ERK-dependent Pathway Research Articles

CCR5 activation stimulates adipocyte differentiation through ERK-dependent pathway

CCR5 activation stimulates adipocyte differentiation through ERK-dependent pathway

Inhibition of Amyloid-β (Aβ)-Induced Cognitive Impairment and Neuroinflammation in CHI3L1 Knockout Mice through Downregulation of ERK-PTX3 Pathway.

Several clinical studies reported that the elevated expression of Chitinase-3-like 1 (CHI3L1) was observed in patients suffering from a wide range of diseases: cancer, metabolic, and neurological diseases. However, the role of CHI3L1 in AD is still unclear. Our previous study demonstrated that 2-({3-[2-(1-Cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}culfanyl)-N-(4-ethylphenyl)butanamide, a CHI3L1 inhibiting compound, alleviates memory and cognitive impairment and inhibits neuroinflammation in AD mouse models. In this study, we studied the detailed correlation of CHI3L1 and AD using serum from AD patients and using CHI3L1 knockout (KO) mice with Aβ infusion (300 pmol/day, 14 days). Serum levels of CHI3L1 were significantly elevated in patients with AD compared to normal subjects, and receiver operating characteristic (ROC) analysis data based on serum analysis suggested that CHI3L1 could be a significant diagnostic reference for AD. To reveal the role of CHI3L1 in AD, we investigated the CHI3L1 deficiency effect on memory impairment in Aβ-infused mice and microglial BV-2 cells. In CHI3L1 KO mice, Aβ infusion resulted in lower levels of memory dysfunction and neuroinflammation compared to that of WT mice. CHI3L1 deficiency selectively inhibited phosphorylation of ERK and IκB as well as inhibition of neuroinflammation-related factors in vivo and in vitro. On the other hand, treatment with recombinant CHI3L1 increased neuroinflammation-related factors and promoted phosphorylation of IκB except for ERK in vitro. Web-based gene network analysis and our results showed that CHI3L1 is closely correlated with PTX3. Moreover, in AD patients, we found that serum levels of PTX3 were correlated with serum levels of CHI3L1 by Spearman correlation analysis. These results suggest that CHI3L1 deficiency could inhibit AD development by blocking the ERK-dependent PTX3 pathway.

Differential modulation of innate immune response by lipopolysaccharide of Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. having more than 300 serovars. These serovars can infect a variety of hosts, some being asymptomatic carriers and others showing varied symptoms of mild to severe infection. Since lipopolysaccharide (LPS) is the major antigen which defines serovar specificity, this different course of infection may be attributed to a differential innate response against this antigen. Previous studies have shown that Leptospira LPS is less endotoxic. However, it is unclear whether there is a difference in the ability of LPS isolated from different serovars to modulate the innate response. In this study, we purified LPS from three widely prevalent pathogenic serovars, i.e. Icterohaemorrhagiae strain RGA, Pomona, Hardjo, and from non-pathogenic L. biflexa serovar semeranga strain Potac 1 collectively termed as L-LPS and tested their ability to modulate innate response in macrophages from both resistant (mice) and susceptible (human and bovine) hosts. L-LPS induced differential response being more proinflammatory in mouse and less proinflammatory in human and bovine macrophages but overall less immunostimulatory than E. coli LPS (E-LPS). Irrespective of serovar, this response was TLR2-dependent in humans, whereas TLR4-dependent/CD14-independent in mouse using MyD88 adapter and signalling through P38 and ERK-dependent MAP kinase pathway. L-LPS-activated macrophages were able to phagocytose Leptospira and this effect was significantly higher or more pronounced when the macrophages were stimulated with L-LPS from the corresponding serovar. L-LPS activated both canonical and non-canonical inflammasome, producing IL-1β without inducing pyroptosis. Further, L-LPS induced both TNF-mediated early and NO-mediated late apoptosis. Altogether, these results indicate that L-LPS induces a differential innate response that is quite distinct from that induced by E-LPS and may be attributed to the structural differences and its atypical nature.

Apoptotic bodies inhibit inflammation by PDL1-PD1-mediated macrophage metabolic reprogramming.

