Interleukin-11 (IL11) inhibits myogenic differentiation of C2C12 cells through activation of extracellular signal-regulated kinase (ERK).

Cancer-associated cachexia (CAC) is a multifactorial wasting syndrome characterized by loss of skeletal muscle. Interleukin-11 (IL11), one of the IL6 family cytokines, is highly expressed in various types of cancer including cancers frequently associated with cachexia. However, the impact of IL11 on muscle metabolism remains to be determined. Since one of the mechanisms of muscle wasting in cachexia is defective muscle regeneration due to impaired myogenic differentiation, we examined the effect of IL11 on the differentiation of C2C12 mouse myoblasts. Treatment of C2C12 cells with recombinant mouse IL11 resulted in decreased myotube formation. In addition, IL11 treatment reduced the protein and mRNA levels of myosin heavy chain (MHC), a marker of myogenic differentiation. Moreover, the levels of myogenic regulatory factors including myogenin and Mrf4 were significantly reduced by IL11 treatment. IL11 treatment increased the number of BrdU-positive cells and the level of phosphorylated retinoblastoma (Rb) protein, while the levels of p21Waf1 and p27Kip1 were reduced by IL11 treatment in differentiating C2C12 cells, suggesting that IL11 interferes with cell cycle exit during the early stages of myogenic differentiation. Consistent with this, IL11 treatment at the late stage of differentiation did not affect myotube formation and MHC expression. IL11 treatment resulted in an activation of ERK, STAT3, and AKT in differentiating C2C12 cells. However, only ERK inhibitors including PD98059 and U0126 were able to ameliorate the suppressive effect of IL11 on the expression of MHC and myogenin. Additionally, pretreatment with PD98059 and U0126 resulted in improved myotube formation and reduced BrdU staining in IL11-treated cells. Together, our results suggest that IL11 inhibits myogenic differentiation through delayed cell cycle exit in an ERK-dependent manner. To our knowledge, this study is the first to demonstrate an inhibitory role of IL11 in myogenic differentiation and identifies the previously unrecognized role of IL11 as a possible mediator of CAC.

Effects of Genistein and Exercise Training on Brain Damage Induced by a High-Fat High-Sucrose Diet in Female C57BL/6 Mice.

In recent decades, a shift in the nutritional landscape to the Western-style diet has led to an unprecedented rise in the prevalence of obesity and neurodegenerative diseases. Consumption of a healthy diet and engaging in regular physical activity represents safe and affordable approaches known to mitigate the adverse consequences of the Western diet. We examined whether genistein treatment, exercise training, and a combination treatment (genistein and exercise training) mitigated the effects of a Western diet-induced by high-fat, high-sugar (HFHS) in brain of female mice. HFHS increased the amyloid-beta (Aß) load and phosphorylation of tau, apoptosis, and decreased brain-derived neurotrophic factor (BDNF) levels. Exercise training and genistein each afforded modest protection on Aß accumulation and apoptosis, and both increased BDNF. The greatest neuroprotective effect occurred with combination treatment. BDNF and all markers of Aß accumulation, phosphorylation of tau, and apoptosis were improved with combined treatment. In a separate series of experiments, PC12 cells were exposed to high glucose (HG) and palmitate (PA) to determine cell viability with genistein as well as in the presence of tamoxifen, an estrogen receptor antagonist, to assess a mechanism of action of genistein on cell apoptosis. Genistein prevented the neurotoxic effects of HG and PA in PC12 cells and tamoxifen blocked the beneficial effects of genistein on apoptosis. Our results indicate the beneficial effects of genistein and exercise training on HFHS-induced brain damage. The benefits of genistein may occur via estrogen receptor-mediated pathways.

Aspirin ameliorates the long-term adverse effects of doxorubicin through suppression of cellular senescence.

