Flavonoids interfere with NLRP3 inflammasome activation.

NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome is a component of innate immunity, and is responsible for producing mature IL-1ß and -18. Several flavonoids were found to affect inflammasome pathway, but the mechanism of action is still obscure. To elucidate the effects on NLRP3 inflammasome pathway and to determine the structure-activity relationships, NLRP3 inflammasome in differentiated THP-1 cells was activated via treatment with monosodium urate (MSU) crystals. Levels of mature IL-1ß, NLRP3 inflammasome components and apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain) (ASC) oligomerization were investigated and the mechanisms of action were also elucidated. Among the 56 flavonoids initially tested, only flavone, 2',4'-dihhydroxyflavone, 3',4'-dichloroflavone, 4',5,7-trihydroxyflavone (apigenin), 3,4',5,7-tetrahydroxyflavone (kaempferol) and 3,3',4',5,7-pentahydroxyflavone (quercetin) significantly inhibited IL-1ß production at 10 µM. Apigenin, kaempferol and 3',4'-dichloroflavone inhibited ASC oligomerization without affecting the ASC level in cell lysates. Apigenin also inhibited absent in melanoma 2 (AIM2) inflammasome-related pathway, but not NLR family CARD domain-containing protein 4 (NLRC4) inflammasome activation. The action of apigenin on NLRP3 inflammasome activation is mediated partly via inhibition of phosphorylation of spleen tyrosine kinase/protein tyrosine kinase 2 (Syk/Pyk2) pathway. Furthermore, orally administered apigenin (100 mg/kg) strongly reduced the number of neutrophils and monocytes in MSU-induced peritonitis in mice. The present study, for the first time, demonstrated the structure-activity profiles of flavonoids in NLRP3 inflammasome activation and mechanisms of cellular action. Certain flavonoids including apigenin are expected to ameliorate the inflammatory symptoms in autoinflammatory diseases associated with NLRP3 inflammasome activation.

Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase.

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 µM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction.

Fyn inhibition rescues established memory and synapse loss in Alzheimer mice.

OBJECTIVE: Currently no effective disease-modifying agents exist for the treatment of Alzheimer disease (AD). The Fyn tyrosine kinase is implicated in AD pathology triggered by amyloid-ß oligomers (Aßo) and propagated by Tau. Thus, Fyn inhibition may prevent or delay disease progression. Here, we sought to repurpose the Src family kinase inhibitor oncology compound, AZD0530, for AD. METHODS: The pharmacokinetics and distribution of AZD0530 were evaluated in mice. Inhibition of Aßo signaling to Fyn, Pyk2, and Glu receptors by AZD0530 was tested by brain slice assays. After AZD0530 or vehicle treatment of wild-type and AD transgenic mice, memory was assessed by Morris water maze and novel object recognition. For these cohorts, amyloid precursor protein (APP) metabolism, synaptic markers (SV2 and PSD-95), and targets of Fyn (Pyk2 and Tau) were studied by immunohistochemistry and by immunoblotting. RESULTS: AZD0530 potently inhibits Fyn and prevents both Aßo-induced Fyn signaling and downstream phosphorylation of the AD risk gene product Pyk2, and of NR2B Glu receptors in brain slices. After 4 weeks of treatment, AZD0530 dosing of APP/PS1 transgenic mice fully rescues spatial memory deficits and synaptic depletion, without altering APP or Aß metabolism. AZD0530 treatment also reduces microglial activation in APP/PS1 mice, and rescues Tau phosphorylation and deposition abnormalities in APP/PS1/Tau transgenic mice. There is no evidence of AZD0530 chronic toxicity. INTERPRETATION: Targeting Fyn can reverse memory deficits found in AD mouse models, and rescue synapse density loss characteristic of the disease. Thus, AZD0530 is a promising candidate to test as a potential therapy for AD.

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism.

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.

SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro.

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor kinase and has been found crucial to cell motility. However, little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer. METHODS: The methylation status of SOCS3 was investigated in HBE and A549 cell lines by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine or transfected with three SOCS3 mutants with various functional domains deleted. Besides, cells were pretreated with a proteasome inhibitor beta-lactacystin where indicated. The effects of SOCS3 up-regulation on PYK2 expression, PYK2 and ERK1/2 phosphorylations were assessed by western blot using indicated antibodies. RT-PCR was used to estimate PYK2 mRNA levels. Transwell experiments were performed to evaluate cell migration. RESULTS: SOCS3 expression was found impaired in A549 cells and higher PYK2 activity was correlated with enhanced cell migration. We identified that SOCS3 was aberrantly methylated in the exon 2, and 5-aza-2'-deoxycytidine restored SOCS3 expression. Reactivation of SOCS3 attenuated PYK2 expression and phosphorylation, cell migration was inhibited as well. Transfection studies indicated that exogenous SOCS3 interacted with PYK2, and both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of SOCS3 contributed to PYK2 binding. Furthermore, SOCS3 was found to inhibit PYK2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of PYK2 in a SOCS-box-dependent manner and interfered with PYK2-related signaling events, such as cell migration. CONCLUSION: These data indicate that SOCS3 negatively regulates cell motility and decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells. These results also suggest a negative role of SOCS3 in PYK2 signaling, and a previously unidentified regulatory mechanism for PYK2 function.

Chronic nicotine-induced switch in Src-family kinase signaling for long-term potentiation induction in hippocampal CA1 pyramidal cells.

Here, we show that chronic nicotine exposure induces changes in Src signaling for the modulation of N-methyl-D-aspartate receptor (NMDAR) function and LTP induction in CA1 pyramidal cells. Activation of muscarinic receptors normally potentiates NMDAR responses in pyramidal cells via a Gq/protein kinase C (PKC)/proline-rich tyrosine kinase 2/Src signaling cascade. However, muscarinic, PKC and Src stimulation had no effect on NMDAR responses after chronic nicotine treatment. The lack of effect was apparently due to enhanced tyrosine phosphorylation, and therefore further stimulation of the signaling cascade caused no effect on NMDAR responses. Interestingly, another Src-family kinase potentiated NMDAR responses after, but not before, chronic nicotine treatment. In control pyramidal cells, Src inhibitor peptides prevented tetanus-induced long-term potentiation (LTP). Conversely, in chronic nicotine-exposed cells, the inhibitor was ineffective in blocking tetanus-induced LTP. Furthermore, in control pyramidal cells, applying exogenous Src and administration of an endogenous Src-family kinase activator increased alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR)-mediated responses. This increase was blocked by Src inhibitor peptides and occluded tetanus-induced LTP, as reported previously. In contrast, in chronic nicotine-treated pyramidal cells, applying exogenous Src had no effect on AMPAR-mediated responses and a tetanus-induced LTP. Interestingly, however, administration of an endogenous Src-family kinase activator enhanced AMPAR-mediated responses, which occluded tetanus-induced LTP. This enhancement was not prevented by co-application of Src inhibitor peptides. Thus, it appears that chronic nicotine exposure recruits another member of the Src-family for the regulation of NMDAR function and LTP induction. The nicotine-induced distinct signaling cascades may be involved in long-lasting memories of nicotine misuse.