Erini Dermitzaki

Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase.

Recent experimental findings involve corticotropin-releasing hormone (CRH) in the cellular response to noxious stimuli and possibly apoptosis. The aim of the present work was to examine the effect of CRH on apoptosis and the Fas/Fas ligand system in an in vitro model, the PC12 rat pheochromocytoma cell line, which is widely used in the study of apoptosis and at the same time expresses the CRH/CRH receptor system. We have found the following. CRH induced Fas ligand production and apoptosis. These effects were mediated by the CRH type 1 receptor because its antagonist antalarmin blocked CRH-induced apoptosis and Fas ligand expression. CRH activated p38 mitogen-activated protein kinase, which was found to be essential for CRH-induced apoptosis and Fas ligand production. CRH also promoted a rapid and transient activation of ERK1/2, which, however, was not necessary for either CRH-induced apoptosis or Fas ligand production. Thus, CRH promotes PC12 apoptosis via the CRH type 1 receptor, which induces Fas ligand production via activation of p38.

Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2

Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin‐Releasing Factor Receptor‐2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose‐ and time‐dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)‐induced apoptosis, the pro‐apoptosis pathway activated by urocortins involved the pro‐apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti‐inflammatory effect via induction of macrophage apoptosis.

Corticotropin releasing factor receptor 1 (CRF1) and CRF2 agonists exert an anti-inflammatory effect during the early phase of inflammation suppressing LPS-induced TNF-α release from macrophages via induction of COX-2 and PGE2†

Corticotropin‐releasing factor (CRF), the principal regulator of the hypothalamus‐pituitary‐adrenal (HPA) axis, also modulates the inflammatory response directly, via its effect on mast cells and macrophages. On macrophages, it augments production of lipopolysaccharide (LPS)‐induced pro‐inflammatory cytokines. CRF and its related peptides may also act as anti‐inflammatory agents. Aim of the present work was to examine the role of macrophages on the anti‐inflammatory effects of CRF‐peptides and the mechanism involved. Thus, we examined if CRF receptor 1 (CRF1) and CRF2 agonists exert any anti‐inflammatory effect on primary mouse macrophages. We have found that: (a) CRF, Urocortin (UCN)1 and UCN2 transiently suppressed the release of Tumor Necrosis Factor‐alpha (TNF‐α) in LPS‐activated macrophages, an effect peaking at 4 h. This effect did not involve changes on TNF‐α transcription. (b) CRF peptide‐induced suppression of TNF‐α release depended on induction of COX‐2 and PGE2 synthesis. (c) Use of specific CRF1 and CRF2 antagonists suggested that this effect involved both CRF receptor types. (d) The effect of CRF‐peptides on COX‐2 was mediated via PI3K and p38MAPK. (e) Longer exposure of macrophages to CRF‐peptides resulted in induction of TNF‐α production via enhancement of its transcription. In conclusion, this is the first report suggesting that CRF1 and CRF2 agonists exert a biphasic effect on macrophages. During the early stages of the inflammatory response, they suppress TNF‐α release via induction of COX‐2/PGE2 while later on they induce TNF‐α transcription. Hence, the reported anti‐inflammatory effect of CRF‐peptides appears to involve macrophages and is confined at the early stage of inflammation. J. Cell. Physiol. 210: 774–783, 2007.

Corticotropin-Releasing Factor and the Urocortins Induce the Expression of TLR4 in Macrophages via Activation of the Transcription Factors PU.1 and AP-1

Corticotropin-releasing factor (CRF) augments LPS-induced proinflammatory cytokine production from macrophages. The aim of the present study was to determine the mechanism by which CRF and its related peptides urocortins (UCN) 1 and 2 affect LPS-induced cytokine production. We examined their role on TLR4 expression, the signal-transducing receptor of LPS. For this purpose, the murine macrophage cell line RAW 264.7 and primary murine peritoneal macrophages were used. Exposure of peritoneal macrophages and RAW 264.7 cells to CRF, UCN1, or UCN2 up-regulated TLR4 mRNA and protein levels. To study whether that effect occurred at the transcriptional level, RAW 264.7 cells were transfected with a construct containing the proximal region of the TLR4 promoter linked to the luciferase gene. CRF peptides induced activation of the TLR4 promoter, an effect abolished upon mutation of a proximal PU.1-binding consensus or upon mutation of an AP-1-binding element. Indeed, all three peptides promoted PU.1 binding to the proximal PU.1 site and increased DNA-binding activity to the AP-1 site. The effects of CRF peptides were inhibited by the CRF2 antagonist anti-sauvagine-30, but not by the CRF1 antagonist antalarmin, suggesting that CRF peptides mediated the up-regulation of TLR4 via the CRF2 receptor. Finally, CRF peptides blocked the inhibitory effect of LPS on TLR4 expression. In conclusion, our data suggest that CRF peptides play an important role on macrophage function. They augment the effect of LPS by inducing Tlr4 gene expression, through CRF2, via activation of the transcription factors PU.1 and AP-1.

The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function

Abstract.Corticotropin-releasing factor (CRF), also termed corticotropin-releasing hormone (CRH) or corticoliberin, is the major regulator of the adaptive response to internal or external stresses. An essential component of the adaptation mechanism is the adrenal gland. CRF regulates adrenal function indirectly through the central nervous system (CNS) via the hypothalamic-pituitary-adrenal (HPA) axis and via the autonomic nervous system by way of locus coeruleus (LC) in the brain stem. Accumulating evidence suggests that CRF and its related peptides also affect the adrenals directly, i.e. not through the CNS but from within the adrenal gland where they form paracrine regulatory loops. Indeed, CRF and its related peptides, the urocortins (UCNs: UCN1, UCN2 and UCN3), their receptors CRF type 1 (CRF1) and 2 (CRF2) as well as the endogenous pseudo-receptor CRF-binding protein (CRF-BP) are all expressed in adrenal cortical, medullary chromaffin and resident immune cells. The intra-adrenal CRF-based regulatory system is complex and depends on the balance between the local concentration of CRF ligands and the availability of their receptors.

