Interorgan Communication Pathways in Physiology: Focus on Drosophila.

Studies in mammals and Drosophila have demonstrated the existence and significance of secreted factors involved in communication between distal organs. In this review, primarily focusing on Drosophila, we examine the known interorgan communication factors and their functions, physiological inducers, and integration in regulating physiology. Moreover, we describe how organ-sensing screens in Drosophila can systematically identify novel conserved interorgan communication factors. Finally, we discuss how interorgan communication enabled and evolved as a result of specialization of organs. Together, we anticipate that future studies will establish a model for metazoan interorgan communication network (ICN) and how it is deregulated in disease.

Discovery of thymosin ß4 as a human exerkine and growth factor.

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in interorgan cross talk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin ß4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in the plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular cross talk.

Direct Comparison of Therapeutic Effects on Diabetic Polyneuropathy between Transplantation of Dental Pulp Stem Cells and Administration of Dental Pulp Stem Cell-Secreted Factors.

Stem cell transplantation is a potential novel therapy for diabetic polyneuropathy. Dental pulp stem cells (DPSCs) are attractive stem cell sources because DPSCs can be isolated from extracted teeth and cryopreserved while retaining viability. In this study, we directly compared the efficacy of the transplantation of DPSCs and the administration of the secreted factors from DPSCs (DPSC-SFs) on diabetic polyneuropathy. Eight weeks after streptozotocin injection, DPSCs (1.0 × 106 cells/rat) or DPSC-SFs (1.0 mL/rat) were administered into the unilateral hindlimb skeletal muscles of diabetic Sprague-Dawley rats. DPSC transplantation and DPSC-SF administration did not affect blood glucose levels and body weights in the diabetic rats. Both DPSC transplantation and DPSC-SF administration significantly ameliorated sciatic nerve conduction velocity and sciatic nerve blood flow, accompanied by increases in muscle bundle size, vascular density in the skeletal muscles and intraepidermal nerve fiber density in the diabetic rats, while there was no difference between the results for DPSCs and DPSC-SFs. These results suggest that the efficacy of both DPSC transplantation and DPSC-SF administration for diabetic polyneuropathy four weeks after transplantation/administration was mainly due to the multiple secretomes secreted from transplanted DPSCs or directly injected DPSC-SFs in the early phase of transplantation/administration.

STAT3 activation confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive breast cancer.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that has been approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. Despite the remarkable efficacy of T-DM1 in many patients, resistance to this therapeutic has emerged as a significant clinical problem. In the current study, we used BT-474/KR cells, a T-DM1-resistant cell line established from HER2-positive BT-474 breast cancer cells, as a model to investigate mechanisms of T-DM1 resistance and explore effective therapeutic regimens. We show here for the first time that activation of signal transducer and activator of transcription 3 (STAT3) mediated by leukemia inhibitory factor receptor (LIFR) overexpression confers resistance to T-DM1. Moreover, secreted factors induced by activated STAT3 in resistant cells limit the responsiveness of cells that were originally sensitive to T-DM1. Importantly, STAT3 inhibition sensitizes resistant cells to T-DM1, both in vitro and in vivo, suggesting that the combination T-DM1 with STAT3-targeted therapy is a potential treatment for T-DM1-refractory patients.

An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umbilical cord, using a culture medium supplemented with human platelet lysate as an alternative source to fetal bovine serum. Our procedure successfully maximizes the yield of viable MSCs that maintain canonical key features. Importantly, under these conditions, the compositional profile and biological effects elicited by the conditioned medium preparations derived from these MSC populations do not depend on donor individuality. Moreover, approximately 120 L of conditioned medium could be obtained from a single umbilical cord, which provides a suitable framework to produce industrial amounts of toxic-free conditioned medium with predictable composition.

Secreted factors from dental pulp stem cells improve Sjögren's syndrome via regulatory T cell-mediated immunosuppression.

BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disease primarily characterized by inflammation in the salivary and lacrimal glands. Activated T cells contribute to disease pathogenesis by producing proinflammatory cytokines, which leads to a positive feedback loop establishment. The study aimed to evaluate the effects of secreted factors derived from dental pulp stem cells (DPSCs) or bone marrow mesenchymal stem cells (BMMSCs) on hyposalivation in SS and to investigate the mechanism involved. METHODS: Eighty percent confluent stem cells were replenished with serum-free Dulbecco's modified Eagle's medium and incubated for 48 h; following which, conditioned media from DPSCs (DPSC-CM) and BMMSCs (BMMSC-CM) were collected. Cytokine array analysis was performed to assess the types of cytokines present in the media. Flow cytometric analysis was performed to evaluate the number of activated T cells cultured in DPSC-CM or BMMSC-CM. Subsequently, DPSC-CM or BMMSC-CM was administered to an SS mouse model. The mice were categorized into the following groups (n = 6 each): non-treatment, Dulbecco's modified Eagle's medium (-), BMMSC-CM, and DPSC-CM. Histological analysis of the salivary glands was performed. The gene and protein expression levels of cytokines associated with T helper subsets in the submandibular glands (SMGs) were evaluated. RESULTS: DPSC-CM contained more secreted factors with tissue-regenerating mechanisms, such as cell proliferation, anti-inflammatory effects, and immunomodulatory effects. DPSC-CM was more effective in suppressing the activated T cells than other groups in the flow cytometric analysis. The stimulated salivary flow rate increased in SS mice with DPSC-CM compared with that in the other groups. In addition, the number of inflammation sites in SMGs of the mice administered with DPSC-CM was lower than that in the other groups. The expression levels of interleukin (Il)-10 and transforming growth factor-ß1 were upregulated in the DPSC-CM group, whereas those of Il-4 and Il-17a were downregulated. The DPSC-CM-administered group presented with a significantly increased percentage of regulatory T (Treg) cells and a significantly decreased percentage of type 17 Th (Th17) cells compared with the other groups. CONCLUSIONS: These results indicated that DPSC-CM ameliorated SS by promoting Treg cell differentiation and inhibiting Th17 cell differentiation in the mouse spleen.

