Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling.

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial-mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (ß-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.

FGF21 promotes thermogenic gene expression as an autocrine factor in adipocytes.

The contribution of adipose-derived FGF21 to energy homeostasis is unclear. Here we show that browning of inguinal white adipose tissue (iWAT) by ß-adrenergic agonists requires autocrine FGF21 signaling. Adipose-specific deletion of the FGF21 co-receptor ß-Klotho renders mice unresponsive to ß-adrenergic stimulation. In contrast, mice with liver-specific ablation of FGF21, which eliminates circulating FGF21, remain sensitive to ß-adrenergic browning of iWAT. Concordantly, transgenic overexpression of FGF21 in adipocytes promotes browning in a ß-Klotho-dependent manner without increasing circulating FGF21. Mechanistically, we show that ß-adrenergic stimulation of thermogenic gene expression requires FGF21 in adipocytes to promote phosphorylation of phospholipase C-γ and mobilization of intracellular calcium. Moreover, we find that the ß-adrenergic-dependent increase in circulating FGF21 occurs through an indirect mechanism in which fatty acids released by adipocyte lipolysis subsequently activate hepatic PPARα to increase FGF21 expression. These studies identify FGF21 as a cell-autonomous autocrine regulator of adipose tissue function.

Levels of ß-klotho determine the thermogenic responsiveness of adipose tissues: involvement of the autocrine action of FGF21.

Fibroblast growth factor-21 (FGF21) is a hormonal regulator of metabolism; it promotes glucose oxidation and the thermogenic capacity of adipose tissues. The levels of ß-klotho (KLB), the co-receptor required for FGF21 action, are decreased in brown (BAT) and white (WAT) adipose tissues during obesity, diabetes, and lipodystrophy. Reduced ß-klotho levels have been proposed to account for FGF21 resistance in these conditions. In this study, we explored whether downregulation of ß-klotho affects metabolic regulation and the thermogenic responsiveness of adipose tissues using mice with total (KLB-KO) or partial (KLB-heterozygotes) ablation of ß-klotho. We herein show that KLB gene dosage was inversely associated with adiposity in mice. Upon cold exposure, impaired browning of subcutaneous WAT and milder alterations in BAT were associated with reduced KLB gene dosage in mice. Cultured brown and beige adipocytes from mice with total or partial ablation of the KLB gene showed reduced thermogenic responsiveness to ß3-adrenergic activation by treatment with CL316,243, indicating that these effects were cell-autonomous. Deficiency in FGF21 mimicked the KLB-reduction-induced impairment of thermogenic responsiveness in brown and beige adipocytes. These results indicate that the levels of KLB in adipose tissues determine their thermogenic capacity to respond to cold and/or adrenergic stimuli. Moreover, an autocrine action of FGF21 in brown and beige adipocytes may account for the ability of the KLB level to influence thermogenic responsiveness.NEW & NOTEWORTHY Reduced levels of KLB (the obligatory FGF21 co-receptor), as occurring in obesity and type 2 diabetes, reduce the thermogenic responsiveness of adipose tissues in cold-exposed mice. Impaired response to ß3-adrenergic activation in brown and beige adipocytes with reduced KLB occurs in a cell-autonomous manner involving an autocrine action of FGF21.

Measurement of 45 cytokine, chemokine and growth factors in established cell culture supernatants and autologous serum from advanced melanoma patients.

Melanoma is one of the most aggressive forms of human cancer and its incidence has significantly increased worldwide over the last decades. This neoplasia has been characterized by the release of a wide variety of soluble factors, which could stimulate tumor cell proliferation and survival in an autocrine and paracrine manner. Consequently, we sought to evaluate the pattern of soluble factors produced by pre-metastatic and metastatic melanoma established cultures, and to determine whether these factors can be detected in the autologous serum of malignant melanoma patients. Our results showed that both melanoma cultures had a common profile of 27 soluble factors mainly characterized by the high expression of VEGF-A, IL-6, MCP-1, IL-8, and SDF-1. In addition, when we compared supernatants, we observed significant differences in VEGF-A, BDNF, FGF-2, and NGF-ß concentrations. As we found in melanoma cultures, serum samples also had their specific production pattern composed by 21 soluble factors. Surprisingly, PDGF-BB and EGF were only found in serum, whereas IL-2, IL-4, IL-8, IL31, FGF2, and GRO-α were only expressed in the supernatant. Significant differences in PDGF-BB, MIP-1ß, HGF, PIGF-1, BDNF, EGF, Eotaxin, and IP-10 were also found after comparing autologous serum with healthy controls. According to this, no correlation was found between culture supernatants and autologous serum samples, which suggests that some factors may act locally, and others systemically. Nonetheless, after validation of our results in an independent cohort of patients, we concluded that PDGF-BB, VEGF-A, and IP-10 serum levels could be used to monitor different melanoma stages.