A number of childhood cancer survivors develop adverse, late onset side effects of earlier cancer treatments, known as the late effects of cancer therapy. As the number of survivors continues to increase, this growing population is at increased risk for a number of health-related problems. In the present study, we have examined the effect of aspirin on the late effects of chemotherapy by treating juvenile mice with doxorubicin (DOX). This novel mouse model produced various long-term adverse effects, some of which resemble premature aging phenotypes. DOX also resulted in the tissue accumulation of senescent cells and up-regulation of cyclooxygenase-2 (COX2) expression. However, treatment with aspirin following juvenile exposure to DOX improved body weight gain, ameliorated the long-term adverse effects, and reduced the levels of senescence markers. Moreover, aspirin reduced p53 and p21 accumulation in DOX-treated human and mouse fibroblasts. However, the suppressive effect of aspirin on DOX-induced p53 accumulation was significantly decreased in COX2 knockout mouse embryonic fibroblasts. Additionally, treatment of senescent fibroblasts with aspirin or celecoxib, a COX2 specific inhibitor, reduced cell viability and decreased the levels of Bcl-xL protein. Taken together, these studies suggest that aspirin may be able to reduce the late effects of chemotherapy through the suppression of cellular senescence.

Jagged-2 (JAG2) enhances tumorigenicity and chemoresistance of colorectal cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality. Recent studies have stated that NOTCH signaling plays an important role in the development and progression of CRC. However, the role of Jagged-2 (JAG2), one of the NOTCH ligands, has not been delineated in colorectal tumorigenesis and drug resistance. In the present study, we have examined the impact of targeting JAG2 on CRC cells. Among all the members of NOTCH ligands, only the expression of JAG2 was found up-regulated in the intestinal tumors of Apc Min /+ mice as compared to the nearby normal mucosa. JAG2 expression was also observed in a panel of human CRC cell lines. Pharmacological inhibition or genetic knockdown of ß-catenin in CRC cell lines suppressed JAG2 expression, suggesting Wnt/ß-catenin regulation of JAG2 expression. In addition, deletion of Apc gene in the intestinal cells of Apc conditional knockout mice resulted in up-regulation of JAG2 expression. Modulation of JAG2 expression significantly affected in vivo tumorigenicity of CRC cell lines. Moreover, knockdown of JAG2 sensitized CRC cells to chemotherapeutic agents, while ectopic expression of JAG2 increased chemoresistance of the CRC cells. Significant down-regulation of p21 was observed in JAG2-knockdown cells. Forced expression of p21 rescued the sensitivity of JAG2-knockdown cells to doxorubicin. In addition, the chemosensitivity of p21-null cells was not affected by JAG2 knockdown. These results suggest that JAG2 modulates the sensitivity of CRC cells to chemotherapeutic agents through p21. Our study identifies JAG2 as a novel target for therapeutic intervention of CRC.

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice.

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated ß-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

Lentivirus-mediated somatic recombination and development of a novel mouse model for sporadic colorectal cancer.

In this study, we have developed a novel mouse model for sporadic colorectal cancer (CRC) by utilizing APC conditional knockout (Apc(CKO) ) mouse and lentivirus encoding Cre recombinase and a reporter gene (EGFP or LacZ). Lentiviral transduction of colonic crypt stem cells allowed for the long-term expression of reporter gene as well as excision of floxed Apc alleles, which resulted in tumor development. Tumors represented adenoma stages along with the nuclear accumulation of ß-catenin. Loss of E-cadherin at the cellular junctions and strong expression of Vimentin suggested the sign of active epithelial-mesenchymal transition. Moreover, nuclear staining of Ki67 inside epithelial cells of aberrant crypts demonstrated their higher proliferative nature. Erratic downstream signaling of activated Wnt/ß-catenin, AKT/mTOR, and Notch pathways provided strong evidence towards the higher proliferative index of epithelial cells inside the aberrant crypts. These results do recapitulate the findings of previous APC mutant mouse models. Our model represents sporadic CRC more precisely as (i) tumors result from somatic mutations but not from germline; (ii) tumors develop in colon not in small intestine; (iii) few tumors develop at the distal end of colons. Additionally, our model allows for the long-term expression of the gene(s), which get integrated into the host cell genome and provides an ability to track the tumor growth. © 2016 Wiley Periodicals, Inc.

