Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.
PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-zeta was not changed by PKC-delta siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-delta transfectants, compared to EV cells, PKC-delta upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-delta specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-delta regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-delta that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-delta in colonic carcinogenesis.
Cerda
S R
SR
Department of Medicine, Division of Gastroenterology, University of Chicago, Chicago, IL 60637, USA. scerda@medicine.bsd.uchicago.edu
Mustafi
R
R
Little
H
H
Cohen
G
G
Khare
S
S
Moore
C
C
Majumder
P
P
Bissonnette
M
M
D017209:Apoptosis; D018938:Caco-2 Cells; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D051745:Protein Kinase C-delta / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D019938:Cyclin D1; D051745:Protein Kinase C-delta; C495894:CDK2 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D013755:Tetradecanoylphorbol Acetate