Impact of Elevated Brain IL-6 in Transgenic Mice on the Behavioral and Neurochemical Consequences of Chronic Alcohol Exposure.

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of the neuroimmune system is known to dysregulate the production of neuroimmune factors, such as the cytokine IL-6, thereby changing the neuroimmune status of the brain, which could impact the actions of alcohol. The consequences of neuroimmune-alcohol interactions are not fully known. In the current studies we investigated this issue in transgenic (TG) mice with altered neuroimmune status relative to IL-6. The TG mice express elevated levels of astrocyte-produced IL-6, a condition known to occur with alcohol exposure. Standard behavioral tests of alcohol drinking and negative affect/emotionality were carried out in homozygous and heterozygous TG mice and control mice to assess the impact of neuroimmune status on the actions of chronic intermittent alcohol (ethanol) (CIE) exposure on these behaviors. The expressions of signal transduction and synaptic proteins were also assessed by Western blot to identify the impact of alcohol-neuroimmune interactions on brain neurochemistry. The results from these studies show that neuroimmune status with respect to IL-6 significantly impacts the effects of alcohol on multiple levels.

MAPKs Are Highly Abundant but Do Not Contribute to α1-Adrenergic Contraction of Rat Saphenous Arteries in the Early Postnatal Period.

Previously, the abundance of p42/44 and p38 MAPK proteins had been shown to be higher in arteries of 1- to 2-week-old compared to 2- to 3-month-old rats. However, the role of MAPKs in vascular tone regulation in early ontogenesis remains largely unexplored. We tested the hypothesis that the contribution of p42/44 and p38 MAPKs to the contraction of peripheral arteries is higher in the early postnatal period compared to adulthood. Saphenous arteries of 1- to 2-week-old and 2- to 3-month-old rats were studied using wire myography and western blotting. The α1-adrenoceptor agonist methoxamine did not increase the phosphorylation level of p38 MAPK in either 1- to 2-week-old or 2- to 3-month-old rats. Accordingly, inhibition of p38 MAPK did not affect arterial contraction to methoxamine in either age group. Methoxamine increased the phosphorylation level of p42/44 MAPKs in arteries of 2- to 3-month-old and of p44 MAPK in 1- to 2-week-old rats. Inhibition of p42/44 MAPKs reduced methoxamine-induced contractions in arteries of 2- to 3-month-old, but not 1- to 2-week-old rats. Thus, despite a high abundance in arterial tissue, p38 and p42/44 MAPKs do not regulate contraction of the saphenous artery in the early postnatal period. However, p42/44 MAPK activity contributes to arterial contractions in adult rats.

Alcohol alters IL-6 Signal Transduction in the CNS of Transgenic Mice with Increased Astrocyte Expression of IL-6.

Neuroimmune factors, including the cytokine interleukin-6 (IL-6), are important chemical regulators of central nervous system (CNS) function under both physiological and pathological conditions. Elevated expression of IL-6 occurs in the CNS in a variety of disorders associated with altered CNS function, including excessive alcohol use. Alcohol-induced production of IL-6 has been reported for several CNS regions including the cerebellum. Cerebellar actions of alcohol occur through a variety of mechanisms, but alcohol-induced changes in signal transduction, transcription, and translation are known to play important roles. IL-6 is an activator of signal transduction that regulates gene expression. Thus, alcohol-induced IL-6 production could contribute to cerebellar effects of alcohol by altering gene expression, especially under conditions of chronic alcohol abuse, where IL-6 levels could be habitually elevated. To gain an understanding of the effects of alcohol on IL-6 signal transduction, we studied activation/expression of IL-6 signal transduction partners STAT3 (Signal Transducer and Activator of Transcription), CCAAT-enhancer binding protein (C/EBP) beta, and p42/p44 mitogen-activated protein kinase (MAPK) at the protein level. Cerebella of transgenic mice that express elevated levels of astrocyte produced IL-6 in the CNS were studied. Results show that the both IL-6 and chronic intermittent alcohol exposure/withdrawal affect IL-6 signal transduction partners and that the actions of IL-6 and alcohol interact to alter activation/expression of IL-6 signal transduction partners. The alcohol/IL-6 interactions may contribute to cerebellar actions of alcohol, whereas the effects of IL-6 alone may have relevance to cerebellar changes occurring in CNS disorders associated with elevated levels of IL-6.

Alcohol Enhances Responses to High Frequency Stimulation in Hippocampus from Transgenic Mice with Increased Astrocyte Expression of IL-6.

Recent studies show that alcohol exposure can induce glial production of neuroimmune factors in the CNS. Of these, IL-6 has gained attention because it is involved in a number of important physiological and pathophysiological processes that could be affected by alcohol-induced CNS production of IL-6, particularly under conditions of excessive alcohol use. For example, IL-6 has been shown to play a role in hippocampal behaviors and synaptic plasticity (long-term potentiation; LTP) associated with memory and learning. Surprisingly, in our in vitro studies of LTP at the Schaffer collateral to CA1 pyramidal neuron synapse in hippocampus from transgenic mice that express elevated levels of astrocyte produced IL-6 (TG), LTP was not altered by the increased levels of IL-6. However, exposure to acute alcohol revealed neuroadaptive changes that served to protect LTP against the alcohol-induced reduction of LTP observed in hippocampus from non-transgenic control mice (WT). Here we examined the induction phase of LTP to assess if presynaptic neuroadaptive changes occurred in the hippocampus of TG mice that contributed to the resistance of LTP to alcohol. Results are consistent with a role for IL-6-induced neuroadaptive effects on presynaptic mechanisms involved in transmitter release in the resistance of LTP to alcohol in hippocampus from the TG mice. These actions are important with respect to a role for IL-6 in physiological and pathophysiological processes in the CNS and in CNS actions of alcohol, especially when excessive alcohol used is comorbid with conditions associated with elevated levels of IL-6 in the CNS.

Intra-Cellular Calcium Signaling Pathways (PKC, RAS/RAF/MAPK, PI3K) in Lamina Cribrosa Cells in Glaucoma.

The lamina cribrosa (LC) is a key site of fibrotic damage in glaucomatous optic neuropathy and the precise mechanisms of LC change remain unclear. Elevated Ca2+ is a major driver of fibrosis, and therefore intracellular Ca2+ signaling pathways are relevant glaucoma-related mechanisms that need to be studied. Protein kinase C (PKC), mitogen-activated MAPK kinases (p38 and p42/44-MAPK), and the PI3K/mTOR axis are key Ca2+ signal transducers in fibrosis and we therefore investigated their expression and activity in normal and glaucoma cultured LC cells. We show, using Western immune-blotting, that hyposmotic-induced cellular swelling activates PKCα, p42/p44, and p38 MAPKs, the activity is transient and biphasic as it peaks between 2 min and 10 min. The expression and activity of PKCα, p38 and p42/p44-MAPKs are significantly (p < 0.05) increased in glaucoma LC cells at basal level, and at different time-points after hyposmotic stretch. We also found elevated mRNA expression of mRNA expression of PI3K, IP3R, mTOR, and CaMKII in glaucoma LC cells. This study has identified abnormalities in multiple calcium signaling pathways (PKCα, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in glaucoma.

Expression and activation of mitogen-activated protein kinases in the optic nerve head in a rat model of ocular hypertension.

BACKGROUND: Glaucoma is a leading cause of irreversible blindness manifesting as an age-related, progressive optic neuropathy with associated retinal ganglion cell (RGC) loss. Mitogen-activated protein kinases (MAPKs: p42/44 MAPK, SAPK/JNK, p38 MAPK) are activated in various retinal disease models and likely contribute to the mechanisms of RGC death. Although MAPKs play roles in the development of retinal pathology, their action in the optic nerve head (ONH), where the initial insult to RGC axons likely resides in glaucoma, remains unexplored. METHODS: An experimental paradigm representing glaucoma was established by induction of chronic ocular hypertension (OHT) via laser-induced coagulation of the trabecular meshwork in Sprague-Dawley rats. MAPKs were subsequently investigated over the following days for expression and activity alterations, using RT-PCR, immunohistochemistry and Western immunoblot. RESULTS: p42/44 MAPK expression was unaltered after intraocular pressure (IOP) elevation, but there was a significant activation of this enzyme in ONH astrocytes after 6-24 h. Activated SAPK/JNK isoforms were present throughout healthy RGC axons but after IOP elevation or optic nerve crush, they both accumulated at the ONH, likely due to RGC axon transport disruption, and were subject to additional activation. p38 MAPK was expressed by a population of microglia which were significantly more populous following IOP elevation. However it was only significantly activated in microglia after 3 days, and then only in the ONH and optic nerve; in the retina it was solely activated in RGC perikarya. CONCLUSIONS: In conclusion, each of the MAPKs showed a specific spatio-temporal expression and activation pattern in the retina, ONH and optic nerve as a result of IOP elevation. These findings likely reflect the roles of the individual enzymes, and the cells in which they reside, in the developing pathology following IOP elevation. These data have implications for understanding the mechanisms of ocular pathology in diseases such as glaucoma.

Decisive role of P42/44 mitogen-activated protein kinase in Δ9-tetrahydrocannabinol-induced migration of human mesenchymal stem cells.

In past years, medical interest in Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient of the Cannabis plant, has been renewed due to the elucidation of the endocannabinoid system and diverse other receptor targets involved in biological cannabinoid effects. The present study therefore investigates the impact of THC on the migration of mesenchymal stem cells (MSCs) which are known to be involved in various regenerative processes such as bone healing. Using Boyden chamber assays, THC was found to increase the migration of adipose-derived MSCs. Migration by THC was almost completely suppressed by the CB1 receptor antagonist AM-251 and to a lesser extent by the CB2 receptor antagonist AM-630. By contrast, the TRPV1 antagonist capsazepine as well as the G protein-coupled receptor 55 (GRP55) agonist O-1602 did not significantly interfere with the promigratory effect of THC. Furthermore, increased migration by THC was fully suppressed by PD98059, an inhibitor of p42/44 mitogen-activated protein kinase (MAPK) activation, and was accompanied by a time-dependent activation of this pathway accordingly. In line with the migration data, additional inhibitor experiments pointed towards a decisive role of the CB1 receptor in conferring THC-induced activation of p42/44 MAPK. Collectively, this study demonstrates THC to exert a promigratory effect on MSCs via a CB1 receptor-dependent activation of p42/44 MAPK phosphorylation. This pathway may be involved in regenerative effects of THC and could be a target of pharmacological intervention.

Cannabidiol upregulates melanogenesis through CB1 dependent pathway by activating p38 MAPK and p42/44 MAPK.

Melanogenesis plays a critical role in the protection of skin against external stresses such as ultraviolet irradiation and oxidative stressors. This study was aimed to investigate the effects of cannabidiol on melanogenesis and its mechanisms of action in human epidermal melanocytes. We found that cannabidiol increased both melanin content and tyrosinase activity. The mRNA levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP) 1, and TRP2 were increased following cannabidiol treatment. Likewise, cannabidiol increased the protein levels of MITF, TRP 1, TRP 2, and tyrosinase. Mechanistically, we found that cannabidiol regulated melanogenesis by upregulating MITF through phosphorylation of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, independent of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. In addition, the melanogenic effect of cannabidiol was found to be mediated by cannabinoid CB1 receptor, not by CB2 receptor. Taken together, these findings indicate that cannabidiol-induced melanogenesis is cannabinoid CB1 receptor-dependent, and cannabidiol induces melanogenesis through increasing MITF gene expression which is mediated by activation of p38 MAPK and p42/44 MAPK. Our results suggest that cannabidiol might be useful as a protective agent against external stresses.

Lactoferrin interacts with SPLUNC1 to attenuate lipopolysaccharide-induced inflammation of human nasal epithelial cells via down-regulated MEK1/2-MAPK signaling.

The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an important innate material in the upper airway, and lactoferrin (LF) aids the innate functions in humans. In this study, a nasal epithelial model was used to investigate how LF modulates SPLUNC1 to reduce the inflammatory process mediated by lipopolysaccharide (LPS). The inflammation of human RPMI-2650 cells was induced with LPS to evaluate SPLUNC1 expression after treating the cells with bovine LF (bLF). The interaction pathway between LF and SPLUNC1 in LPS-induced inflammation was further investigated. Our study reveals that the addition of bLF results in the recovery of SPLUNC1 expression in nasal epithelial cells under LPS-induced inflammation. MAPK is involved in the main pathway for the SPLUNC1 and bLF interaction. Decreased SPLUNC1 function could be recovered by addition of bLF. The MEK1/2-MAPK signaling pathway is crucial for the SPLUNC1 and bLF interaction. Therefore, LF could support SPLUNC1 in the innate immunity recovery process.

Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells.

BACKGROUND: It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta. METHODS: We used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta-producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells. RESULTS: We report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression. CONCLUSIONS: These data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression.

Increase of mesenchymal stem cell migration by cannabidiol via activation of p42/44 MAPK.

Migration and differentiation of mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as bone healing. However, little is known about the pharmacotherapeutical options aiming at the mobilization and differentiation of MSCs. The present study therefore focussed on cannabinoids which have been demonstrated to exhibit tissue healing properties. Using Boyden chamber assays, the non-psychoactive phytocannabinoid cannabidiol (CBD) was found to increase the migration of adipose-derived MSCs in a time- and concentration-dependent manner. CBD-induced migration was inhibited by AM-630 (CB2 receptor antagonist) and O-1602 (G protein-coupled receptor 55 [GRP55] agonist). Moreover, the promigratory effect of CBD was antagonized by inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway which became activated upon CBD treatment. In line with this data, AM-630 and O-1602 attenuated CBD-induced p42/44 MAPK phosphorylation. A p42/44 MAPK-dependent promigratory effect was likewise demonstrated for the GPR55 antagonist O-1918 and the selective CB2 receptor agonist JWH-133. Additional evidence for a functional effect of CBD on MSCs was provided by experiments demonstrating long-term stimulation with CBD to induce differentiation of MSCs into the osteoblastic lineage as evidenced by increased mineralization assessed by cresolphthalein complexone assay and enhanced activity of alkaline phosphatase. Collectively, this study demonstrates CBD to promote the migration of MSCs via activation of the CB2 receptor and inhibition of GPR55 and to induce osteoblastic differentiation. CBD may therefore recruit MSCs to sites of calcifying tissue regeneration and subsequently support bone regeneration via an osteoanabolic action on MSCs.

Molecular interactions of serotonin (5-HT) and endothelin-1 in vascular smooth muscle cells: in vitro and ex vivo analyses.

Elevated levels of serotonin (5-HT) and endothelin-1 (ET-1) may be involved in cardiovascular complications of diabetes mellitus. Data suggest supraphysiological concentrations of 5-HT (10(-6) M) potentiate the ability of ET-1 to stimulate DNA synthesis and vascular smooth muscle cell (VSMC) proliferation in vitro via activation of mitogen-activated protein kinase (p42/44 MAPK) and Janus kinase 2 (JAK2) pathways. Additionally, 5-HT enhances agonist-induced contractions via p42/44 MAPK and an unknown tyrosine kinase. However, the exact mechanisms of the 5-HT/ET-1 interactions and whether these effects occur at physiological levels (10(-9) M) are unknown. Therefore, we hypothesized that interactions between 5-HT and ET-1 at physiological concentrations in VSMC enhanced activation of both p42/44 MAPK and JAK2 pathways contributing to vascular growth and contractile responses. With the use of rat VSMC and Western blot analysis, our data suggest no effect of acute (30 min) preincubation with 5-HT (10(-9) M) and/or ET-1 (10(-9) M) on the activation of either pathway in normal or high glucose conditions. To determine if there was altered vascular reactivity in intact vessels we tested the effects of 5-HT and ET-1 interaction using myographs to measure isometric contractions of rat thoracic aortic rings. 5-HT (10(-9) M) and ET-1 (10(-12) M) stimulate enhanced contractile responses to each other that were inhibited by JAK2 and p42/44 MAPK antagonists. Our findings demonstrate that both 5-HT and ET-1 at physiological concentrations could interact with each other and activate p42/44 MAPK and JAK2 signaling pathways to cause an increase in smooth muscle contraction that could lead to altered vascular function.