Corticotropin-releasing hormone activates protein kinase C in an isoenzyme-specific manner

Protein kinase C (PKC) has recently emerged as mediator of corticotropin-releasing hormone (CRH) effects. Aim of the present study was to study the effects of CRH on each PKC isoenzyme. As a model we have used the PC12 rat pheochromocytoma cell line, expressing the CRH type 1 receptor (CRHR1). Our data were as follows: (a) CRH-induced rapid phosphorylation of conventional PKCalpha and PKCbeta, accompanied by parallel increase of their concentration within nucleus. (b) CRH suppressed the phosphorylation of novel PKCdelta and PKCtheta;, which remained in the cytosol. (c) CRH-induced transient phosphorylation of atypical PKClambda and had no effect on PKCmu. (d) The effect of CRH on each PKC isoenzyme was blocked by a CRHR1 antagonist. (e) Blockade of conventional PKC phosphorylation inhibited CRH-induced calcium ion mobilization from intracellular stores as well as the CRH-induced apoptosis and Fas ligand production. In conclusion, our findings suggest that CRH via its CRHR1 receptor differentially regulates PKC-isoenzyme phosphorylation, an apparently physiologically relevant effect since blockade of conventional PKC phosphorylation abolished the biological effect of CRH.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

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

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 delta- and kappa-opioid agonists and their receptors. Our results were as follows: The kappa- and delta2-opioid receptor agonists had a rapid but transient effect on apoptosis at 3 h, whereas mu 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 approximately 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.     

Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

Chronic activation of innate immunity takes place in obesity and initiated by the hypertrophic adipocytes which obtain a pro-inflammatory phenotype. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRF₁ and CRF₂) affect stress response and innate immunity. Adipose tissue expresses a complete CRF system. The aim of this study was to examine the role of CRF neuropeptides in the immune phenotype of adipocytes assessed by their expression of the toll-like receptor-4 (TLR4), the production of inflammatory cytokines IL-6, TNF-α and IL-1β, chemokines IL-8, monocyte attractant protein-1 (MCP-1) and of the adipokines adiponectin, resistin and leptin. Our data are as follows: (a) CRF, UCN2 and UCN3 are expressed in human white adipocytes as well as CRFR_1a, CRFR_2a and CRFR_2b but not CRFR_2c. 3T3L1 pre-adipocytes and differentiated adipocytes expressed both CRF₁ and CRF₂ receptors and UCN3, while UCN2 was detected only in differentiated adipocytes. CRF₂ was up-regulated in mouse mature adipocytes. (b) CRF₁ agonists suppressed media- and LPS-induced pre-adipocyte differentiation while CRF₂ receptor agonists had no effect. (c) In mouse pre-adipocytes, CRF₂ agonists suppressed TLR4 expression and the production of IL-6, CXCL1 and adiponectin while CRF₁ agonists had no effect. (d) In mature mouse adipocytes LPS induced IL-6 and CXCL1 production and suppressed leptin. (e) In human visceral adipocytes LPS induced IL-6, TNF-α, IL-8, MCP-1 and leptin production and suppressed adiponectin and resistin. (f) In mouse mature adipocytes CRF₁ and CRF₂ agonists suppressed basal and LPS-induced production of inflammatory cytokines, TLR4 expression and adiponectin production, while in human visceral adipocytes CRF and UCN1 suppressed basal and LPS-induced IL-6, TNF-α, IL-8 and MCP-1 production. In conclusion, the effects of the activation of CRF₁ and CRF₂ may be significant in ameliorating the pro-inflammatory activity of adipocytes in obesity.     

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.     

Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids

Amino Acids - Neurotensin (NT) (pGlu–Leu–Tyr–Glu–Asn–Lys–Pro–Arg–Arg–Pro–Tyr–Ile–Leu) exerts a dual function as a...     

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.     

Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.