Angiotensin II effects on STAT3 phosphorylation in cardiomyocytes: evidence for Erk-dependent Tyr705 dephosphorylation

Experiments were performed to define the basis for negative regulation of STAT3 activation (i.e., Tyr705 phosphorylation) by angiotensin II in cardiomyocytes. Treatment of cardiomyocytes with angiotensin II resulted in rapid and sustained phosphorylation of STAT3 on Ser727; in contrast, STAT3 Tyr705 phosphorylation was decreased, with dephosphorylation being most pronounced at 30 minutes. Angiotensin II-induced STAT3 Tyr705 dephosphorylation was not prevented by inhibiting protein synthesis, but was blocked by vanadate or the MEK inhibitor PD98059. PD98059 was found to inhibit angiotensin II-induced Erk activation and STAT3 Ser727 phosphorylation. Angiotensin II also attenuated LIF-induced STAT3 Tyr705 phosphorylation, and this effect could be blocked with PD89059. These results are consistent with Erk-mediated STAT3 Ser727 phosphorylation leading to STAT3 Tyr705 dephosphorylation, and accounting for angiotensin II-mediated STAT3 inhibition in cardiomyocytes. We propose that Erk serves as a scaffolding protein in recruiting either a protein tyrosine or MAP kinase phosphatase to STAT3.     

Chronic alcohol feeding impairs mTOR(Ser 2448) phosphorylation in rat hearts

Background: Chronic alcohol administration impairs protein synthesis ultimately causing a loss of proteins in cardiac muscle. Inhibition of protein synthesis resides in the process of mRNA translation. The present set of experiments were designed to examine the potential regulatory effect of chronic alcohol consumption on mammalian target of rapamycin (mTOR), a serine/threonine kinase important in controlling signaling cascades in the mRNA translation initiation pathway in rat hearts. Methods: Rats were fed a diet containing ethanol for 20 to 26 weeks. Pair‐fed rats served as controls. Rates of protein synthesis were measured following intravenous infusion of [³H]‐l ‐phenylalanine (150 mM, 30 μCi/ml; 1 ml/100 g body weight). The phosphorylation state of mTOR, eukaryotic initiation factor 4G (eIF4G), protein kinase B (PKB) and S6K1 in heart were measured using immunoblot techniques with phospho‐specific antibodies. Results: Protein synthesis was reduced by 35% in animals consuming a diet containing ethanol. The fall in protein synthesis was accompanied by diminished S6K1(Thr³⁸⁹) and eIF4G (Ser¹¹⁰⁸) phosphorylation, both downstream effectors of mTOR signaling. These changes in phosphorylation of S6K1 and eIF4G were not associated with differences in the distribution of mTOR between TORC1 and TORC2. Instead, phosphorylation of mTOR on Ser²⁴⁴⁸ but not on Ser²⁴⁸¹ was significantly reduced following feeding rats an ethanol containing diet. Decreased phosphorylation of mTOR(Ser²⁴⁴⁸) was not associated with a corresponding lessening of tumor suppressor complex 2 phosphorylation or expression of regulated in development and DNA damage 1, both upstream regulators of mTOR. Likewise, phosphorylation of PKB on either Ser⁴⁷³ or Thr³⁰⁸ was unaffected by long‐term alcohol consumption. Conclusions: Chronic ethanol consumption does not alter the distribution of mTOR between TORC1 and TORC2, but instead diminishes mTOR phosphorylation on Ser²⁴⁴⁸ independent of changes in tumor suppressor complex 2 and PKB phosphorylation. Furthermore, the data suggest that protein synthesis in rats fed a diet containing ethanol is limited by mTOR‐dependent reduction in phosphorylation of S6K1(Thr³⁸⁹) and eIF4G(Ser¹¹⁰⁸) secondary to reduced phosphorylation of mTOR(Ser²⁴⁴⁸).     

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

Activation of thromboxane A2 receptors mediates endothelial dysfunction in diabetic mice

Background: Diabetes is one of high-risk factors for cardiovascular disease. Improvement of endothelial dysfunction in diabetes reduces vascular complications. However, the underlying mechanism needs to be uncovered. This study was conducted to elucidate whether and how thromboxane A2 receptor (TPr) activation contributes to endothelial dysfunction in diabetes. Methods and Results: Exposure of human umbilical vein endothelial cells (HUVECs) to either TPr agonists, two structurally related thromboxane A₂ (TxA₂) mimetics, significantly reduced phosphorylations of endothelial nitric oxide synthase (eNOS) at Ser¹¹⁷⁷ and Akt at Ser⁴⁷³. These effects were abolished by pharmacological or genetic inhibitors of TPr. TPr-induced suppression of eNOS and Akt phosphorylation was accompanied by upregulation of PTEN (phosphatase and tension homolog deleted on chromosome 10) and Ser³⁸⁰/Thr^382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt–eNOS signaling in the face of TPr activation. The small GTPase, Rho, was also activated by TPr stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued TPr-impaired Akt–eNOS signaling. In mice, streptozotocin-induced diabetes was associated with aortic PTEN upregulation, PTEN-Ser³⁸⁰/Thr^382/383 phosphorylation, and dephosphorylation of Akt (at Ser⁴⁷³) and eNOS (at Ser¹¹⁷⁷). Importantly, administration of TPr antagonist blocked these changes. Conclusion: We conclude that TPr activation impairs endothelial function by selectively inactivating the ROCK–PTEN–Akt–eNOS pathway in diabetic mice.     