COX-2 inhibitor NS-398 suppresses doxorubicin-induced p53 accumulation through inhibition of ROS-mediated Jnk activation.

Cyclooxygenase-2 (COX-2) is one of the isoforms of cyclooxygenase, a rate-limiting enzyme in the arachidonic acid cascade. COX-2 protein expression is highly induced by numerous factors and it has been reportedly overexpressed in various human malignancies. Although anti-tumorigenic effects of COX-2 inhibitors have been shown, several lines of evidence suggest that COX-2 inhibitors antagonize the cytotoxicity of chemotherapeutic agents. In this study, we investigated the effect of NS-398, a COX-2 inhibitor, on modulation of doxorubicin (DOX)-induced p53 accumulation. Non-selective and selective COX-2 inhibitors attenuated DOX-induced accumulation of wild type (WT) but not mutant p53. Nutlin-3α or MG132 abolished the suppressive effect of a COX-2 inhibitor on DOX-induced p53 increase. Moreover, the DOX-induced increase in p53 protein levels was reduced in COX-2 knockout (KO) mouse embryonic fibroblasts (MEFs) compared to those in WT or COX-1 KO MEFs. DOX-induced accumulation of p53 was attenuated by a specific inhibitor or knockdown of Jun-N-terminal kinase (Jnk). In addition, DOX-induced Jnk activation was decreased in COX-2 KO MEFs or by COX-2 inhibition, suggesting that Jnk stabilizes p53 by a mechanism that involves COX-2. Pre-treatment with a reactive oxygen species (ROS) scavenger, N-acetylcysteine, attenuated DOX-induced Jnk activation and subsequent p53 accumulation. Furthermore, the absence or inhibition of COX-2 resulted in suppression of DOX-induced increase in ROS levels. These results suggest that COX-2 activates Jnk through modulation of ROS levels, leading to accumulation of p53. Our study identifies a putative novel cross-talk between COX-2 and p53. © 2016 Wiley Periodicals, Inc.

EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells.

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, binds to four different prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of PGE2 induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and ß-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway.

Prostaglandin F2α receptor (FP) signaling regulates Bmp signaling and promotes chondrocyte differentiation.

Prostaglandins are a group of lipid signaling molecules involved in various physiological processes. In addition, prostaglandins have been implicated in the development and progression of diseases including cancer, cardiovascular disease, and arthritis. Prostaglandins exert their effects through the activation of specific G protein-coupled receptors (GPCRs). In this report, we examined the role of prostaglandin F2α receptor (FP) signaling as a regulator of chondrocyte differentiation. We found that FP expression was dramatically induced during the differentiation of chondrocytes and was up-regulated in cartilages. Forced expression of FP in ATDC5 chondrogenic cell line resulted in the increased expression of differentiation-related genes and increased synthesis of the extracellular matrix (ECM) regardless of the presence of insulin. Similarly, PGF2α treatment induced the expression of chondrogenic marker genes. In contrast, knockdown of endogenous FP expression suppressed the expression of chondrocyte marker genes and ECM synthesis. Organ culture of cartilage rudiments revealed that PGF2α induces chondrocyte hypertrophy. Additionally, FP overexpression increased the levels of Bmp-6, phospho-Smad1/5, and Bmpr1a, while knockdown of FP reduced expression of those genes. These results demonstrate that up-regulation of FP expression plays an important role in chondrocyte differentiation and modulates Bmp signaling.

The H6D variant of NAG-1/GDF15 inhibits prostate xenograft growth in vivo.

BACKGROUND: Non-steroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor-beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo. METHODS: Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type (WT) NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis. RESULTS: The H6D variant was more potent than the WT NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the WT NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the WT NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin, and IGF-1 serum levels was observed in the tumor-bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant. CONCLUSION: Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and cyclin D1 expression but likely additional mechanisms are operative.

Embryonic expression of cyclooxygenase-2 causes malformations in axial skeleton.

