi,Hideyuki Mukai

doi:10.1016/j.bbrep.2020.100895

Abstract

The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2flox/flox mouse embryos. PKN2flox/flox cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2flox/flox cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre;PKN2flox/flox cell motility was significantly lower than that of PKN2flox/flox in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2flox/flox cells. Conversely, Cre;PKN2flox/flox cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2flox/flox cells with decreased roundness, although PKN2flox/flox cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both PKN2flox/flox and Cre;PKN2flox/flox cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2flox/flox cells compared to PKN2flox/flox cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2flox/flox cells was significantly lower than that of PKN2flox/flox cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of N-cadherin in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture.

Highlights

Many types of cells can form aggregates and multicellular spheroids when cultured in suspension or in a non-adhesive environment
We report that PKN2 is important for fibroblast aggregate/spheroid formation in suspension culture through its involvement in cell motility and gene expression of N-cadherin
PKN2flox/flox cells in adherent condition (Fig. 3F). These results indicate the possibility that the amount of N-cadherin is key for the compaction process difference observed between PKN2flox/flox and Cre;PKN2flox/flox cells, and that PKN2 is involved in the expression of N-cadherin during the recovery phase after trypsin-EDTA treatment, and in normal adherent condition

Summary

Introduction

Many types of cells can form aggregates and multicellular spheroids when cultured in suspension or in a non-adhesive environment. PKN1 kinase-negative mutant (PKN1[T778A]) mice develop to adulthood without apparent external abnormalities [7], the isolated primary fibroblasts from PKN1[T778A] mouse embryo showed impaired aggregates/spheroid formation in suspension culture with lower cell motility and surface expression of N-cadherin and integrins for unknown reasons [8]. PKN2flox/flox cells were incubated for 48 h after treatment with mock adenovirus (“PKN2flox/flox”) and. Avi) and Cre;PKN2flox/flox (Movie 2.avi) cells in suspension culture in poly hema-coated flat bottom well plate. PKN2 was deleted by the expression of Cre recombinase in these cells, which were subjected to suspension culture. We report that PKN2 is important for fibroblast aggregate/spheroid formation in suspension culture through its involvement in cell motility and gene expression of N-cadherin.

Preparation of cell suspension

Materials and methods

Immunoblotting

Statistical analysis

Findings

Discussion