Induction of premature senescence and a less-fibrogenic phenotype by programmed cell death 4 knockdown in the human hepatic stellate cell line Lieming Xu-2.

Although programmed cell death 4 (PDCD4) was initially reported as a tumor suppressor and has been shown to inhibit cancer cell growth and metastasis, recent studies have demonstrated that loss of PDCD4 expression also induces growth inhibition by inducing apoptosis and/or cellular senescence. At present, the roles of PDCD4 in the activation and profibrogenic properties of myofibroblasts, which are critically involved in organ fibrosis, such as that in the liver, are unclear. We, therefore, investigated the roles of PDCD4 in myofibroblasts using human hepatic stellate cell line Lieming Xu-2 (LX-2). PDCD4 knockdown inhibited LX-2 proliferation and induced a senescent phenotype with increased ß-galactosidase staining and p21 expression in a p53-independent manner together with downregulation of the notch signaling mediator RBJ-κ/CSL. During PDCD4 knockdown, alpha smooth muscle actin (α-SMA; an activation marker of myofibroblasts), matrix metalloproteinases MMP-1 and MMP-9, and collagen IV were upregulated, but the expression of collagen1α1 and collagen III was markedly downregulated without any marked change in the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). These results demonstrated that knockdown of PDCD4 induced the cellular senescence phenotype and activated myofibroblasts while suppressing the profibrogenic phenotype, suggesting roles of PDCD4 in cellular senescence and fibrogenesis in the liver.

Degradation of the Tumor Suppressor PDCD4 Is Impaired by the Suppression of p62/SQSTM1 and Autophagy.

PDCD4 (programmed cell death 4) is a tumor suppressor that plays a crucial role in multiple cellular functions, such as the control of protein synthesis and transcriptional control of some genes, the inhibition of cancer invasion and metastasis. The expression of this protein is controlled by synthesis, such as via transcription and translation, and degradation by the ubiquitin-proteasome system. The mitogens, known as tumor promotors, EGF (epidermal growth factor) and TPA (12-O-tetradecanoylphorbol-13-acetate) stimulate the degradation of PDCD4 protein. However, the whole picture of PDCD4 degradation mechanisms is still unclear, we therefore investigated the relationship between PDCD4 and autophagy. The proteasome inhibitor MG132 and the autophagy inhibitor bafilomycin A1 were found to upregulate the PDCD4 levels. PDCD4 protein levels increased synergistically in the presence of both inhibitors. Knockdown of p62/SQSTM1 (sequestosome-1), a polyubiquitin binding partner, also upregulated the PDCD4 levels. P62 and LC3 (microtubule-associated protein 1A/1B-light chain 3)-II were co-immunoprecipitated by an anti-PDCD4 antibody. Colocalization particles of PDCD4, p62 and the autophagosome marker LC3 were observed and the colocalization areas increased in the presence of autophagy and/or proteasome inhibitor(s) in Huh7 cells. In ATG (autophagy related) 5-deficient Huh7 cells in which autophagy was impaired, the PDCD4 levels were increased at the basal levels and upregulated in the presence of autophagy inhibitors. Based on the above findings, we concluded that after phosphorylation in the degron and ubiquitination, PDCD4 is degraded by both the proteasome and autophagy systems.

Mechanisms of PKC-Mediated Enhancement of HIF-1α Activity and its Inhibition by Vitamin K2 in Hepatocellular Carcinoma Cells.

Hypoxia-inducible factor 1 (HIF-1) plays important roles in cancer cell biology. HIF-1α is reportedly activated by several factors, including protein kinase C (PKC), in addition to hypoxia. We investigated the role of PKC isoforms and the effects of vitamin K2 (VK2) in the activation process of HIF-1α. Human hepatocellular carcinoma (HCC)-derived Huh7 cells were cultured under normoxic and hypoxic (1% O2) conditions with or without the PKC stimulator TPA. The expression, transcriptional activity and nuclear translocation of HIF-1α were examined under treatment with PKC inhibitors, siRNAs against each PKC isoform and VK2. Hypoxia increased the expression and activity of HIF-1α. TPA increased the HIF-1α activity several times under both normoxic and hypoxic conditions. PKC-δ siRNA-mediated knockdown, PKC-δ inhibitor (rottlerin) and pan-PKC inhibitor (Ro-31-8425) suppressed the expression and transcriptional activity of HIF-1α. VK2 significantly inhibited the TPA-induced HIF-1α transcriptional activity and suppressed the expression and nuclear translocation of HIF-1α induced by TPA without altering the HIF-1α mRNA levels. These data indicate that PKC-δ enhances the HIF-1α transcriptional activity by increasing the nuclear translocation, and that VK2 might suppress the HIF-1α activation through the inhibition of PKC in HCC cells.

PDCD4 Knockdown Induces Senescence in Hepatoma Cells by Up-Regulating the p21 Expression.

While the over-expression of tumor suppressor programmed cell death 4 (PDCD4) induces apoptosis, it was recently shown that PDCD4 knockdown also induced apoptosis. In this study, we examined the cell cycle regulators whose activation is affected by PDCD4 knockdown to investigate the contribution of PDCD4 to cell cycle regulation in three types of hepatoma cells: HepG2, Huh7 (mutant p53 and p16-deficient), and Hep3B (p53- and Rb-deficient). PDCD4 knockdown suppressed cell growth in all three cell lines by inhibiting Rb phosphorylation via down-regulating the expression of Rb itself and CDKs, which phosphorylate Rb, and up-regulating the expression of the CDK inhibitor p21 through a p53-independent pathway. We also found that apoptosis was induced in a p53-dependent manner in PDCD4 knockdown HepG2 cells (p53+), although the mechanism of cell death in PDCD4 knockdown Hep3B cells (p53-) was different. Furthermore, PDCD4 knockdown induced cellular senescence characterized by ß-galactosidase staining, and p21 knockdown rescued the senescence and cell death as well as the inhibition of Rb phosphorylation induced by PDCD4 knockdown. Thus, PDCD4 is an important cell cycle regulator of hepatoma cells and may be a promising therapeutic target for the treatment of hepatocellular carcinoma.