Apoptosis triggers immunoregulation to prevent and suppress inflammation and autoimmunity. However, the mechanism by which apoptotic cells modulate immune responses remains largely elusive. In the context of allogeneic mesenchymal stem cells (MSCs) transplantation, long-term immunoregulation is observed in the host despite the short survive of the injected MSCs. In this study, utilizing a mouse model of acute lung injury (ALI), we demonstrate that apoptotic bodies (ABs) released by transplanted human umbilical cord MSCs (UC-MSCs) convert the macrophages from a pro-inflammatory to an anti-inflammatory state, thereby ameliorating the disease. Mechanistically, we identify the expression of programmed cell death 1 ligand 1 (PDL1) on the membrane of UC-MSCs-derived ABs, which interacts with programmed cell death protein 1 (PD1) on host macrophages. This interaction leads to the reprogramming of macrophage metabolism, shifting from glycolysis to mitochondrial oxidative phosphorylation via the Erk-dependent pathway in ALI. Importantly, we have reproduced the PDL1-PD1 effects of ABs on metabolic switch using alveolar macrophages from patients with ALI, suggesting the potential clinical implications of developing therapeutic strategies for the patients.

Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway.

Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its efficacy is severely limited by cell aging and senescence. Gremlin-1 (GREM1), a member of the bone morphogenetic protein antagonist family, has been implicated in cell proliferation and survival. However, GREM1's role in cell aging and senescence has never been investigated in human cardiac mesenchymal progenitor cells (hMPCs). Therefore, this study assessed the hypothesis that overexpression of GREM1 rejuvenates the cardiac regenerative potential of aging hMPCs to a youthful stage and therefore allows better capacity for myocardial repair. We recently reported that a subpopulation of hMPCs with low mitochondrial membrane potential can be sorted from right atrial appendage-derived cells in patients with cardiomyopathy and exhibit cardiac reparative capacity in a mouse model of myocardial infarction. In this study, lentiviral particles were used to overexpress GREM1 in these hMPCs. Protein and mRNA expression were assessed through Western blot and RT-qPCR. FACS analysis for Annexin V/PI staining and lactate dehydrogenase assay were used to assess cell survival. It was observed that cell aging and cell senescence led to a decrease in GREM1 expression. In addition, overexpression of GREM1 led to a decrease in expression of senescence genes. Overexpression of GREM1 led to no significant change in cell proliferation. However, GREM1 appeared to have an anti-apoptotic effect, with an increase in survival and decrease in cytotoxicity evident in GREM1-overexpressing hMPCs. Overexpressing GREM1 also induced cytoprotective properties by decreasing reactive oxidative species and mitochondrial membrane potential. This result was associated with increased expression of antioxidant proteins, such as SOD1 and catalase, and activation of the ERK/NRF2 survival signal pathway. Inhibition of ERK led to a decrease in GREM1-mediated rejuvenation in terms of cell survival, which suggests that an ERK-dependent pathway may be involved. Taken altogether, these results indicate that overexpression of GREM1 can allow aging hMPCs to adopt a more robust phenotype with improved survival capacity, which is associated with an activated ERK/NRF2 antioxidant signal pathway.

Three-dimensional growth reveals fine-tuning of 5-lipoxygenase by proliferative pathways in cancer.

The leukotriene (LT) pathway is positively correlated with the progression of solid malignancies, but the factors that control the expression of 5-lipoxygenase (5-LO), the central enzyme in LT biosynthesis, in tumors are poorly understood. Here, we report that 5-LO along with other members of the LT pathway is up-regulated in multicellular colon tumor spheroids. This up-regulation was inversely correlated with cell proliferation and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways. Furthermore, we found that E2F1 and its target gene MYBL2 were involved in the repression of 5-LO during cell proliferation. Importantly, we found that this PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO is also existent in tumor cells from other origins, suggesting that this mechanism is widely applicable to other tumor entities. Our data show that tumor cells fine-tune 5-LO and LT biosynthesis in response to environmental changes repressing the enzyme during proliferation while making use of the enzyme under cell stress conditions, implying that tumor-derived 5-LO plays a role in the manipulation of the tumor stroma to quickly restore cell proliferation.

ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50,upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

ABSTRACT The cold-shock protein Y-box-binding protein (YB)-1 regulates the expression of various chemokines and their receptors at the transcriptional level. Expression of the orphan chemokine CXCL14 is repressed by EGF induced signaling. The possible links between EGF-mediated YB-1 and CXCL14 as well as the functions of critical kinase pathways in the progression of prostate cancer have remained unexplored. Here we examined the correlation between YB-1 and CXCL14, and the ERK/AKT/mTOR pathways in prostate cancer. Knockdown of YB-1 decreased cyclinD1 expression with an upregulation of cleaved-PARP in human prostate cancer cells. EGF treatment upregulated phospho-YB-1 expression in a time-dependent manner, while treatment with an ERK inhibitor completely silenced its expression in prostate cancer cells. EGF treatment stimulates CyclinD1 and YB-1 phosphorylation in an ERK-dependent pathway. Positive and negative regulation of YB-1 and CXCL14 was observed after EGF treatment in prostate cancer cells, respectively. EGF rescues cell cycle and apoptosis via the AKT and ERK pathways. Furthermore, YB-1 silencing induces G1 arrest and apoptosis, while knockdown of CXCL14 facilitates cell growth and inhibits apoptosis in prostate cancer cells. YB-1 and CXCL14 were inversely correlated in prostate cancer cells and tissues. A significant association between poor overall survival and High YB-1 expression was observed in human prostate cancer patients. In conclusion, our data reveal the functional relationship between YB-1 and CXCL14 in EGF mediated ERK signaling, and YB-1 expression is a significant prognostic marker to predict prostate cancer.

P–801 Effects of Lonomia obliqua venom components on human endometrial stromal cells: A potential source for new cytoprotective biomolecules against recurrent pregnancy loss

Abstract Study question Could new molecules like Lonomia obliqua lipocalins and hemolins have cytoprotective effects on endometrial stem cells (hESC)? Summary answer Lonomia obliqua venom-induced hESC viability, proliferation and migration occurred mainly by protection against oxidative damage and ERK-dependent pathway activation What is known already Recurrent pregnancy loss (RPL) is associated with severe physical and psychological morbidity, for which there is no treatment options. The pathophysiology involves deficiency in proliferation and migration capacities of endometrial stromal cells (hESCs) impairing embryo implantation and development. Animal venoms are rich sources of bioactive molecules and despite its known toxic effects, they also have protective components such as pro-proliferative molecules, growth factor-like, anti-apoptotic and anti-oxidant. Study design, size, duration This study was an experimental in vitro with endometrial stem cells. Treatment duration was 8–72h. Every assay had control cells exposed to phosphate buffered saline (PBS). Participants/materials, setting, methods hESCs were isolated from fresh human endometrial biopsies and characterized according standard protocols. Then the effects of L. obliqua venomous secretions on cell viability, proliferation and migration were determined using MTT, wound-healing assay, sulforhodamine B (SRB) assay and measuring the immunocontent of Ki67. Venom components involved in cell enhancing effects were also identified by classical chromatographic methods and proteomic analysis. Assays were conducted in triplicate. Main results and the role of chance The hESCs in culture showed adhesiveness properties, presented a fusiform fibroblastoid morphology and ability to in vitro differentiate into adipocytes, osteocytes and chondrocytes. The expression of cell surface markers was also characterized by flow cytometry. hESCs were positive for mesenchymal markers (CD105, CD90 and CD73) and negative for hematopoietic markers (CD45 and CD11), indicating that isolated cells have potential for multilineage differentiation. L. obliqua bristle extract (LOBE) increased dose-dependently hESCs viability in a concentration range varying from 0.001 to 0.1 µg/mL, independently of the cell isolation bath. For some cell isolates (patient ID 1, 3, 4, 6 and 7) it was observed a slightly reduction in hESC viability at highest LOBE concentrations (10 µg/mL). Treatment increased hESC viability in the presence of low concentrations of fetal bovine serum (1% FBS) and even in its complete absence. This effect was long lasting, being significant up to 72 h of incubation with LOBE in serum deprivation conditions. r to identify the potential molecules involved in the cytoprotective action, a mass spectrometry-based proteomic analysis was performed. It was identified a total number of 430 proteins in LOBE and 312 proteins in L. obliqua hemolymph. Limitations, reasons for caution This study was only conducted in vitro. Wider implications of the findings: In this work we reported the identification of at least six protein classes with cytoprotective properties through proteomic analysis and isolated one fraction enriched in this cytoprotective factors. L. obliqua secretions induced increase in hESCs viability, proliferation and migration mainly by the protection against oxidative damage and ERK-dependent pathway activation. Trial registration number Not applicable

