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 translocation of protein kinase C (PKC) isozymes from their inactive cell locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specificity. We have utilized confocal microscopy to compare phorbol 12, 13 dibutyrate (PDB) - stimulated translocation of PKCalpha in cultured cells derived from rat vascular smooth muscle. In enzymatically dispersed, passaged smooth muscle cells, PKCalpha was uniformly distributed throughout the unstimulated cell. PDB stimulation resulted in extensive association of the PKCalpha into filamentous strands with subsequent accumulation of the isoform in the peri-nuclear region of the cell. Dual immunostaining indicated that PKCalpha was extensively colocalized with microtubules in the interval immediately following PDB stimulation but was largely disassociated from microtubules at 10 min, at which time the translocation of PKCalpha to the peri-nucleus/nucleus was nearly complete. It was further found that the use of colchicine to disrupt the microtubules caused the loss of PKCalpha translocation to the peri-nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The data suggest that PDB stimulation results in a transient association of PKCalpha with cell microtubules and that the microtubules play an important role in the translocation of PKCalpha from the cytosol in passaged cells derived from rat aortic smooth muscle.

Reports from numerous laboratories suggest that protein kinase C (PKC) translocation to substrate target sites may vary depending on cell type and experimental conditions. We have proposed that acutely variable targeting of PKC to different substrate sites could greatly expand the functional properties of individual isoforms in individual cell types (Li et al., 2001). Confocal microscopy and PKC alpha-enhanced green fluorescent protein (PKC alpha-EGFP) fusion protein expression were utilized to investigate the spatial and temporal pattern of PKC alpha translocation to different stimulating agents in A7r5 smooth muscle cells. Phorbol 12, 13 dibutyrate (PDBu 10(-8) M) caused a slow but irreversible relocation of the fusion protein from the cytosol to the plasmalemma. By comparison, thapsigargin (10(-5) M) and A23 187 (2 x 10(-5) M) induced a rapidly transient translocation to the cell membrane which was completed within 4 min. In contrast to these agents, angiotensin II (Ang II, 10(-6) M) caused only partial relocalization of cytosolic PKC alpha-EGFP to brightly fluorescing patches at the cell periphery. Localization at peripheral patches was completed within seconds and the fusion protein returned to the cytosol within 2 min. The PKC inhibitor staurosporine blocked cellular contraction to PDBu but not A(23 187) and had no effect on PKC alpha-EGFP translocation. By comparison, the calcium chelators EDTA and BAPTA-AM blocked the contraction to A(23 187), attenuated the contraction to PDBu, and abolished the translocation of PKC alpha-EGFP by both agents. The results show that in a single cell type the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly. This further suggests the existence of potentially complex mechanisms which regulate the rate and location of target site availability.

It has been proposed that the reorganization of components of the actin cytomatrix could contribute to force development and the low energy cost of sustained contraction in contractile cells which lack a structured sarcomere (A.S. Battistella-Patterson, S. Wang and G.L. Wright (1997) Can J Physiol Pharmacol 75: 1287-1299). However, there has been no direct evidence of an apropos actin reorganization specifically linked to the contractile response in cells of this type. Remodeling of the alpha- and beta-actin domains was studied in A7r5 smooth muscle cells during phorbol 12,13 dibutyrate (PDB)-induced contraction using immunohistologic staining and beta-actin-green fluorescent protein (beta-actin-GFP) fusion protein expression. Cell stained with phalloidin as well as cells expressing beta-actin-GFP showed densely packed actin stress cables, arranged in parallel and extending across the cell body. PDB caused approximately 85% of cells to contract with evidence of forcible detachment from peripheral adhesion sites seen in many cells. The contraction of the cell body was not uniform but occurred along a principal axis parallel to the system of densely packed beta-actin cables. During the interval of contraction, the beta-actin cables shortened without evidence of disassembly or new cable formation. The use of cytochalasin to inhibit actin polymerization resulted in the dissolution of the actin cables at the central region of the cell and caused the elongation of precontracted cells. In unstimulated cells, alpha-actin formed cables similar in arrangement to the cell spanning beta-actin cables. Within a short interval after PDB addition; however, the majority of alpha-actin cables disassembled and reformed into intensely fluorescing column-like structures extending vertically from the cell base at the center of clusters of alpha-actin filaments. The alpha-actin columns of contracting cells showed strong colocalization of alpha-actinin suggesting they could be structurally analogous to the dense bodies of highly differentiated smooth muscle cells. The results indicate that the alpha- and beta-actin domains of A7r5 cells undergo a highly structured reorganization during PDB-induced contraction. The extent and nature of this restructuring suggest that remodeling could play a role in contractile function.

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the alpha-actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775-781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca2+]i by A23187 and thapsigargin on alpha- and beta-actin remodeling. The effects of A23187 and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the alpha- and beta-actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, beta-actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca2+]i resulted in partial or complete dissolution of alpha-actin cables without further remodeling. In addition, PDBu-mediated alpha-actin remodeling was blocked in the presence of A23187. Increased [Ca2+]i also caused dispersal of alpha-actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for alpha-actin cytoskeletal structure. The incubation of cells in calcium-free media prevented alpha-actin dissolution by A23187/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of alpha-actin cables by increased [Ca2+]i. The results suggest that the sustained elevation of [Ca2+]i beyond a threshold level initiates depolymerization of alpha-actin but not beta-actin. It further appears that PDBu-induced alpha-actin remodeling requires Ca2+ but increases of [Ca2+]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits alpha-actin dissolution in the presence of A23187/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing alpha-actin structure in the activated cell.