Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR‐108 osteoblastic cells

Abstract

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR-108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC-alpha (11.6-fold), nPKC-delta (5.3-fold), and nPKC-epsilon (22.0-fold) but not aPKC-zeta. Northern blot analysis indicated a significant (2.3-fold) increase in the 10 kb transcript of cPKC-alpha, a slight (1.3-fold) increase in that of nPKC-epsilon but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC-alpha, PKC-delta, and PKC-epsilon and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR-108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme-selective increases in the protein levels of PKC isozymes of osteoblastic UMR-108 cells upon growth to confluence which may be regulated at the transcriptional or the post-transcriptional level. The results from UMR-108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999-1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

The expression profile of PKC isoforms during MC3T3-E1 differentiation

“…In primary rat OBC there is a continuous increase in PKC · expression from early to late cell cultures. Geng et al also demonstrated a marked increase in the level of cPKC-· expression in rat osteogenic sarcoma cells (UMR-108) upon cellular confluency (32).…”

Bioluminescence imaging in bone tissue engineering

“…In addition, we observed that rottlerin treatment changed hMSC morphology from its typical spindle-shaped fibroblastic morphology observed in proliferating hMSCs to a triangular shape (Fig. 5C), which is consistent with reports stating that inhibition of PKCδ changes the morphology of hMSCs by altering the cytoskeleton [137,138]. To elucidate which isozyme is involved in this effect, we exposed hMSCs to a PKCθ pseudosubstrate, which inhibits PKCθ activity but does not affect PKCδ activity.…”

Protein kinase Cδ in tumorigenesis of human malignant fibrous histiocytoma

“…Several PKC isoforms have been reported to act as regulators of cell differentiation, growth, survival, migration, invasion, and apoptosis in various human malignancies (7,10,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Therefore, PKC may be an attractive candidate for targeting using novel anticancer therapies.…”

“…Therefore, PKC may be an attractive candidate for targeting using novel anticancer therapies. Among the PKC isoforms, PKCα, β, and γ are the most abundant isoforms in various tissues (19), and in general, PKCα and PKCβ have been linked to increased invasion, proliferation, drug resistance and genetic instability in tumor cells (9,19,21,(24)(25)(26)(27)(28)(29)(30)(31)(32), while PKCδ is thought to promote apoptosis (7,10,33). Koivunen et al theorized that increased proportional activation of PKCα/β to PKCδ is an important factor in the aggressiveness of cancers (9), and other studies demonstrated that PKCβ plays an important role in the signal transduction pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis in human malignancies (26,27).…”

“…Dysregulated PKC activity leads to tumor progression in a variety of human sarcomas, including rhabdomyosarcomas (28), osteosarcomas (29), fibrosarcomas (30) and leiomyosarcomas (31). Keller et al reported that the PKC isoforms α, γ and β1, but not β2 or δ, are involved in the regulation of cell shape and locomotion of human fibrosarcoma HT1080 cells (30).…”