Non-Tumor CCAAT/Enhancer-Binding Protein Delta Potentiates Tumor Cell Extravasation and Pancreatic Cancer Metastasis Formation.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a transcription factor involved in apoptosis and proliferation, which is downregulated in pancreatic ductal adenocarcinoma (PDAC) cells. Loss of nuclear C/EBPδ in PDAC cells is associated with decreased patient survival and pro-tumorigenic properties in vitro. Interestingly however, next to C/EBPδ expression in tumor cells, C/EBPδ is also expressed by cells constituting the tumor microenvironment and by cells comprising the organs and parenchyma. However, the functional relevance of systemic C/EBPδ in carcinogenesis remains elusive. Here, we consequently assessed the potential importance of C/EBPδ in somatic tissues by utilizing an orthotopic pancreatic cancer model. In doing so, we show that genetic ablation of C/EBPδ does not significantly affect primary tumor growth but has a strong impact on metastases; wildtype mice developed metastases at multiple sites, whilst this was not the case in C/EBPδ-/- mice. In line with reduced metastasis formation in C/EBPδ-/- mice, C/EBPδ-deficiency also limited tumor cell dissemination in a specific extravasation model. Tumor cell extravasation was dependent on the platelet-activating factor receptor (PAFR) as a PAFR antagonist inhibited tumor cell extravasation in wildtype mice but not in C/EBPδ-/- mice. Overall, we show that systemic C/EBPδ facilitates pancreatic cancer metastasis, and we suggest this is due to C/EBPδ-PAFR-dependent tumor cell extravasation.

Constitutive activation of p46JNK2 is indispensable for C/EBPδ induction in the initial stage of adipogenic differentiation.

Adipogenic differentiation plays a vital role in energy homeostasis and endocrine system. Several transcription factors, including peroxisome proliferator-activated receptor gamma 2 and CCAAT-enhancer-binding protein (C/EBP) α, ß, and δ, are important for the process, whereas the stage-specific intracellular signal transduction regulating the onset of adipogenesis remains enigmatic. Here, we explored the functional role of c-jun N-terminal kinases (JNKs) in adipogenic differentiation using in vitro differentiation models of 3T3-L1 cells and primary adipo-progenitor cells. JNK inactivation with either a pharmacological inhibitor or JNK2-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the down-regulation of Adiponectin, fatty acid protein 4 (Fabp4), Pparg2, and C/ebpa expressions. Conversely, increased adipogenesis was observed by the inducible overexpression of p46JNK2 (JNK2-1), whereas it was not observed by that of p54JNK2 (JNK2-2), indicating a distinct role of p46JNK2. The essential role of JNK appears restricted to the early stage of adipogenic differentiation, as JNK inhibition in the later stages did not influence adipogenesis. Indeed, JNK phosphorylation was significantly induced at the onset of adipogenic differentiation. As for the transcription factors involved in early adipogenesis, JNK inactivation significantly inhibited the induction of C/ebpd, but not C/ebpb, during the initial stage of adipogenic differentiation. JNK activation increased C/ebpd mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the C/ebpd gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes.

Stratified control of IGF-I expression by hypoxia and stress hormones in osteoblasts.

Bone cells respond to the integrated effects of local and systemic regulation. Here we show that hypoxia and the stress hormones PGE2 and glucocorticoid interact in complex ways in osteoblasts, converging on insulin like growth factor I (IGF-I) expression. Whereas hypoxia alone rapidly increased transcription factor HIF activity, it suppressed DNA synthesis, had no significant effects on protein synthesis or alkaline phosphatase activity, and drove discrete changes in a panel of osteoblast mRNAs. Notably, hypoxia increased expression of the acute phase response transcription factor C/EBPδ which can induce IGF-I in response to PGE2, but conversely prevented the stimulatory effect of PGE2 on IGF-I mRNA. However, unlike its effect on C/EBPδ, hypoxia suppressed expression of the obligate osteoblast transcription factor Runx2, which can activate an upstream response element in the IGF-I gene promoter. Hypoxic inhibition of IGF-I and Runx2 were enforced by glucocorticoid, and continued with prolonged exposure. Our studies thus reveal that IGF-I expression is stratified by two critical transcriptional elements in osteoblasts, which are resolved by the individual and combined effects of hypoxic stress and stress hormones. In so doing, hypoxia suppresses Runx2, limits the enhancing influence of PGE2, and interacts with glucocorticoid to reduce IGF-I expression by osteoblasts.

