Regulation of the expression of prostate apoptosis response protein 4 (Par-4) in rat granulosa cells

The par-4 gene, directs the expression of a protein in the rat ventral prostate after apoptotic stimuli but not growth stimulatory, growth arresting or necrotic signals. Since Par-4 expression appears to be ubiquitous we investigated the possibility of Par-4 having a role in the rat ovary granulosa cells apoptotic death. Par-4 mRNA was detected by RT-PCR with oligonucleotides designed to prime Par-4 leucine zipper in the ovaries of 12 day old rats and reached the higher levels in 24 days old rats. In situ hybridization analysis revealed that Par-4 expression is restricted to granulosa cells. PMSG priming of 24 day old rats for 2 days greatly reduced Par-4 expression in granulosa cells as determined by in situ hybridization, RT-PCR of mRNA and protein immunodetection with Western blot. Granulosa cells placed in serum-fee culture, exhibited increased levels of Par-4 mRNA and protein, in good correlation with the degree of apoptosis. The culture-induced increases in Par-4 are significantly prevented by FSH. Transient transfection of granulosa cells with Par-4 leucine zipper domain that functions as a dominant-negative regulator of Par-4 activity resulted in lower rates of apoptosis while overexpression of the full length Par-4 counteracted FSH effects on apoptosis. Par-4 association with PKCzeta which is supposed to inhibit this kinase mediated antiapoptotic way is also prevented by FSH and, FSH antiapoptotic effects are counteracted by a PKCzeta specific inhibitor. These findings indicate that FSH by suppressing Par-4 expression in the ovary activates PKCzeta-dependent antiapoptotic pathway and suggest that Par-4 is part of the mechanism underlying granulosa cells apoptotic demise.     

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.     

Mammalian melanocytes do not use phenylalanine for melanin synthesis

Hamster melanoma cells (RPMI 3460) were examined for their ability to utilize phenylalanine for melanin biosynthesis. There was a small but significant incorporation of L-[1-1414C] phenylalanine into hot acid-insoluble cellular material in the presence of cycloheximide. However, this radioactivity was removable from the acid-insoluble fraction by pronase digestion. A similar percentage of L-[U-14C] leucine incorporation was likewise resistant to cycloheximide inhibition. Residual protein synthesis is apparently responsible for the incorporation of both amino acids. Cycloheximide did not inhibit melanin synthesis. These results suggest that mammalian melanocytes do not use phenylalanine for melanin synthesis. Phenylalanine is not incorporated directly into melanin, nor do the cells appear to convert it to tyrosine via a phenylalanine hydroxylase.     

Phosphorylation of Par-4 by protein kinase A is critical for apoptosis

Despite distinct dissimilarities, diverse cancers express several common protumorigenic traits. We present here evidence that the proapoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated protein kinase A (PKA) activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC (selective for apoptosis induction in cancer cells) domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide-, or small interfering RNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA- and phosphorylated T155-dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a procancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.     

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.     

Crosstalk between PKCzeta and the IL4/Stat6 pathway during T-cell-mediated hepatitis

PKCzeta is required for nuclear factor kappa-B (NF-kappaB) activation in several cell systems. NF-kappaB is a suppressor of liver apoptosis during development and in concanavalin A (ConA)-induced T-cell-mediated hepatitis. Here we show that PKCzeta-/- mice display inhibited ConA-induced NF-kappaB activation and reduced damage in liver. As the IL-4/Stat6 pathway is necessary for ConA-induced hepatitis, we addressed here the potential role of PKCzeta in this cascade. Interestingly, the loss of PKCzeta severely attenuated serum IL-5 and liver eotaxin-1 levels, two critical mediators of liver damage. Stat6 tyrosine phosphorylation and Jak1 activation were ablated in the liver of ConA-injected PKCzeta-/- mice and in IL-4-stimulated PKCzeta-/- fibroblasts. PKCzeta interacts with and phosphorylates Jak1 and PKCzeta activity is required for Jak1 function. In contrast, Par-4-/- mice have increased sensitivity to ConA-induced liver damage and IL-4 signaling. This unveils a novel and critical involvement of PKCzeta in the IL-4/Stat6 signaling pathway in vitro and in vivo.     

Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) zeta to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCzeta.PAR-4 complex. Ceramide and its analogs activated PKCzeta prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCzeta and glycogen synthase kinase-3beta and emergence of a PAR-4-to-phosphorylated PKCzeta fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCzeta increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCzeta counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-kappaB unless PAR-4-dependent inhibition of PKCzeta suppresses NF-kappaB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCzeta. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3beta and NF-kappaB.     

Apoptosis Mediated by a Novel Leucine Zipper Protein Par-4

The prostate apoptosis response-4 (par-4) gene was isolated in a differential screen for immediate-early genes that are up-regulated during apoptosis of prostate cancer cells. Unlike most other immediate-early genes, par-4 is exclusively induced during apoptosis. The expression or induction of par-4 is not restricted to prostatic cells. The par-4 gene is widely expressed in diverse normal tissues and cell types and conserved during evolution. Par-4 protein contains a leucine zipper domain that is essential for sensitization of cells to apoptosis. Functional studies indicate that par-4 expression is necessary to induce apoptosis. Par-4 protein may induce apoptosis by a p53-independent pathway that involves cytoplasmic inactivation of atypical protein kinase C isoforms resulting in down-regulation of MAP kinase activity and an up-regulation of p38 kinase activity. However, Par-4 is detected in the cytoplasm and in the nucleus, suggesting both cytoplasmic and nuclear roles for the pro-apoptotic protein. Interestingly, Par-4 is predicted to contain a death domain homologous to that of Fas or TRADD, and may therefore trigger a death cascade analogous to that of the death domain proteins. Par-4-dependent apoptosis is abrogated by Bcl-2 and by caspase inhibitors. Identification of the components of the p53-independent apoptosis pathway induced by Par-4 may help to further elucidate the mechanism of Par-4 action. Moreover, in view of the pro-apoptotic function of Par-4, its role in diseases, such as cancer and neurogenerative disorders, whose pathophysiology involves apoptotic cell death needs further investigation.     

EphrinB1 controls cell-cell junctions through the Par polarity complex

A body of evidence is emerging that shows a requirement for ephrin ligands in the proper migration of cells, and the formation of cell and tissue boundaries. These processes are dependent on the cell-cell adhesion system, which plays a crucial role in normal morphogenetic processes during development, as well as in invasion and metastasis. Although ephrinB ligands are bi-directional signalling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. Here, we present evidence that ephrinB1 associates with the Par polarity complex protein Par-6 (a scaffold protein required for establishing tight junctions) and can compete with the small GTPase Cdc42 for association with Par-6. This competition causes inactivation of the Par complex, resulting in the loss of tight junctions. Moreover, the interaction between ephrinB1 and Par-6 is disrupted by tyrosine phosphorylation of the intracellular domain of ephrinB1. Thus, we have identified a mechanism by which ephrinB1 signalling regulates cell-cell junctions in epithelial cells, and this may influence how we devise therapeutic interventions regarding these molecules in metastatic disease.     

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

Par-4 links dopamine signaling and depression

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4DeltaLZ. Primary neurons from Par-4DeltaLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4DeltaLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.     

Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system

Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.     

mGPDH Deficiency leads to melanoma metastasis via induced NRF2

Oxidative stress critically influences carcinogenesis and the progression of melanoma, and aggressive malignant melanoma activity is due to its high metastatic ability. Some findings in several cancer cell lines have indicated that mGPDH, a component of the mitochondrial respiratory chain, also modulates oxidative stress. However, the role of mGPDH in melanoma remains elusive. Here, we report that the mGPDH protein level is decreased in human skin melanoma compared to normal skin and decreased in metastatic melanoma compared to primary melanoma. Our in vivo and in vitro experiments indicated that mGPDH depletion accelerated melanoma migration and invasion without affecting proliferation or apoptosis. Mechanistically, we found elevated NRF2 protein levels in human skin melanoma and mGPDH-knockout (ko) metastatic xenografts in the lungs of nude mice. Moreover, in A375 melanoma cells, the loss of mGPDH-induced NRF2 expression but did not affect NRF2 protein degradation. Additionally, melanoma metastasis induced by the loss of mGPDH was rescued by the further down-regulation of NRF2 in vivo and in vitro. Consistently, mGPDH overexpression (oe) depressed NRF2 expression and attenuated the malignant properties of melanoma cells. In conclusion, our findings suggest that mGPDH suppresses melanoma metastasis by inhibiting NRF2 and downstream oxidative signals, highlighting the therapeutic potential of mGPDH for melanoma treatment.     

Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.     

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.