Survival Pathways Are Differently Affected by Microgravity in Normal and Cancerous Breast Cells.

Metazoan living cells exposed to microgravity undergo dramatic changes in morphological and biological properties, which ultimately lead to apoptosis and phenotype reprogramming. However, apoptosis can occur at very different rates depending on the experimental model, and in some cases, cells seem to be paradoxically protected from programmed cell death during weightlessness. These controversial results can be explained by considering the notion that the behavior of adherent cells dramatically diverges in respect to that of detached cells, organized into organoids-like, floating structures. We investigated both normal (MCF10A) and cancerous (MCF-7) breast cells and found that appreciable apoptosis occurs only after 72 h in MCF-7 cells growing in organoid-like structures, in which major modifications of cytoskeleton components were observed. Indeed, preserving cell attachment to the substrate allows cells to upregulate distinct Akt- and ERK-dependent pathways in MCF-7 and MCF-10A cells, respectively. These findings show that survival strategies may differ between cell types but cannot provide sufficient protection against weightlessness-induced apoptosis alone if adhesion to the substrate is perturbed.

Stimulation of hepatocarcinogenesis by activated cholangiocytes via Il17a/f1 pathway in kras transgenic zebrafish model

It has been well known that tumor progression is dependent on secreted factors not only from tumor cells but also from other surrounding non-tumor cells. In the current study, we investigated the role of cholangiocytes during hepatocarcinogenesis following induction of oncogenic krasV12 expression in hepatocytes using an inducible transgenic zebrafish model. Upon induction of carcinogenesis in hepatocytes, a progressive cell proliferation in cholangiocytes was observed. The proliferative response in cholangiocytes was induced by enhanced lipogenesis and bile acids secretion from hepatocytes through activation of Sphingosine 1 phosphate receptor 2 (S1pr2), a known cholangiocyte receptor involving in cholangiocyte proliferation. Enhancement and inhibition of S1pr2 could accelerate or inhibit cholangiocyte proliferation and hepatocarcinogenesis respectively. Gene expression analysis of hepatocytes and cholangiocytes showed that cholangiocytes stimulated carcinogenesis in hepatocytes via an inflammatory cytokine, Il17a/f1, which activated its receptor (Il17ra1a) on hepatocytes and enhanced hepatocarcinogenesis via an ERK dependent pathway. Thus, the enhancing effect of cholangiocytes on hepatocarcinogenesis is likely via an inflammatory loop.

Zinc deficiency induces epithelial barrier dysfunction and altered intestinal ion transport in novel murine model of Shigella flexneri diarrhea