Control of a tumor suppressor PDCD4: Degradation mechanisms of the protein in hepatocellular carcinoma cells.

In this study, we demonstrate that EGF inhibits the TGF-ß1-induced apoptosis of Huh7 cells. TGF-ß1 up-regulates the expression of PDCD4 causing apoptosis, by stimulating the synthesis of PDCD4 mRNA via the Smad signaling pathway. TGF-ß1 also inhibits the activation of S6 kinase 1 which phosphorylates the serine 67 residue of PDCD4 and leads to the phosphorylation of serine 71 and serine 76 in the ß-TRCP binding sequence. This phosphorylation sequence causes the protein to be degraded in the ubiquitin-proteasome system. EGF activates S6 kinase 1 via the PI3K-Akt-mTOR signaling pathway and stimulates the degradation of PDCD4. EGF also suppresses PDCD4 mRNA levels. As the mTOR inhibitor rapamycin up-regulated PDCD4 mRNA levels, the PI3K-Akt-mTOR signaling pathway may control the transcription of the PDCD4 gene as well as the degradation of the protein. TPA also inhibited the TGF-ß1-induced apoptosis of Huh7 cells, stimulating the degradation of the PDCD4-protein. Analyses using PDCD4 mutants with changes of serines 67, 71 and 76 to alanine revealed that the phosphorylation of serine 67 is not essential for the TPA-induced suppression of the protein. The mitogens could not suppress the PDCD4-mutant proteins with changes of serine 71 and/or serine 76 to alanine, however, indicating that phosphorylations at these residues are necessary for the proteasome-mediated degradation of PDCD4. The phosphor-mimic S71/D and S76/D mutants were able to be degraded in the ubiquitin-proteasome system unlike the mutants with changes of serine to alanine. The expression of S71/D mutant was suppressed with EGF but that of S76/D mutant was not indicating that at least partly the phosphorylation of both sites was mediated by different enzymes.

The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells.

Vitamin K (VK) has diverse protective effects against osteoporosis, atherosclerosis and carcinogenesis. We recently reported that menatetrenone, a VK2 analogue, suppressed nuclear factor (NF)-κB activation in human hepatoma cells. Although NF-κB is regulated by isoforms of protein kinase C (PKC), the involvement of PKCs in VK2-mediated NF-κB inhibition remains unknown. Therefore, the effects of VK2 on the activation and the kinase activity of each PKC isoform were investigated. The human hepatoma Huh7 cells were treated with PKC isoform-specific inhibitors and/or siRNAs against each PKC isoform with or without 12-O-tetradecanoylphorbol-13-acetate (TPA). VK2 inhibited the TPA-induced NF-κB activation in Huh7 cells. NF-κB activity was inhibited by the pan-PKC inhibitor Ro-31-8425, but not by the PKCα-specific inhibitor Gö6976. The knockdown of individual PKC isoforms including PKCα, δ and ɛ showed only marginal effects on the NF-κB activity. However, the knockdown of both PKCδ and PKCɛ, together with treatment with a PKCα-specific inhibitor, depressed the NF-κB activity. VK2 suppressed the PKCα kinase activity and the phosphorylation of PKCɛ after TPA treatment, but neither the activation nor the enzyme activity of PKCδ was affected. The knockdown of PKCɛ abolished the TPA-induced phosphorylation of PKD1, and the effects of PKD1 knockdown on NF-κB activation were similar to those of PKCɛ knockdown. Collectively, all of the PKCs, including α, δ and ɛ, and PKD1 are involved in the TPA-mediated activation of NF-κB. VK2 inhibited the NF-κB activation through the inhibition of PKCα and ɛ kinase activities, as well as subsequent inhibition of PKD1 activation.

Regulation of tumor suppressor PDCD4 by novel protein kinase C isoforms.

Transforming growth factor-beta1 (TGF-beta1) induces apoptosis in normal hepatocytes and hepatoma cells. PDCD4 is involved in TGF-beta1-induced apoptosis via the Smad pathway. The tumor promoter 12-O-tetradecanoylphorbor-13-acetate (TPA), a protein kinase C stimulator, inhibits TGF-beta1-induced apoptosis. However, the mechanisms of TPA action on PDCD4 expression remain to be elucidated. Therefore. the regulatory mechanism of PDCD4 expression by PKC was investigated. The treatment of the human hepatoma cell line, Huh7 with TPA suppressed PDCD4 protein expression and TGF-beta1 failed to increase the PDCD4 protein expression. PKC inhibitors Ro-31-8425 or bisindolylmaleimide-1-hydrocholoride (pan-PKC inhibitors) and rottlerin (PKCdelta inhibitor), but not Go6976 (PKCalpha inhibitor), enhanced the induction of PDCD4 protein by TGF-beta1. Furthermore, siRNA-mediated knockdown of PKCdelta and epsilon, but not PKCalpha, augmented the TGF-beta1-stimulated PDCD4 protein expression. However, TPA or pan-PKC inhibitor did not alter the PDCD4 mRNA expression either under basal- and TGF-beta1-treated conditions. The down-regulation of PDCD4 by TPA was restored by treatment with the proteasome inhibitor MG132. These data suggest that two isoforms of PKCs are involved in the regulation of the PDCD4 protein expression related to the proteasomal degradation pathway.