Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

Nonmotor Symptoms in LRRK2 G2019S Associated Parkinson’s Disease

Background

Idiopathic Parkinson’s disease (IPD) and LRRK2-associated PD (LRRK2-PD) might be expected to differ clinically since the neuropathological substrate of LRRK2-PD is heterogeneous. The range and severity of extra-nigral nonmotor features associated with LRRK2 mutations is also not well-defined.

Objective

To evaluate the prevalence and time of onset of nonmotor symptoms (NMS) in LRRK2-PD patients.

Methods

The presence of hyposmia and of neuropsychiatric, dysautonomic and sleep disturbances was assessed in 33 LRRK2-G2019S-PD patients by standardized questionnaires and validated scales. Thirty-three IPD patients, matched for age, gender, duration of parkinsonism and disease severity and 33 healthy subjects were also evaluated.

Results

University of Pennsylvania Smell Identification Test (UPSIT) scores in LRRK2-G2019S-PD were higher than those in IPD (23.5±6.8 vs 18.4±6.0; p = 0.002), and hyposmia was less frequent in G2019S carriers than in IPD (39.4% vs 75.8%; p = 0.01). UPSIT scores were significantly higher in females than in males in LRRK2-PD patients (26.9±4.7 vs 19.4±6.8; p<0.01). The frequency of sleep and neuropsychiatric disturbances and of dysautonomic symptoms in LRRK2-G2019S-PD was not significantly different from that in IPD. Hyposmia, depression, constipation and excessive daytime sleepiness, were reported to occur before the onset of classical motor symptoms in more than 40% of LRRK2-PD patients in whom these symptoms were present at the time of examination.

Conclusion

Neuropsychiatric, dysautonomic and sleep disturbances occur as frequently in patients with LRRK2-G2019S-PD as in IPD but smell loss was less frequent in LRRK2-PD. Like in IPD, disturbances such as hyposmia, depression, constipation and excessive daytime sleepiness may antedate the onset of classical motor symptoms in LRRK2-G2019S-PD.     

PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-δ hydrophobic motif phosphorylation. The late phase is mediated by Gα(12), which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-δ, but not AKT, results in PKC-δ hydrophobic motif phosphorylation and activation. This Gα(12)-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.     

Nur77 promotes cerebral ischemia–reperfusion injury via activating INF2-mediated mitochondrial fragmentation

Mitochondrial fragmentation drastically regulates mitochondrial homeostasis in brain illness. However, the role of mitochondrial fragmentation in cerebral ischemia–reperfusion (IR) injury remains unclear. Nur77, a regulator of mitochondrial homeostasis, is associated with heart and liver IR injury, but its effects on mitochondrial function in cerebral IR injury has not been studied intensively. The aim of our study is to explore whether cerebral IR injury is modulated by Nur77 via modification of mitochondrial homeostasis. Our results indicated that Nur77 was upregulated in reperfused brain tissues. Genetic ablation of Nur77 reduced infarction area and promoted neuron survival under IR burden. Biochemical analysis demonstrated that Nur77 deletion protected mitochondrial function, attenuated mitochondrial oxidative stress, preserved mitochondrial potential, and blocked mitochondria-related cell apoptosis. In addition, we illustrated that Nur77 mediated mitochondrial damage via evoking mitochondrial fragmentation that occurred through increased mitochondrial fission and decreased fusion. Besides, our results also demonstrated that Nur77 controlled mitochondrial fragmentation via upregulating INF2 in a manner dependent on the Wnt/β-catenin pathway; inhibition of the Wnt pathway abrogated the protective effect of Nur77 deletion on reperfused-mediated neurons. Altogether, our study highlights that the pathogenesis of cerebral IR injury is associated with Nur77 activation followed by augmented mitochondrial fragmentation via an abnormal Wnt/β-catenin/INF2 pathway. Accordingly, Nur77-dependent mitochondrial fragmentation and the Wnt/β-catenin/INF2 axis may represent novel therapeutic targets to reduce cerebral IR injury.     

IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and its pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction and oxidative stress play central roles in the pathophysiology of PD, through activation of mitochondria-dependent apoptotic molecular pathways. Several mitochondrial internal regulating factors act to maintain mitochondrial function. However, the mechanism by which these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2), has been implicated in the regulation of mitochondrial redox balance and reduction of oxidative stress-induced cell injury. Here we report that IDH2 regulates mitochondrial dysfunction and cell death in MPP⁺/MPTP-induced DA neuronal cells, and in a mouse model of PD. Down-regulation of IDH2 increased DA neuron sensitivity to MPP⁺; lowered IDH2 levels facilitated induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Deficient IDH2 also promoted loss of DA SNpc neurons in an MPTP mouse model of PD. Interestingly, Mito-TEMPO, a mitochondrial ROS-specific scavenger, protected degeneration of SNpc DA neurons in the MPTP model of PD. These findings demonstrate that IDH2 contributes to degeneration of the DA neuron in the neurotoxin model of PD and establish IDH2 as a molecular target of potential therapeutic significance for this disabling neurological illness.     

LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.     

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

α2,6-Sialylation promotes hepatocellular carcinoma cells migration and invasion via enhancement of nSmase2-mediated exosomal miRNA sorting

Journal of Physiology and Biochemistry - Exosomes have a critical role in the intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). Recently, our group showed...     

Differences in Gα12- and Gα13-mediated plasma membrane recruitment of p115-RhoGEF

Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein α subunits to the small GTPase Rho. Stimulation of G_12/13-coupled GPCRs or expression of constitutively activated forms of α₁₂ and α₁₃ has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for α₁₃-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent α₁₂-induced PM translocation of p115-RhoGEF. Additional differences between α₁₃ and α₁₂ in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated α₁₂ effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas α₁₃ only weakly does. On the other hand, α₁₃ strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, α₁₂ is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of α_12/13-mediated PM recruitment of p115-RhoGEF.     

Nitrated fatty acids prevent TNFα-stimulated inflammatory and atherogenic responses in endothelial cells

Nitration products (nitroalkenes) of linoleic acid (LNO₂) and oleic acid (OA-NO₂) can act as endogenous PPARγ ligands with electrophilic properties to exert anti-inflammatory effects on atherosclerotic plaques in the vasculature. Here, we show that OA-NO₂ and LNO₂ prevent tumor necrosis factor α (TNFα)-stimulated inflammatory and atherogenic responses in human umbilical vein endothelial cells (HUVECs). Both OA-NO₂ and LNO₂ prevented TNFα-stimulated release of the cytokines, IL-6, IL-8, IL-12/p40, IFNγ, MCP-1, and IP-10, and inhibited NF-κB activation. OA-NO₂ and LNO₂ also blocked TNFα-induced expression of the adhesion molecules, ICAM-1, VCAM-1, and E-selectin, and suppressed monocyte adhesion to HUVECs. In each case, OA-NO₂ was more potent and efficacious than was LNO₂, possibly due to increased stability in aqueous media. Collectively, these results substantiate a new functional role for nitrated fatty acids, demonstrating that OA-NO₂ and LNO₂ exert an anti-inflammatory function against the inflammatory cascade initiated by the representative pro-inflammatory cytokine, TNFα.     

The interaction between importin-α and Nup153 promotes importin-α/β-mediated nuclear import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.     

Porphyromonas gingivalis promotes malignancy and chemo-resistance via GSK3β-mediated mitochondrial oxidative phosphorylation in human esophageal squamous cell carcinoma

Our prior studies have confirmed that long-term colonization of Porphyromonas gingivalis (Pg) and overexpression of the inflammatory factor glycogen synthase kinase 3β (GSK3β) promote the malignant evolution of esophageal squamous cell carcinoma (ESCC). We aimed to investigate the functional mechanism by which Pg could promote ESCC malignancy and chemo-resistance through GSK3β-mediated mitochondrial oxidative phosphorylation (mtOXPHOS), and the clinical implications. The effects of Pg and GSK3β on mtOXPHOS, malignant behaviors and response to paclitaxel and cisplatin treatment of ESCC cells were evaluated by in vitro and in vivo studies. The results showed that Pg induced high expression of the GSK3β protein in ESCC cells and promoted the progression and chemo-resistance via GSK3β-mediated mtOXPHOS in human ESCC. Then, Pg infection and the expression of GSK3β, SIRT1 and MRPS5 in ESCC tissues were detected, and the correlations between each index and postoperative survival of ESCC patients were analysed. The results showed that Pg-positive ESCC patients with high-expression of GSK3β, SIRT1 and MRPS5 have significant short postoperative survival. In conclusion, we demonstrated that the effective removal of Pg and inhibition of its promotion of GSK3β-mediated mtOXPHOS may provide a new strategy for ESCC treatment and new insights into the aetiology of ESCC.     