Background: Zinc (Zn) deficiency has been linked with etiology for wide range of gastrointestinal diseases. Although low serum Zn has been associated with diarrhea and growth retardation, however the underlying mechanism of Zn deficiency aggravating the diarrheal manifestation remains elusive. Here, we have studied the physiological cascade elicited during Zn deficiency in both colonic cell lines and murine model leading to altered intestinal barrier function and ion transport. Methods and materials: T84 were grown onto transwell inserts in Zn deficient conditions for measurements of transepithelial electrical resistance (TER) and short-circuit current (Isc). Adult BALB/c mice fed with Zn deficient diet for 14 days and were intraperitoneally challenged with virulent S.flexneri 2a for following assessments of electrophysiological properties of the colonized colon in ussing chamber. Colonization evaluation from feces and colon homogenate were further analyzed for bacterial translocation assay. Results: Zn deficient cells infected with Shigella flexneri 2a showed significant increase in transmigration of the pathogens (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) for 6 h post infection along with poor formation of transepithelial resistance (1014 + 165 Ωcm2 vs 3663 + 293 Ωcm2). Ultrastructure of T84 cells grown in Zn deficient medium showed widened TJ complex and due to few membrane fusion proteins showing distorted TJ morphology and shorthened microvilli. Immunoblotting analysis further showed increased expression of Claudin-2 leaky tight junction protein in Zn deficient cells by modulating ERK dependent pathways. Following studies with Ussing chambers further demonstrated increase in Ca2+ dependent electrogenic Cl− secretion in Zn deficient cells. In vivo studies showed Zn deficient condition in mice increased bacterial translocation, colonization, bacterial shedding and pronounced disruption in intestinal permeability. Conclusion: Our results suggest that in T84 cells and murine of Shigellosis, Zn deficiency causes (1) pronounced barrier dysfunction and (2) altered Cl− secretion leading to heightened diarrheal pathogenesis thus linking micronutrient deficiency with bacterial infection and inflammation.

K284-6111 alleviates memory impairment and neuroinflammation in Tg2576 mice by inhibition of Chitinase-3-like 1 regulating ERK-dependent PTX3 pathway

BackgroundAlzheimer’s disease (AD) is one of the most prevalent neurodegenerative disorders characterized by gradual memory loss and neuropsychiatric symptoms. We have previously demonstrated that the 2-({3-[2-(1-cyclohexene-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111), the inhibitor of CHI3L1, has the inhibitory effect on memory impairment in Αβ infusion mouse model and on LPS-induced neuroinflammation in the murine BV-2 microglia and primary cultured astrocyte.MethodsIn the present study, we investigated the inhibitory effect of K284-6111 on memory dysfunction and neuroinflammation in Tg2576 transgenic mice, and a more detailed correlation of CHI3L1 and AD. To investigate the effects of K284-6111 on memory dysfunction, we administered K284-6111 (3 mg/kg, p.o.) daily for 4 weeks to Tg2576 mice, followed by behavioral tests of water maze test, probe test, and passive avoidance test.ResultsAdministration of K284-6111 alleviated memory impairment in Tg2576 mice and had the effect of reducing the accumulation of Aβ and neuroinflammatory responses in the mouse brain. K284-6111 treatment also selectively inactivated ERK and NF-κB pathways, which were activated when CHI3L1 was overexpressed, in the mouse brain and in BV-2 cells. Web-based gene network analysis and our results of gene expression level in BV-2 cells showed that CHI3L1 is closely correlated with PTX3. Our result revealed that knockdown of PTX3 has an inhibitory effect on the production of inflammatory proteins and cytokines, and on the phosphorylation of ERK and IκBα.ConclusionThese results suggest that K284-6111 could improve memory dysfunction by alleviating neuroinflammation through inhibiting CHI3L1 enhancing ERK-dependent PTX3 pathway.

Elaphuri Davidiani Cornu Improves Depressive-Like Behavior in Mice and Increases Neurotrophic Factor Expression in Mouse Primary Astrocytes via cAMP and ERK-Dependent Pathways.

Elaphuri Davidiani Cornu (EDC) is the natural shedding horn of Elaphurus davidiauus Millne-Edwards that was used by people in ancient China for maintaining physical and mental health. We evaluated the antidepressant effect of EDC using depression-like animal models and explored possible mechanisms in mouse primary astrocyte cultures. We found that aqueous extracts of EDC significantly improved depression-like behavior in a mouse model of depression. The extracts enhanced expression of nerve growth factor and brain-derived neurotrophic factor neurotrophic factors in mouse prefrontal cortex and hippocampus tissues. In the mouse primary astrocyte cultures, the EDC aqueous extracts significantly increased the neurotrophic factor expression both at the transcriptional and protein levels. EDC extracts might exhibit these functions by regulating matrix metalloprotein-9 of the nerve growth factor and brain-derived neurotrophic factor metabolic pathways and might enhance expression of neurotrophic factors via the cAMP- and ERK-dependent pathways. We confirmed this possibility by showing the effects of related inhibitors, providing scientific evidence that supports the utility of EDC in the development of drugs to treat major depressive disorders.