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

A GRN Autocrine-Dependent FAM135B/AKT/mTOR Feedforward Loop Promotes Esophageal Squamous Cell Carcinoma Progression.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and deadly diseases. In our previous comprehensive genomics study, we found that family with sequence similarity 135 member B (FAM135B) was a novel cancer-related gene, yet its biological functions and molecular mechanisms remain unclear. In this study, we demonstrate that the protein levels of FAM135B are significantly higher in ESCC tissues than in precancerous tissues, and high expression of FAM135B correlates with poorer clinical prognosis. Ectopic expression of FAM135B promoted ESCC cell proliferation in vitro and in vivo, likely through its direct interaction with growth factor GRN, thus forming a feedforward loop with AKT/mTOR signaling. Patients with ESCC with overexpression of both FAM135B and GRN had worse prognosis; multivariate Cox model analysis indicated that high expression of both FAM135B and GRN was an independent prognostic factor for patients with ESCC. FAM135B transgenic mice bore heavier tumor burden than wild-type mice and survived a relatively shorter lifespan after 4-nitroquinoline 1-oxide treatment. In addition, serum level of GRN in transgenic mice was higher than in wild-type mice, suggesting that serum GRN levels might provide diagnostic discrimination for patients with ESCC. These findings suggest that the interaction between FAM135B and GRN plays critical roles in the regulation of ESCC progression and both FAM135B and GRN might be potential therapeutic targets and prognostic factors in ESCC. SIGNIFICANCE: These findings investigate the mechanisms of FAM135B in promoting ESCC progression and suggest new potential prognostic biomarkers and therapeutic targets in patients with ESCC.

A feed-forward regulatory loop in adipose tissue promotes signaling by the hepatokine FGF21.

The cJun NH2-terminal kinase (JNK) signaling pathway is activated by metabolic stress and promotes the development of metabolic syndrome, including hyperglycemia, hyperlipidemia, and insulin resistance. This integrated physiological response involves cross-talk between different organs. Here we demonstrate that JNK signaling in adipocytes causes an increased circulating concentration of the hepatokine fibroblast growth factor 21 (FGF21) that regulates systemic metabolism. The mechanism of organ crosstalk is mediated by a feed-forward regulatory loop caused by JNK-regulated FGF21 autocrine signaling in adipocytes that promotes increased expression of the adipokine adiponectin and subsequent hepatic expression of the hormone FGF21. The mechanism of organ cross-talk places circulating adiponectin downstream of autocrine FGF21 expressed by adipocytes and upstream of endocrine FGF21 expressed by hepatocytes. This regulatory loop represents a novel signaling paradigm that connects autocrine and endocrine signaling modes of the same hormone in different tissues.

Abnormal expression of Rab27B in prostatic epithelial cells of benign prostatic hyperplasia alters intercellular communication.

Abnormal intraglandular stromal-epithelial interactions have been known as a main key contributing factor for development of Benign Prostatic Hyperplasia (BPH). However, the underlying mechanism for the dysregulated intercellular communication remains unclear. In this study we compared the proteomic profiles of hyperplastic tissue with adjacent normal tissue of BPH and identified Rab27B small GTPase, a key regulator of exocytosis, as a protein that was overexpressed in the epithelium of BPH tissue. Overexpression of Rab27B in prostatic epithelial cells strongly increased the signaling activities of the PI3K/AKT and ERK1/2 pathways, whereas, downregulation of Rab27B expression in the epithelial cells of BPH reduced the signaling activities and decreased cell proliferation. The elevated Rab27B expression caused an overall increase in cell surface presentation of growth factor receptors without affecting their expression. However, the small GTPase also possesses an inhibitory activity against mTORC1 independent of its role in cell surface presentation of growth factor receptors. Our findings demonstrate a pivotal role of the small GTPase in autocrine and paracrine signaling and suggest that its abnormal expression underlies the dysregulated stromal-epithelial interactions in BPH.

Autocrine IL-6/STAT3 signaling aids development of acquired drug resistance in Group 3 medulloblastoma.