ß-Arrestin signal complex plays a critical role in adipose differentiation.

ß-Arrestins were identified as scaffold-proteins that have the capacity to desensitize G protein-coupled receptors. However, it has been found that ß-arrestins activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized for receptor tyrosine kinase. The aim of the present study was to validate the ß-arrestin-dependent signaling mechanism(s) responsible for regulation of adipogenesis. Two signal models were selected, ghrelin and insulin, based on its ß-arrestin-associated Akt activity. Herein, we found that ß-arrestin 1 and 2 were essential molecules for adipocyte differentiation. More specifically, the role of these scaffolding proteins was demonstrated by depletion of ß-arrestin 1 and 2 during ghrelin-induced adipogenesis in 3T3-L1 cells, which decreased the adipocyte differentiation and the expression levels of master regulators of early, the CCAAT/enhancer-binding protein ß (C/EBPß) and the CCAAT/enhancer-binding protein δ (C/EBPδ), and terminal, the peroxisome proliferator-activated receptor (PPARγ) and the CCAAT/enhancer-binding protein α (C/EBPα), adipogenesis. Accordingly ghrelin-induced Akt activity and its downstream targets, the mammalian target of rapamycin complex 1 (mTORC1) and the ribosomal protein S6 kinase beta-1 (S6K1), were inhibited by ß-arrestin 1 and 2 siRNAs. By contrast, assays performed during insulin-activated adipogenesis showed an intensifying effect on the adipocyte differentiation as well as on the expression of C/EBPß, C/EBPδ, PPARγ and C/EBPα. The increase in insulin-induced adipogenesis by ß-arrestin knock-down was concomitant to a decrease in the insulin receptor susbtrate-1 (IRS-1) serine phosphorylation, proving the loss of the negative feedback loop on IRS-1/phosphoinositide 3-kinase (PI3K)/Akt. Therefore, ß-arrestins control the extent and intensity of the lipogenic and adipogenic factors associated to Akt signaling, although the mechanistic and functional principles that underlie the connection between signaling and ß-arrestins are specifically associated to each receptor type.

Artemisinic acid is a regulator of adipocyte differentiation and C/EBP δ expression.

Adipocyte dysfunction is associated with the development of obesity. In this study, artemisinic acid, which was isolated from Artemisia annua L., inhibited adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) and its mechanism of action was determined. The mRNA levels of peroxidase proliferation-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, late adipogenic factors, were reduced by artemisinic acid. Moreover, the mRNA levels of the PPAR γ target genes lipoprotein lipase, CD36, adipocyte protein, and liver X receptor were down-regulated by artemisinic acid. Artemisinic acid reduced expression of the C/EBP δ gene without impacting C/EBP ß. In addition, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that reduced expression of the C/EBP δ gene was mediated by inhibiting Jun N-terminal kinase (JNK). Additionally, artemisinic acid also reduced the expression of the adipogenesis-associated genes glucose transporter-4 and vascular endothelial growth factor. In addition to the interference of artemisinic acid with adipogenesis, artemisinic acid significantly attenuated tumor necrosis factor-α-induced secretion of interleukin-6 by undifferentiated hAMSCs, thus influencing insulin resistance and the inflammatory state characterizing obesity. Taken together, these findings indicate that inhibiting adipogenic differentiation of hAMSCs by artemisinic acid occurs primarily through reduced expression of C/EBP δ, which is mediated by the inhibition of JNK and suggest that aremisinic acid may be used as a complementary treatment option for obesity associated with metabolic syndrome.

Acute heat stress and thermal acclimation induce CCAAT/enhancer-binding protein delta in the goby Gillichthys mirabilis.