A G-protein coupled receptor 39 agonist stimulates proliferation of keratinocytes via an ERK-dependent pathway

Keratinocyte proliferation serves as a crucial process in skin wound healing. The zinc-sensing G-protein coupled receptor 39 (GPR39), which is highly expressed in keratinocytes, has been shown to promote skin wound healing. The aim of this study was to investigate the effect of GPR39 activation on proliferation of keratinocytes and its underlying mechanism using immortalized human keratinocytes (HaCaT) as an in vitro model. GPR39 was functionally expressed in HaCaT cells. BrdU proliferation assays showed that treatment with GPR39 agonist TC-G 1008 (100 nM and 1 μM) increased cell proliferation. TC-G 1008 also enhanced ERK phosphorylation in time- and concentration-dependent manners. This effect was suppressed by co-treatment with wortmannin (PI3K inhibitor) and U0126 (MKK inhibitor). Of note, neither inhibition of Gαq-phospholipase C (PLC)-[Ca2+]i nor Gαs-PKA pathway affected GPR39 stimulation-induced ERK phosphorylation. Similarly, barbadin, an inhibitor of G-protein-independent β-arrestin pathway, did not suppress ERK phosphorylation induced by GPR39 activation. Of particular importance, wortmannin, U0126, and FR180204 (ERK inhibitor) abrogated the effect of TC-G 1008-induced cell proliferation. Taken together, this study reveals novel insights into the role of GPR39 in regulating keratinocyte proliferation via a PI3K-MKK-ERK-dependent mechanism. GPR39 agonists may be used in enhancing keratinocyte proliferation, which may be beneficial for the cutaneous wound treatment.

Irisin Prevents Disuse-Induced Osteocyte Apoptosis.

Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research.

Sonic hedgehog signaling instigates high-fat diet–induced insulin resistance by targeting PPARγ stability

Obesity is a major risk for patients with chronic metabolic disorders including type 2 diabetes. Sonic hedgehog (Shh) is a morphogen that regulates the pancreas and adipose tissue formation during embryonic development. Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily and one of the most important regulators of insulin action. Here, we evaluated the role and mechanism of Shh signaling in obesity-associated insulin resistance and characterized its effect on PPARγ. We showed that Shh expression was up-regulated in subcutaneous fat from obese mice. In differentiated 3T3-L1 and primary cultured adipocytes from rats, recombinant Shh protein and SAG (an agonist of Shh signaling) activated an extracellular signal-regulated kinase (ERK)-dependent noncanonical pathway and induced PPARγ phosphorylation at serine 112, which decreased PPARγ activity. Meanwhile, Shh signaling degraded PPARγ protein via binding of PPARγ to neural precursor cell-expressed developmentally down-regulated protein 4-1 (NEDD4-1). Furthermore, vismodegib, an inhibitor of Shh signaling, attenuated ERK phosphorylation induced by a high fat diet (HFD) and restored PPARγ protein level, thus ameliorating glucose intolerance and insulin resistance in obese mice. Our finding suggests that Shh in subcutaneous fat decreases PPARγ activity and stability via activation of an ERK-dependent noncanonical pathway, resulting in impaired insulin action. Inhibition of Shh may serve as a potential therapeutic approach to treat obesity-related diabetes.

Curcumin inhibits hypoxia inducible factor-1α-induced inflammation and apoptosis in macrophages through an ERK dependent pathway.

Atherosclerosis, a kind of peripheral arterial disease with chronic inflammation, leads to the dysfunction of the vascular system and many other diseases. Hypoxia has been proven to participate in the progression of atherosclerosis, while curcumin can inhibit hypoxia-inducible factor 1α (HIF-1α). However, the underlying mechanisms are still elusive. qRT-PCR was used to examine the expression of HIF-1α, IL-6 and TNFα of macrophages under hypoxic condition. Western blot was applied to examine the changes of HIF-1α, ERK and p-ERK after treatment with curcumin. Oli Red O staining and enzymatic assay were used to examine the lipid and total cholesterol in macrophages, respectively. ELISA was used to examine the release of IL-6 and TNFα by macrophages. FACS and MTT assays were applied to examine the apoptosis and proliferation of macrophages. Here, we found curcumin inhibited the expression of HIF-1α at the protein level in macrophages under hypoxic condition and curcumin and HIF-1α inhibitors repressed the total cholesterol and lipid level in macrophage under hypoxic condition. Moreover, curcumin also decreased the expression of HIF-1α downstream genes, VEGF, HMOX1, ROS and PDGF. Then, the data show the HIF-1α-induced apoptosis and inflammation of macrophages were inhibited by curcumin. Curcumin also rescued the proliferation defect of macrophages caused by hypoxia. Furthermore, we found it inhibited the expression of HIF-1α via ERK signaling pathway. We describe that curcumin inhibited the HIF-1α-induced apoptosis and inflammation of macrophages via ERK signaling pathways. These results suggest curcumin can be used for the treatment of atherosclerosis.

Cigarette Smoke Extract Promotes TIM4 Expression in Murine Dendritic Cells Leading to Th2 Polarization through ERK-Dependent Pathways

Smoking is considered to be the main source of indoor pollution, and it has been identified as an important environmental factor contributing to asthma onset. We know that T helper 2 (Th2) response plays a crucial role in the process of asthma disease. We have investigated the reaction of cigarette smoke extract (CSE) on Th polarization which is controlled by dendritic cells (DCs). Stimulated by CSE, immature DCs from murine bone marrow showed upregulated levels of TIM4. Cocultured with CD4+ T cells, stimulated DCs increased the ratio of IL-4+ versus IFN-γ+ of CD4+ T cells. This suggests a differentiation towards Th2 response. Moreover, antibodies against TIM4 reversed the upexpression of the IL-4+/IFN-γ+ ratio provoked by CSE, indicating that the Th2 polarization which was induced by CSE is via TIM4 mechanisms. CSE could activate mitogen-activated protein kinase pathways like ERK and p38. Upregulation of TIM4 expression by CSE stimulation was found to be inhibited by an ERK inhibitor but not p38 and JNK. In conclusion, DC-induced Th2 polarization is a hallmark of CSE allergy, and this aspect can be explained by CSE-induced TIM4 expression.

RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway.

Members of the ribonuclease A (RNase A) superfamily regulate various physiological processes. RNase A, the best-studied member of the RNase A superfamily, is widely expressed in different tissues, including brains. We unexpectedly found that RNase A can trigger proliferation of neuronal progenitor cells (NPC) both in vitro and in vivo. RNase A treatment induced cell proliferation in dissociated neuronal cultures and increased cell mass in neurosphere cultures. BrdU (5-Bromo-2'-Deoxyuridine) labeling confirmed the effect of RNase A on cell proliferation. Those dividing cells were Nestin- and SOX2-positive, suggesting that RNase A triggers NPC proliferation. The proliferation inhibitor Ara-C completely suppressed the effect of RNase A on NPC counts, further supporting that RNase A increases NPC number mainly by promoting proliferation. Moreover, we found that RNase A treatment increased ERK phosphorylation and blockade of the ERK pathway inhibited the effect of RNase A on NPC proliferation. Intracerebroventricular injection of RNase A into mouse brain increased the population of 5-ethynyl-2'-deoxyuridine (EdU) or BrdU-labeled cells in the subventricular zone. Those RNase A-induced NPCs were able to migrate into other brain areas, including hippocampus, amygdala, cortex, striatum, and thalamus. In conclusion, our study shows that RNase A promotes proliferation of NPCs via an ERK-dependent pathway and further diversifies the physiological functions of the RNase A family.