Medulloblastoma (MB) is a high-grade pediatric brain malignancy that originates from neuronal precursors located in the posterior cranial fossa. In this study, we evaluated the role of STAT3 and IL-6 in a tumor microenvironment mediated drug resistance in human MBs. We established that the Group 3 MB cell line, Med8A, is chemosensitive (hence Med8A-S), and this is correlated with a basal low phosphorylated state of STAT3, while treatment with IL-6 induced robust increases in pY705-STAT3. Via incremental selection with vincristine, we derived the stably chemoresistant variant, Med8A-R, that exhibited multi-drug resistance, enhanced IL-6 induced pY705-STAT3 levels, and increased IL6R expression. Consequently, abrogation of STAT3 or IL6R expression in Med8A-R led to restored chemosensitivity to vincristine, highlighting a prominent role for canonical IL-6/STAT3 signaling in acquired drug resistance. Furthermore, Med8A-S subjected to conditioning exposure with IL-6, termed Med8A-IL6+ cells, exhibited enhanced vincristine resistance, increased expression of pY705-STAT3 and IL6R, and increased secretion of IL-6. When cocultured with Med8A-IL6+ cells, Med8A-S cells exhibited increased pY705-STAT3 and increased IL-6 secretion, suggesting a cytokine feedback loop responsible for amplifying STAT3 activity. Similar IL-6 induced phenomena were also observed in the Group 3 MB cell lines, D283 and D341, including increased pY705-STAT3, drug resistance, IL-6 secretion and IL6R expression. Our study unveiled autocrine IL-6 as a promoter of STAT3 signaling in development of drug resistance, and suggests therapeutic benefits for targeting the IL-6/STAT3 signaling axis in Group 3 MBs.

miR-21a in exosomes from Lewis lung carcinoma cells accelerates tumor growth through targeting PDCD4 to enhance expansion of myeloid-derived suppressor cells.

In patients with lung cancer, myeloid-derived suppressor cells (MDSCs) have been reported to be significantly increased. Tumor-derived exosomes (TDEs) from various cancers played a critical role in MDSC induction. However, studies on the molecular mechanism underlying MDSC expansion induced by exosomes from lung cancer cells are still limited. In this study, we demonstrated that LLC-Exo accelerated tumor growth along with a significant accumulation of MDSCs in mouse tumor model. miRNA profiling showed that miR-21a was enriched in LLC-Exo. The depletion of miR-21a in LLC-Exo leads to the loss of their ability to induce MDSC expansion. Further results showed that miR-21a of LLC-Exo induced MDSC expansion via downregulation of the programmed cell death protein 4 (PDCD4) protein. The results of gain-and loss-of-function experiments validated that PDCD4 function as a critical inhibitor to negatively regulate expansion of MDSCs via inhibition Il-6 production in bone marrow cells. In addition, our data showed that exosomes derived from human lung cancer cell lines expressing miR-21a, also induced expansion of MDSCs in human CD14+ monocytes in vitro. Overall, our results demonstrated that miR-21a enriched in lung carcinoma cell-derived exosomes could promote functional expansion of MDSCs through targeting PDCD4.

The LTB4-BLT1 axis regulates actomyosin and ß2-integrin dynamics during neutrophil extravasation.

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and ß2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons.

Protein kinase type C-ε (PKCε) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKCε is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKCε, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKCε via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain.

Signaling pathways and promoter regions that mediate pituitary adenylate cyclase activating polypeptide (PACAP) self-regulation in gonadotrophs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a role in the development and regulation of gonadotrophs. PACAP levels are very high in the rodent fetal pituitary, and decline substantially and rapidly at birth, followed by a significant rise in FSHß and GnRH-R expression. Because there is evidence that PACAP stimulates its own transcription, we propose that this self-regulation is interrupted around the time of birth. To begin to examine the mechanisms for PACAP self-regulation, we used two well-established gonadotroph cell lines, αT3-1 cells and the more mature LßT2 cells which were transfected with a PACAP promoter-reporter construct As in vivo, the basal PACAP transcription level is significantly lower in the more mature LßT2 cells in which basal cAMP signaling is also much reduced. The PACAP promoter was stimulated by PACAP in both cell lines. Treatment with inhibitors of second messenger pathways implicated PKA, PKC and MAPK in PACAP transcription. Three regions of the PACAP promoter were found to confer inhibition or stimulation of PACAP transcription. By inhibiting cAMP response element binding (CREB) activity and mutating a proximal CREB binding site, we found that CREB is essential for promoter activation. Finally, overexpression of PACAP receptor HOP1 isoform, to increase the level in LßT2 cells to that of αT3-1 cells and simulate the E19 pituitary, increased PACAP- stimulated sensitivity and significantly altered downstream gene transcription. These results provide novel insight into the feed-forward regulation of PACAP expression that may help initiate gonadotroph function at birth.

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain­of­function (treatment with recombinant activin A) or loss­of­function [treatment with activin A­antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA­mediated depletion of activin A was also tested. The profile of pro­ and anti­angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription­qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30­4.71; P=0.006) for disease­specific survival and 2.09 (95% CI, 1.07­4.08l: P=0.03) for disease­free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A­depleted OSCC cells. Activin A­knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro­angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

Involvement of CXCL12/CXCR4 in the motility of human first-trimester endometrial epithelial cells through an autocrine mechanism by activating PI3K/AKT signaling.