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.     

CCL2 increases αvβ3 integrin expression and subsequently promotes prostate cancer migration

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family which is associated with the disease status and outcomes of cancers. Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. However, the effect of CCL2 on human prostate cancer cells is largely unknown. The aim of this study was to examine the role of CCL2 in integrin expression and migratory activity in prostate cancers.

Methods

Prostate cancer migration was examined using Transwell, wound healing, and invasion assay. The PKCδ and c-Src phosphorylations were examined by using western blotting. The qPCR was used to examine the mRNA expression of integrins. A transient transfection protocol was used to examine AP-1 activity.

Results

Stimulation of prostate cancer cell lines (PC3, DU145, and LNCaP) induced migration and expression of integrin αvβ3. Treatment of cells with αvβ3 antibody or siRNA abolished CCL2-increased cell migration. CCL2-increased migration and integrin expression were diminished by CCR2 but not by CCR4 inhibitors, suggesting that the CCR2 receptor is involved in CCL2-promoted prostate cancer migration. CCL2 activated a signal transduction pathway that includes PKCδ, c-Src, and AP-1. Reagents that inhibit specific components of this pathway each diminished the ability of CCL2 to effect cell migration and integrin expression.

Conclusions

Interaction between CCL2 and CCR2 enhances migration of prostate cancer cells through an increase in αvβ3 integrin production.

General significance

CCL2 is a critical factor of prostate cancer metastasis.     

A new non-canonical pathway of Gαq protein regulating mitochondrial dynamics and bioenergetics

Contrary to previous assumptions, G proteins do not permanently reside on the plasma membrane, but are constantly monitoring the cytoplasmic surfaces of the plasma membrane and endomembranes. Here, we report that the Gα_q and Gα₁₁ proteins locate at the mitochondria and play a role in a complex signaling pathway that regulates mitochondrial dynamics. Our results provide evidence for the presence of the heteromeric G protein (Gα_q/11βγ) at the outer mitochondrial membrane and for Gα_q at the inner membrane. Both localizations are necessary to maintain the proper equilibrium between fusion and fission; which is achieved by altering the activity of mitofusin proteins, Drp1, OPA1 and the membrane potential at both the outer and inner mitochondrial membranes. As a result of the absence of Gα_q/11, there is a decrease in mitochondrial fusion rates and a decrease in overall respiratory capacity, ATP production and OXPHOS-dependent growth. These findings demonstrate that the presence of Gα_q proteins at the mitochondria serves as a physiological function: stabilizing elongated mitochondria and regulating energy production in Drp1 and Opa1 dependent mechanisms. This thereby links organelle dynamics and physiology.     

Gαs promotes EEA1 endosome maturation and shuts down proliferative signaling through interaction with GIV (Girdin)

The organization of the endocytic system into biochemically distinct subcompartments allows for spatial and temporal control of the strength and duration of signaling. Recent work has established that Akt cell survival signaling via the epidermal growth factor receptor (EGFR) occurs from APPL early endosomes that mature into early EEA1 endosomes. Less is known about receptor signaling from EEA1 endosomes. We show here that EGF-induced, proliferative signaling occurs from EEA1 endosomes and is regulated by the heterotrimeric G protein Gαs through interaction with the signal transducing protein GIV (also known as Girdin). When Gαs or GIV is depleted, activated EGFR and its adaptors accumulate in EEA1 endosomes, and EGFR signaling is prolonged, EGFR down-regulation is delayed, and cell proliferation is greatly enhanced. Our findings define EEA1 endosomes as major sites for proliferative signaling and establish that Gαs and GIV regulate EEA1 but not APPL endosome maturation and determine the duration and strength of proliferative signaling from this compartment.