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

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 PKN2 flox/flox mouse embryos. PKN2 flox/flox cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2 flox/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;PKN2 flox/flox cell motility was significantly lower than that of PKN2 flox/flox in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2 flox/flox cells. Conversely, Cre;PKN2 flox/flox cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2 flox/flox cells with decreased roundness, although PKN2 flox/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 PKN2 flox/flox and Cre;PKN2 flox/flox cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2 flox/flox cells compared to PKN2 flox/flox cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2 flox/flox cells was significantly lower than that of PKN2 flox/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.