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor-beta superfamily, is involved in many cellular processes, such as inflammation, apoptosis/survival, and tumorigenesis. Vitamin E succinate (VES) is the succinate derivative of alpha-tocopherol and has antitumorigenic activity in a variety of cell culture and animal models. In the current study, the regulation and role of NAG-1 expression in PC-3 human prostate carcinoma cells by VES was examined. VES treatment induced growth arrest and apoptosis as well as an increase in NAG-1 protein and mRNA levels in a time- and concentration-dependent manner. VES treatment induced nuclear translocation and activation of p38 kinase. Pretreatment with p38 kinase inhibitor blocked the VES-induced increase in NAG-1 protein and mRNA levels, whereas an inhibition of protein kinase C, Akt, c-Jun NH(2)-terminal kinase, or MEK activity had no effect on VES-induced NAG-1 levels. Forced expression of constitutively active MKK6, an upstream kinase for p38, induced an increase in NAG-1 promoter activity, whereas p38 kinase inhibitor blocked MKK6-induced increase in NAG-1 promoter activity. VES treatment resulted in >3-fold increase in the half-life of NAG-1 mRNA in a p38 kinase-dependent manner and transient transfection experiment showed that VES stabilizes NAG-1 mRNA through AU-rich elements in 3'-untranslated region of NAG-1 mRNA. The inhibition of NAG-1 expression by small interfering RNA significantly blocked VES-induced poly(ADP-ribose) polymerase cleavage, suggesting that NAG-1 may play an important role in VES-induced apoptosis. These results indicate that VES-induced expression of NAG-1 mRNA/protein is regulated by transcriptional/post-transcriptional mechanism in a p38 kinase-dependent manner and NAG-1 can be chemopreventive/therapeutic target in prostate cancer.

NSAID activated gene (NAG-1), a modulator of tumorigenesis.

The NSAID activated gene (NAG-1), a member of the TGF-beta superfamily, is involved in tumor progression and development. The over-expression of NAG-1 in cancer cells results in growth arrest and increase in apoptosis, suggesting that NAG-1 has anti-tumorigenic activity. This conclusion is further supported by results of experiments with transgenic mice that ubiquitously express human NAG-1. These transgenic mice are resistant to the development of intestinal tumors following treatment with azoxymethane or by introduction of a mutant APC gene. In contrast, other data suggest a pro-tumorigenic role for NAG-1, for example, high expression of NAG-1 is frequently observed in tumors. NAG-1 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of NAG-1 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. Most notable is the increase in NAG-1 expression by the inhibitors of cyclooxygenases that prevent human colorectal cancer development. The regulation of NAG-1 is complex, but these agents act through either p53 or EGR-1 related pathways. In addition, an increase in NAG-1 is observed in inhibition of the AKT/GSK-3beta pathway, suggesting NAG-1 alters cell survival. Thus, NAG-1 expression is regulated by tumor suppressor pathways and appears to modulate tumor progression.

Prostate derived factor in human prostate cancer cells: gene induction by vitamin D via a p53-dependent mechanism and inhibition of prostate cancer cell growth.

The secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1,25D) has been shown to regulate the growth and differentiation of human prostate cancer (PCa) cells, although the precise molecular mechanisms mediating these effects have not been defined. Previous studies in our laboratory demonstrated that the antiproliferative effects of 1,25D on PCa cells are mediated through the nuclear vitamin D receptor (VDR). In the present study, we performed gene profiling of LNCaP human PCa cells following 1,25D treatment and identified the antitumorigenic gene, prostate derived factor (PDF), as being highly induced by 1,25D. PDF is a member of the TGF-beta superfamily and has been implicated in a variety of functions directly related totumorigenicity including antiproliferative and pro-apoptotic effects. Gene expression studies using 1,25D analogs and a VDR antagonist demonstrate that 1,25D-mediated induction of PDF message and protein in PCa cells is dependent on VDR action. PDF is a transcriptional target of the tumor suppressor, p53. Here we show that the expression of PDF in nine PCa cell lines is dependent on functional p53. Additionally, transfection of p53-null ALVA-31 PCa cells with a p53 expression plasmid, and expression of dominant negative p53 in LNCaP PCa cells, show that the ability of VDR to induce PDF requires functional p53. Importantly, forced PDF expression in PC-3 cells results in decreased cell proliferation, soft agar cloning, and xenograft tumor size. These data demonstrate that PDF exerts antitumorigenic properties on PCa cells and its regulation by 1,25D may provide insights into the action of 1,25D in PCa.