Tyr323‐dependent p38 activation is associated with rheumatoid arthritis and correlates with disease activity

Objective

The p38 MAPK is important in the pathogenic immune response in rheumatoid arthritis (RA). The p38 molecule can be activated through phosphorylation on Thr¹⁸⁰–Tyr¹⁸² by upstream MAPK kinases and via an alternative pathway through phosphorylation on Tyr³²³. We undertook this study to quantify the phosphorylation of Tyr³²³ p38 and of Thr¹⁸⁰–Tyr¹⁸² p38 on T cells from healthy controls and patients with RA or ankylosing spondylitis (AS) to identify variables associated with p38 phosphorylation and disease activity.

Methods

We measured p38 phosphorylation on Tyr³²³ and Thr¹⁸⁰–Tyr¹⁸² by flow cytometry and Western blotting on T cells from 30 control subjects, 33 AS patients, 30 patients with RA in remission, and 79 patients with active RA. We collected the clinical characteristics and analyzed correlations between clinical variables, the Disease Activity Score in 28 joints (DAS28), and p38 phosphorylation levels. Multivariate regression analysis was performed to identify variables associated with p38 phosphorylation on Tyr³²³ and Thr¹⁸⁰–Tyr¹⁸².

Results

Phosphorylation of p38 on Tyr³²³ was higher in T cells from patients with active RA (P = 0.008 versus healthy controls) than in patients with RA in remission or in patients with AS. Tyr³²³ p38 phosphorylation was associated with disease activity determined by the DAS28 (P = 0.017). Enhanced p38 phosphorylation was linked to Lck‐mediated activation of the Tyr³²³‐dependent pathway in the absence of upstream MAPKK activation.

Conclusion

Our results indicate that phosphorylation status on Tyr³²³ p38 correlates with RA disease activity and suggest that the Tyr³²³‐dependent pathway is an attractive target for down‐regulation of p38 activity in RA patients.

     

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ and MT_2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT₁ (MT₁^−/−), or MT₂ (MT₂^−/−), or both MT₁/MT₂ (MT₁^−/−/MT₂^−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT₁^−/− display no differences compared to their wild-type littermates (CTL), whereas both MT₂^−/− and MT₁^−/−/MT₂^−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT₂ partial agonist UCM924 induced an antinociceptive effect in MT₁^−/− but not in MT₂^−/− and MT₁^−/−/MT₂^−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT₂^−/− mice. Our results show that the genetic inactivation of melatonin MT₂, but not MT₁ receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT₂ receptor subtype is selectively involved in the regulation of pain responses.     

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB,STAT‐3 signaling,and dysregulated apoptosis of macrophages

Objective

To elucidate the role of tumor necrosis factor α–induced adipose‐related protein (TIARP; or tumor necrosis factor α–induced protein 9 [TNFAIP‐9]) in the development and pathogenesis of arthritis.

Methods

We generated TIARP‐deficient (TIARP^−/−) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF‐κB signal transduction were analyzed. We also examined the susceptibility of young TIARP^−/− mice to collagen‐induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti–type II collagen (anti‐CII) antibodies, interleukin‐6 (IL‐6), and TNFα on day 60 were measured. We further investigated the effects of anti–IL‐6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP^−/− mice. IL‐6/STAT‐3 signaling was also analyzed using TIARP^−/− macrophages.

Results

TIARP^−/− mice developed spontaneous enthesitis and synovitis, had high serum levels of IL‐6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS‐ and TNFα‐induced IL‐6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP^−/− macrophages. CIA was clearly exacerbated in TIARP^−/− mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti‐CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP^−/− mice. Treatment with anti–IL‐6 receptor mAb prevented the development of CIA in TIARP^−/− mice, and TIARP^−/− macrophages showed increased IL‐6–induced STAT‐3 phosphorylation.

Conclusion

These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL‐6 production, its signaling and TNFα‐induced NF‐κB signaling, resulting in enhanced apoptosis in macrophages.