BACKGROUND: CXCL12(chemokine ligand 12, CXCL12) and its receptors CXCR4 are widely expressed in maternal-fetal interface and plays an adjust role in materno-fetal dialogue and immune tolerance during early pregnancy. This study aimed to evaluate the role and mechanism of self-derived CXCL12 in modulating the functions of human first-trimester endometrial epithelial cells (EECs) and to identify the potential protein kinase signaling pathways involved in the CXCL12/CXCR4's effect on EECs. METHODS: The expression of CXCL12 and CXCR4 in EECs was measured by using immunohistochemistry, immunofluorescence, real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The effects of EEC-conditioned medium (EEC-CM) and recombinant human CXCL12 (rhCXCL12) on EEC migration and invasion in vitro were evaluated with migration and invasion assays. In-cell western blot analysis was used to examine the phosphorylation of protein kinase B (AKT), extracellular regulated protein kinases (ERKs) and phosphatidylinositol 3-kinase (PI3K) after CXCL12 treatment. RESULTS: CXCL12 and CXCR4 were both expressed in human first-trimester EECs at the mRNA and protein level. Both EEC-CM and rhCXCL12 significantly increased the migration and invasion of EECs (P < 0.05), which could be blocked by neutralizing antibodies against CXCR4 (P < 0.05) or CXCL12 (P < 0.05), respectively. CXCL12 activated both PI3K/AKT and ERK1/2 signaling and CXCR4 neutralizing antibody effectively reduced CXCL12-induced phosphorylation of AKT and ERK1/2. LY294002, a PI3K-AKT inhibitor, was able to reverse the promotive effect of EEC-CM or rhCXCL12 on EEC migration and invasion. CONCLUSIONS: Human first-trimester EECs promoted their own migration and invasion through the autocrine mechanism with CXCL12/CXCR4 axis involvement by activating PI3K/AKT signaling. This study contributes to a better understanding of the epithelium function mediated by chemokine and chemokine receptor during normal pregnancy.

IFNγ and TNFα mediate CCL22/MDC production in alveolar macrophages after hemorrhage and resuscitation.

Acute lung injury is a major complication of hemorrhagic shock and the required resuscitation with large volumes of crystalloid fluids and blood products. We previously identified a role of macrophage-derived chemokine (CCL22/MDC) pulmonary inflammation following hemorrhage and resuscitation. However, further details regarding the induction of CCL22/MDC and its precise role in pulmonary inflammation after trauma remain unknown. In the current study we used in vitro experiments with a murine alveolar macrophage cell line, as well as an in vivo mouse model of hemorrhage and resuscitation, to identify key regulators in CCL22/MDC production. We show that trauma induces expression of IFNγ, which leads to production of CCL22/MDC through a signaling mechanism involving p38 MAPK, NF-κB, JAK, and STAT-1. IFNγ also activates TNFα production by alveolar macrophages, potentiating CCL22/MDC production via an autocrine mechanism. Neutralization of IFNγ or TNFα with specific antibodies reduced histological signs of pulmonary injury after hemorrhage and reduced inflammatory cell infiltration into the lungs.

Tumor cell-derived angiopoietin-like protein 2 establishes a preference for glycolytic metabolism in lung cancer cells.

We previously revealed that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates the metastatic capacity of tumors in an autocrine/paracrine manner by activating tumor cell motility and invasiveness and the epithelial-mesenchymal transition. However, the effects of ANGPTL2 on cancer cell glycolytic metabolism, which is a hallmark of tumor cells, are unknown. Here we report evidence supporting a role for tumor cell-derived ANGPTL2 in establishing a preference for glycolytic metabolism. We report that a highly metastatic lung cancer cell subline expressing abundant ANGPTL2 showed upregulated expression of the glucose transporter GLUT3 as well as enhanced glycolytic metabolism relative to a less metastatic parental line. Most notably, ANGPTL2 overexpression in the less metastatic line activated glycolytic metabolism by increasing GLUT3 expression. Moreover, ANGPTL2 signaling through integrin α5ß1 increased GLUT3 expression by increasing transforming growth factor-ß (TGF-ß) signaling and expression of the downstream transcription factor zinc finger E-box binding homeobox 1 (ZEB1). Conversely, ANGPTL2 knockdown in the highly metastatic subline decreased TGF-ß1, ZEB1, and GLUT3 expression and antagonized glycolytic metabolism. In primary tumor cells from patients with lung cancer, ANGPTL2 expression levels correlated with GLUT3 expression. Overall, this work suggests that tumor cell-derived ANGPTL2 accelerates activities associated with glycolytic metabolism in lung cancer cells by activating TGF-ß-ZEB1-GLUT3 signaling.