Members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors have regulatory control over numerous processes related to cell fate determination, including differentiation, proliferation, cell cycle arrest and apoptosis. In mammals, abnormalities in the expression of some isoforms of C/EBPs are pathogenic and are implicated as being involved in myeloid leukemia and breast cancers. Next to nothing is known about their regulation, function or stress-responsiveness in poikilotherms. Here, both acute heat stress and thermal acclimation were demonstrated to induce the expression of one isoform, C/EBP-δ, in the liver, white muscle and gill of the eurythermal estuarine goby, Gillichthys mirabilis. The established role of C/EBP-δ in causing cell cycle arrest and/or promoting apoptosis in other vertebrates suggests that the heat-inducibility of this protein in poikilotherms may be part of the conserved cellular stress response with the hypothesized role of causing temporary cessation of cell growth and/or programmed cell death during bouts of environmental stress. The observed regulation of c/ebp-δ during hyperthermia represents a novel, heat-inducible signaling pathway in fishes.

Transcriptional up-regulation of SOD1 by CEBPD: a potential target for cisplatin resistant human urothelial carcinoma cells.

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of this and other cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPdelta, NF-IL6beta) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduced cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter transactivation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as anti-angiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

Myc interacts with Max and Miz1 to repress C/EBPdelta promoter activity and gene expression.

BACKGROUND: "Loss of function" alterations in CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPdelta gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPdelta exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing. We previously reported that C/EBPdelta expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPalpha, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPdelta expression. RESULTS: Myc represses C/EBPdelta promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPdelta promoter in proliferating cells, when C/EBPdelta expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPdelta promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPdelta promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPdelta promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPdelta promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPdelta expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPdelta promoter activity. CONCLUSION: Myc represses C/EBPdelta expression by associating with the C/EBPdelta proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPdelta promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPdelta, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.

The cell growth inhibitory transcription factor C/EBPdelta is expressed in human meningiomas in association with low histological grade and proliferation index.

CCAAT/enhancer binding protein (C/EBP) delta is a transcription factor which has been demonstrated to mediate the growth arrest of mammary and prostate cancer cell lines. It is induced by several stimuli including inflammatory cytokines. In this study, C/EBPdelta immunohistochemical expression was assessed in 49 meningiomas of different histotype and grade and correlated with a variety of clinico-pathological data and with the overall and recurrence-free survival of the patients. Positive staining was observed in the nuclei of neoplastic cells in 22 out of the 49 cases analyzed. C/EBPdelta expression was significantly associated with a low histological grade and proliferation index, reflected by low Ki-67 labeling index (LI) and mitotic activity, and with the presence of intra-tumoral inflammatory infiltrate and the absence of necrosis. In addition, the absence of C/EBPdelta was significantly correlated with a shorter disease-free interval. Our findings suggest that C/EBPdelta expression may prevent the development of recurrences by inhibition of neoplastic growth in meningiomas. If further studies confirm its induction by inflammatory mediators, this might be exploited in novel therapies to prevent recurrences in meningiomas.

CELF-mediated alternative splicing is required for cardiac function during early, but not later, postnatal life.

During the transition from juvenile to adult life, the heart undergoes programmed remodeling at the levels of transcription and alternative splicing. Members of the CUG-BP and ETR-3-like factor (CELF) family have been implicated in driving developmental transitions in alternative splicing of cardiac transcripts during maturation of the heart. Here, we investigated the timing of the requirement for CELF activity in the postnatal heart using a previously described transgenic mouse model (MHC-CELFDelta). In MHC-CELFDelta mice, nuclear CELF activity has been disrupted specifically in the heart by cardiac-specific expression of a dominant negative CELF protein. Longitudinal analyses of two lines of MHC-CELFDelta mice with differing levels of dominant negative protein expression demonstrate that CELF splicing activity is required for healthy cardiac function during juvenile, but not adult, life. Cardiac function, chamber dilation, and heart size all recover with age in the mild line of MHC-CELFDelta mice without a loss of dominant negative protein expression or change in expression of endogenous CELF proteins or known CELF antagonists. This is the first example of a mouse model with genetically induced cardiomyopathy that spontaneously recovers without intervention. Our results suggest that CELF proteins are key players in the integrated gene expression program involved in postnatal cardiac remodeling and maturation.

