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.

PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes.

Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.

PKN3 is the major regulator of angiogenesis and tumor metastasis in mice.

PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin ß1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes.