PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF‐β1 and HIF‐1α in Ang II‐mediated fibrosis after myocardial infarction

Transforming growth factor (TGF)‐β1 is a known factor in angiotensin II (Ang II)‐mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor‐1 (Hif‐1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif‐1α contributed to the Ang II‐mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif‐1α and TGF‐β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague–Dawley rats with MI daily for 1 week; saline and hydralazine (another anti‐hypertensive agent like valsartan) was used as control. The fibrosis‐related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up‐regulation of Ang II, TGF‐β/Smad and Hif‐1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up‐regulation of TGF‐β/Smad and Hif‐1α was through the Ang II‐mediated pathway. By administering TGF‐β or dimethyloxalylglycine, we determined that both TGF‐β/Smad and Hif‐1α contributed to Ang II‐mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF‐β/Smad, Hif‐1α and fibrosis‐related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II‐induced cardiac fibrosis as well as into the cardiac protection of valsartan.     

α6β2*‐subtype nicotinic acetylcholine receptors are more sensitive than α4β2*‐subtype receptors to regulation by chronic nicotine administration

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*‐nAChR are down‐regulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose–responses and quantitative ligand‐binding autoradiography were used to define nicotine sensitivity of changes in α4β2*‐nAChR and α6β2*‐nAChR expression. α6β2*‐nAChR down‐regulation by chronic nicotine exposure in dopaminergic and optic‐tract nuclei was ≈three‐fold more sensitive than up‐regulation of α4β2*‐nAChR. In contrast, nAChR‐mediated [³H]‐dopamine release from dopamine‐terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR‐mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [³H]‐DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function.

     

Production of Tumor Necrosis Factor-α in Granulocytopenic Mice with Pulmonary Candidiasis and Its Modification with Granulocyte Colony-Stimulating Factor

In the present study, a lethal model of pulmonary candidiasis was established using granulocytopenic mice with cyclophosphamide. These mice started to die 1 day after infection and had all died within the next 48 hr. The counts of live C. albicans in the lung gradually increased with time, while the organisms were quickly eliminated in the normal mice. From the histology and measurements on bronchoalveolar lavage fluid (BALF), polymorphonuclear cells (PMN) response was almost zero up to 24 hr, and then a weak but significant response was observed at 48 hr, while a marked accumulation of PMN was detected from as early as 6 hr in normal mice. In contrast, macrophages had accumulated in BALF by 48 hr in granulocytopenic mice, but not in normal mice. Both in serum and BALF, a considerable level of tumor necrosis factor-α (TNF-α) was detected from 6 hr, peaking at 24 to 48 hr, while in normal mice the quantity was under the detection limit in serum and very low in BALF. The effects of administering granulocyte colony-stimulating factor (G-CSF) on these parameters were next examined. G-CSF significantly prolonged the survival time of granulocytopenic mice, promoted the clearance of organisms through increasing the counts of PMN in the lung, and strongly inhibited the production of TNF-α both in BALF and serum. These results suggest that this cytokine does not protect them, but plays some role in their death due to candidial infection in granulocytopenic mice.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Tumor necrosis factor-α induces substance P in sympathetic ganglia through sequential induction of interleukin-1 and leukemia inhibitory factor

The present study establishes that tumor necrosis factor-α (TNF-α) induction of sympathetic substance P (SP) requires sequential induction of both interleukin (IL-1) and leukemia inhibitory factor (LIF). TNF-α dose-dependently induces SP, an induction that is secondory to an increase in the SP precursor, preprotachykinin (PPT), mRNA. Since TNF-α conditioned medium (CM) mimics the effect of TNF-α by raising SP, actions that are not antagonized by a neutralizing TNF-α antibody, TNF-α induction of SP is mediated by a soluble intermediate or intermediates. The blockade of TNF-α action by a specific IL-1 receptor antagonist and the induction of IL-1 mRNA by TNF-α suggest that IL-1 is one of the intermediates. Moreover, because immunoprecipitation with LIF antibodies decreases SP-inducing activity of TNF-α CM, and because LIF mRNA is also induced by TNF-α, LIF is a second intermediate. Furthermore, TNF-α-induced LIF mRNA is blocked by the IL1 receptor antagonist, whereas IL-1-induced LIF mRNA is not affected by TNF-α antibodies, suggesting that TNF-α first induces IL-1, and IL-1 subsequently induces LIF. These data suggest that TNF-α induces SP in sympathetic ganglia through the sequential inductions of IL1 and LIF. © 1995 John Wiley & Sons, Inc.     

Relationship between conformation and geometry as evidenced by molecular dynamics simulation of Cα,α-dialkylated glycines

The relationship between the local backbone conformation and bond angles at C^α of symmetrically substituted C^α,α-dialkylated glycines (C^α,α-dimethylglycine or α-aminoisobutyric acid, Aib; C^α,α-diethylglycine, Deg; C^α,α-di-n-propylglycine, Dpg) has been investigated by molecular dynamics (MD) simulation adopting flat bottom harmonic potentials, instead of the usual harmonic restraints, for the C^α bond angles. The MD simulations show that the C^α bond angles are related to the local backbone conformation, irrespectively of the side-chain length of Aib, Deg, and Dpg residues. Moreover, the N-C^α-C′ (τ) angle is the most sensitive conformational parameter and, in the folded form, is always larger and more flexible than in the extended one. © 1998 John Wiley & Sons, Inc. Biopoly 46: 239–244, 1998     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.     

Cold pre-conditioning neuroprotection depends on TNF-α and is enhanced by blockade of interleukin-11

Cold pre-conditioning reduces subsequent brain injury in small animals but the underlying mechanisms remain undefined. As hypothermia triggers systemic macrophage tumor necrosis factor alpha (TNF-α) production and other neural pre-conditioning stimuli depend on this cytokine, we reasoned that microglia and TNF-α would be similarly involved with cold pre-conditioning neuroprotection. Also, as slice cultures closely approximate their in vivo counterpart and include quiescent microglia, we used rat hippocampal slice cultures to confirm this hypothesis. Furthermore, inflammatory cytokine gene screening with subsequent PCR and immunostaining confirmation of targeted mRNA and related protein changes showed that cold pre-conditioning triggered a significant rise in TNF-α that localized to microglia and a significant rise in interleukin (IL)-11 that localized mainly to hippocampal pyramidal neurons and, more rarely, astrocytes. Importantly, co-stimulation with cold and IL-11, an anti-inflammatory cytokine that inhibits TNF-α expression, abrogated the otherwise evident protection. Instead, cold pre-conditioning coupled with blockade of IL-11 signaling further enhanced neuroprotection from that seen with cold pre-conditioning alone. Thus, physiological activation of brain pro-inflammatory cytokine signaling, and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning.     

Regulation of TNF-α release from bone marrow–derived macrophages by vitamin D

The calcium-regulating hormone 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] is recognized as an immuno-modulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D–deficient mice (–D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells (+D MPs), and that bone marrow–derived macrophage (BMDM) differentiation is modulated by 1,25(OH)₂D₃. The release of tumor necrosis factor–α (TNF-α) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1,25(OH)₂D₃ in TNF-α synthesis and release. BMDMs were harvested from +D and ?D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF-α. BMDMs did not release measurable amounts of TNF-α without stimulation. Addition of endotoxin (LPS) to the cultures resulted in a marked stimulation of TNF-α release. 1,25(OH)₂D₃ increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon-γ (IFN-γ), yielded essentially similar results. +D and ?D mice were injected with LPS and TNF-α levels in the serum were measured. A marked reduction (～ fourfold) in the TNF-α levels was observed in the serum of ?D mice as compared with +D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1,25(OH)₂D₃ is on TNF-α synthesis. Our findings suggest that 1,25(OH)₂D₃ plays a role in the regulation of TNF-α secretion by mononuclear phagocytes. © 1994 Wiley-Liss, Inc.     

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

Inhibition by Bacterial Lipopolysaccharide of Spontaneous and TNF-α-Induced Human Neutrophil Apoptosis In Vitro

In the previous paper (Takeda et al, Int. Immunol., 5, 691-694, 1993), we demonstrated that tumor necrosis factor-α (TNF-α) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF-α-induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Effects of tumor necrosis factor α (TNFα) on the phospholipid metabolism of Tetrahymena pyriformis

The effect of (0·05 ng ml⁻¹ and 0·1 ng ml⁻¹) TNFα on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP₂) as well, as sphingomyelin (SM) content was lower in TNFα-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [³H]-palmitic acid or ³²P into the phospholipids. To control the functional effects of TNFα in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNFα at a low level of evolution, what is realized by changes in the phospolipid metabolism participating in signalling pathways. © 1998 John Wiley & Sons, Ltd.     

Tumor necrosis factor-α induces changes in the phosphorylation,cellular localization,and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine

The stress protein hsp27 is constitutively expressed in several human cells and shows a rapid phosphorylation following treatment with tumor necrosis factor-α (TNF-α). hsp27 usually displays native molecular mass ranging from 100 to 700 kDa. Here, we have analyzed the TNF-α-mediated changes in the phosphorylation, cellular localization, and structural organization of hsp27 in HeLa cells. We report that the TNF-α-mediated hsp27 phosphorylation is a long-lasting phenomenon that correlates with the cytostatic effect of this cytokine. Following TNF-α treatment, the rapid phosphorylation of hsp27 occurred concomitantly with complex changes in the intracellular distribution and structural organization of this protein. This resulted in the quantitative redistribution of hsp27 toward the soluble phase of the cytoplasm. In addition, during the first 2 h of TNF-α treatment, a transient increase in the native molecular mass of most hsp27 molecules (≤ 700 kDa) occurred. Then, by 4 h of TNF-α treatment, the native size of this stress protein drastically regressed (< 200 kDa). During this phenomenon, the phosphorylated isoforms of hsp27 remained concentrated in the small or medium-sized oligomers (< 300 kDa) of this protein. We also analyzed the properties of human hsp27 in transfected murine L929 cell lines that constitutively express this protein. In these cells, TNF-α induced modifications in the phosphorylation, intracellular distribution, and oligomerization of human hsp27 similar to those observed in HeLa cells. Moreover, the expression of hsp27 in L929 cells was found to correlate with a reduced cytotoxicity of this cytokine. Hence, the complex changes in the phosphorylation, intracellular locale and structural organization of human hsp27 may be related to the protective activity of this protein against the deleterious effects induced by TNF-α.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.