Ultraviolet radiation (UVR) activates p38 MAP kinase and induces post-transcriptional stabilization of the C/EBPdelta mRNA in G0 growth arrested mammary epithelial cells.

The G(0) growth arrest (quiescent) state is highly conserved in evolution to promote survival under adverse environmental conditions. To maintain viability, G(0) growth arrested cells limit gene expression to essential growth control and pro-survival genes. CCAAT enhancer binding protein delta (C/EBPdelta), a member of the C/EBP family of nuclear proteins, is highly expressed in G(0) growth arrested mammary epithelial cells (MECs). Although C/EBPdelta gene transcription is elevated during G(0) growth arrest, C/EBPdelta mRNA and protein are relatively short lived, suggesting tight control of the cellular C/EBPdelta content in unstressed, quiescent cells. Treatment of G(0) growth arrested MECs with ultraviolet radiation (UVR) dramatically increases the C/EBPdelta mRNA half-life (approximately 4-fold) and protein content (approximately 3-fold). The mRNA stabilizing effects of UVR treatment are mediated by the C/EBPdelta mRNA 3'untranslated region, which contains an AU rich element. UVR increased p38 MAP kinase (MAPK) activation and SB203580, a p38 MAPK inhibitor, blocked UVR-induced C/EBPdelta mRNA stabilization. UVR increased the nuclear to cytoplasmic translocation of HuR, an ARE-binding protein that functions in mRNA stabilization. Finally, HuR siRNA treatment blocked UVR-induced stabilization of the C/EBPdelta and C/EBPbeta mRNAs but had no effect on C/EBPzeta (CHOP) mRNA stability. In summary, G(0) growth arrested MECs respond to UVR treatment by activating p38 MAPK, increasing HuR translocation and HuR/C/EBPdelta mRNA binding and stabilizing the C/EBPdelta mRNA. These results identify post-transcriptional stabilization of the C/EBPdelta mRNA as a mechanism to increase C/EBPdelta levels in the stress response of quiescent cells to UVR.

Gene expression profiling identifies C/EBPdelta as a candidate regulator of endotoxin-induced disseminated intravascular coagulation.

RATIONALE: A runaway inflammatory response to systemic infection or severe trauma is characterized by the activation of a diversity of pathways, ultimately resulting in the development of disseminated intravascular coagulation (DIC) and multiorgan failure. OBJECTIVES: Despite increased fundamental knowledge of the pathogenesis of DIC, the exact molecular mechanisms remain elusive. We aimed therefore to improve our understanding of the molecular pathways underlying endotoxin-induced DIC. METHODS: We performed large-scale gene expression profiling in the liver of mice during the onset of endotoxin-induced DIC. The relevance of an identified candidate gene involved in endotoxin-induced DIC was subsequently assessed in the generalized Shwartzman reaction. MEASUREMENTS AND MAIN RESULTS: Approximately 5% of over 20,000 genes were differentially regulated. In addition to well-established sepsis-associated genes, such as macrophage inflammatory protein 1, plasminogen activator inhibitor 1, CD14, and A20, we identified several novel candidates for inflammatory disease of which the transcription factor C/EBPdelta (CAAT/enhancer binding protein delta) was studied further. Induction of DIC in C/EBPdelta-deficient mice decreased endotoxin-induced systemic inflammation as compared with wild-type mice, as evident from decreased plasma levels of tumor necrosis factor-alpha and IL-6. In addition, C/EBPdelta deficiency partly protected against DIC-induced mortality. Interestingly, C/EBPdelta deficiency seemed mainly protective by improving renal function. This latter notion was confirmed in an experimental model of renal ischemia/reperfusion injury in which C/EBPdelta deficiency reduced ischemia/reperfusion-induced creatinine and urea levels. CONCLUSIONS: Our results endorse the usefulness of gene expression profiling in identifying novel mediators of DIC by showing that C/EBPdelta regulates specific pathologic features of this endotoxin-induced syndrome.

STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.