Peripheral Factors in the Metabolic Syndrome The Pivotal Role of Adiponectin

Abstract:  Several recently published reports, including ours, suggest that adiponectin is a strong proinflammatory agent. Indeed, exposure of human placenta and adipose tissue to adiponectin induces the production of interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor α (TNF‐α), and prostaglandin E2 (PGE2). We have previously shown that adiponectin is a powerful inducer of proinflammatory cytokines production by macrophages. The reported anti‐inflammatory effect of adiponectin may be due to the induction of macrophage tolerance to further adiponectin exposure or to other proinflammatory stimuli including the Toll‐like receptor (TLR) 3 ligand polyI:C and the TLR4 ligand lipopolysaccharide (LPS). We now present additional data supporting the hypothesis that adiponectin is a strong proinflammatory adipokine. More specifically, we demonstrate that adiponectin induces IL‐1β and IL‐8 from THP‐1 macrophage cell line. The effect of adiponectin is not restricted to differentiated THP‐1 macrophages but it is evident at lower levels in undifferentiated THP‐1 monocytes promoting TNF‐α, IL‐6, and IL‐8 production. Thus, its high levels in the circulation of lean subjects render their macrophages resistant to several proinflammatory stimuli including its own thus acting in effect as an anti‐inflammatory agent. Lowering of its high levels, as a consequence of increased body mass index (BMI), renders macrophages sensitive to any proinflammatory insult.

Opioids transiently prevent activation of apoptotic mechanisms following short periods of serum withdrawal.

Abstract: Opioids exert a proapoptotic effect on several normal and tumoral cells. The aim of the present article was to examine the effect of opioids on the PC12 rat pheochromocytoma cell line, a model for the study of chromaffin cell apoptosis. These cells produce δ‐ and κ‐opioid agonists and their receptors. Our results were as follows: The κ‐ and δ2‐opioid receptor agonists had a rapid but transient effect on apoptosis at 3 h, whereas μ opioids did not. The effect of opioids was reversible by the opioid antagonists naloxone and nor‐binaltorphimine. The effect of opioids was protective, suppressing serum deprivation‐induced apoptosis to ∼50% of controls. The protective effect of opioids on PC12 apoptosis was measurable only under serum deprivation. The effect of opioids was remarkably reproducible and highly constant in timing, which did not appear to depend on the duration of the preceding serum deprivation. Finally, opioids prevented the elevation of the Bcl‐2 and Bak proteins following serum deprivation to the levels attained by serum supplementation. Our combined data suggest that opioids protect PC12 cells from entering a state of induced apoptosis following serum deprivation.

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

IntroductionCancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells.MethodsIn the present study we report our findings regarding the impact of CRF on breast cancer cell motility and invasiveness. For this purpose we used the MCF7 breast cancer cell line and evaluated the effect of CRF on motility and invasiveness using the wound-healing and boyden-chamber assays. In addition, we measured the effect of CRF on molecules that mediate motility by western blot, immunofluorescence, ELISA and RT-PCR.ResultsOur findings show that: 1. CRF transiently inhibited the apoptosis of MCF7 cells. 2. CRF enhanced MCF7 cell motility in a wound healing assay and their invasiveness through extracellular matrix. 3. CRF increased actin polymerization, phosphorylation of Focal Adhesion Kinase (FAK), providing a potential mechanism for the observed induction of MCF7 motility. 4. CRF induced the expression of Cox-1 but not Cox-2 in MCF7 cells as well as the production of prostaglandins, factors known to promote invasiveness and metastasis.ConclusionOverall, our data suggest that CRF stimulates cell motility and invasiveness of MCF7 cells most probably via induction of FAK phosphorylation and actin filament reorganization and production of prostaglandins via Cox1. Based on these findings we postulate that the stress neuropeptide CRF present in the vicinity of tumors (either produced locally by the tumor cells themselves or by nearby normal cells or secreted from the innervations of surrounding tissues) may play an important role on breast tumor growth and metastatic capacity, providing a potential link between stress and tumor progression.

The impact of stress on tumor growth: peripheral CRF mediates tumor-promoting effects of stress

IntroductionStress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo.MethodsFor this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis.ResultsArray analysis revealed among other genes that CRF induced the expression of SMAD2 and β-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- β-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-β action on proliferation confirming its impact on TGFβ/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo.ConclusionThis is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and β-catenin expression.

Chronic low-grade inflammation

Abstract: Obesity is associated with chronic low-grade inflammation which results in insulin resistance, type 2 diabetes, vascular disease, chronic renal failure, several cancers and endocrine and behavioural abnormalities. The enlarged white adipocytes of the obese are now considered to be the initiators of chronic low-grade inflammation. Indeed, the gradual accumulation of triglycerides within adipocytes shifts their phenotype towards a pro-inflammatory one and at the same time accelerates their apoptosis. The hyperplastic white trunkal adipocytes of obesity produce large amounts of pro-inflammatory cytokines, acute-phase proteins and chemokines which attract circulating monocytes into adipose tissue and induce their activation to macrophages which intensify further and expand the adipose tissue inflammation.