A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L.

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

Protein kinase C-dependent regulation of NAG-1/placental bone morphogenic protein/MIC-1 expression in LNCaP prostate carcinoma cells.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor beta superfamily, is involved in cellular processes such as inflammation, apoptosis/survival, and tumorigenesis and is regulated by p53, Sp1, and Egr-1. In the current study, the regulation of NAG-1 expression in LNCaP human prostate carcinoma cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined. TPA treatment increased NAG-1 protein and mRNA levels in a time- and concentration-dependent manner as well as NF-kappa B binding/transcriptional activity in LNCaP cells. Pretreatment with protein kinase C (PKC) inhibitor blocked the TPA-induced increase in NAG-1 protein levels and NF-kappa B binding/transcriptional activity, whereas an inhibition of p38, JNK, MEK activity had no effect on TPA-induced NAG-1 levels and NF-kappa B transcriptional activity. Expression of constitutively active PKCs induced an increase in NF-kappa B transcriptional activity and NAG-1 protein levels in LNCaP cells. The expression of NF-kappa B p65 induced NAG-1 promoter activity, and chromatin immunoprecipitation assay for p65 showed that NF-kappa B binds the NAG-1 promoter in LNCaP cells. Inhibition of TPA-induced NAG-1 expression by NAG-1 short interfering RNA blocked TPA-induced apoptosis in LNCaP cells, suggesting induction of NAG-1 negatively affects LNCaP cell survival. These results demonstrate that NAG-1 expression is up-regulated by TPA in LNCaP cells through a PKC-dependent pathway involving the activation of NF-kappa B.

Diminished expression of C/EBPalpha in skin carcinomas is linked to oncogenic Ras and reexpression of C/EBPalpha in carcinoma cells inhibits proliferation.

The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is involved in mitotic growth arrest and has been implicated as a human tumor suppressor in acute myeloid leukemia. We have previously shown that C/EBPalpha is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBPalpha was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBPalpha mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPbeta levels were not dramatically changed. Reexpression of C/EBPalpha in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBPalpha expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBPalpha in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBPalpha protein expression and normal C/EBPbeta levels compared with the epidermis and all 14 carcinomas contained mutant Ras. To determine whether oncogenic Ras is involved in the down-regulation of C/EBPalpha, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBPalpha protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBPalpha protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBPalpha expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBPalpha levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBPalpha expression and the loss of C/EBPalpha expression may contribute to the development of skin SCCs.

Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBPalpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity.

CCAAT/enhancer-binding protein alpha (C/EBPalpha), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBPalpha protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBPalpha is a short-lived protein with a half-life of approximately 1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBPalpha protein. Higher molecular weight species of ubiquitinated C/EBPalpha were detected in BALB/MK2, and in vitro studies confirmed that C/EBPalpha is degraded by the proteasome in an ATP- and ubiquitin-dependent manner. GSK3 is a known C/EBPalpha kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBPalpha protein levels, (ii) increased electrophoretic mobility of C/EBPalpha, and (iii) no increase in C/EBPalpha mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBPalpha may target it for proteasomal degradation. However, a mutant C/EBPalpha containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBPalpha levels indicating the effects of LiCl on C/EBPalpha are GSK3-independent. LiCl treatment of BALB/MK2 cells inhibited C/EBPalpha degradation and produced a 6-fold increase in the half-life of C/EBPalpha protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBPalpha. These results demonstrate C/EBPalpha is degraded via a ubiquitin-dependent proteasomal pathway, and LiCl stabilizes C/EBPalpha through a GSK3-independent pathway involving direct inhibition of proteasome activity.