Immune Cell Types and Secreted Factors Contributing to Inflammation-to-Cancer Transition and Immune Therapy Response.

Although chronic inflammation increases many cancers' risk, how inflammation facilitates cancer development is still not well studied. Recognizing whether and when inflamed tissues transition to cancerous tissues is of utmost importance. To unbiasedly infer molecular events, immune cell types, and secreted factors contributing to the inflammation-to-cancer (I2C) transition, we develop a computational package called "SwitchDetector" based on liver, gastric, and colon cancer I2C data. Using it, we identify angiogenesis associated with a common critical transition stage for multiple I2C events. Furthermore, we infer infiltrated immune cell type composition and their secreted or suppressed extracellular proteins to predict expression of important transition stage genes. This identifies extracellular proteins that may serve as early-detection biomarkers for pre-cancer and early-cancer stages. They alone or together with I2C hallmark angiogenesis genes are significantly related to cancer prognosis and can predict immune therapy response. The SwitchDetector and I2C database are publicly available at www.inflammation2cancer.org.

Mapping the molecular signatures of diet-induced NASH and its regulation by the hepatokine Tsukushi.

OBJECTIVE: Nonalcoholic steatohepatitis (NASH) is closely associated with metabolic syndrome and increases the risk for end-stage liver disease, such as cirrhosis and hepatocellular carcinoma. Despite this, the molecular events that influence NASH pathogenesis remain poorly understood. The objectives of the current study are to delineate the transcriptomic and proteomic signatures of NASH liver, to identify potential pathogenic pathways and factors, and to critically assess their role in NASH pathogenesis. METHODS: We performed RNA sequencing and quantitative proteomic analyses on the livers from healthy and diet-induced NASH mice. We examined the association between plasma levels of TSK, a newly discovered hepatokine, and NASH pathologies and reversal in response to dietary switch in mice. Using TSK knockout mouse model, we determined how TSK deficiency modulates key aspects of NASH pathogenesis. RESULTS: RNA sequencing and quantitative proteomic analyses revealed that diet-induced NASH triggers concordant reprogramming of the liver transcriptome and proteome in mice. NASH pathogenesis is linked to elevated plasma levels of the hepatokine TSK, whereas dietary switch reverses NASH pathologies and reduces circulating TSK concentrations. Finally, TSK inactivation protects mice from diet-induced NASH and liver transcriptome remodeling. CONCLUSIONS: Global transcriptomic and proteomic profiling of healthy and NASH livers revealed the molecular signatures of diet-induced NASH and dysregulation of the liver secretome. Our study illustrates a novel pathogenic mechanism through which elevated TSK in circulation promotes NASH pathologies, thereby revealing a potential target for therapeutic intervention.

Lovastatin promotes myelin formation in NPC1 mutant oligodendrocytes.

Niemann-Pick Type C (NPC) disease is a rare neurovisceral disorder caused by mutations of either NPC1 or NPC2 gene and characterized by defective intracellular transport of cholesterol and glycosphingolipids, leading to neuron loss and myelin aberration in the central nervous system. In this study, by comparing protein expression in the cortical white matter tracts from mice at different postnatal days, we identified that in the NPC1 mutant (NPC1-/-) mice, the onset of myelination is delayed and the amount of the major myelin protein MBP and PLP, and oligodendrocyte regulatory factor Olig1 and Olig2, but not NG2 and Sox10, decreased significantly, suggesting a disruption of oligodendrocyte differentiation. Furthermore, in in vitro oligodendrocyte cultivation, NPC1-/- oligodendrocytes showed less response to the stimulation of neuron-conditioned medium (CdM), indicating a defect of oligodendrocyte per se. Interestingly, lovastatin restores the number of mature myelin-forming oligodendrocytes by increasing Olig1 and Olig2 expressions. Our data suggest a potential strategy for improving myelination using lovastatin in NPC disease.

The BMP inhibitor DAND5 in serum predicts poor survival in breast cancer.

BACKGROUND & AIMS: Breast cancer (BC) is prevalent worldwide malignant cancer. Improvements in timely and effective diagnosis and prediction are needed. As reported, secreted DAND5 is contributed to BC metastasis. We aim to assess whether DAND5 in peripheral blood serum could determine BC-specific mortality. METHODS: We used immunohistochemistry staining to detect DAND5 expression in our BC tissue array including 250 samples. Angiogenesis assay and xenograft mice model were used to examine the secreted DAND5 function in BC progression. Serum concentration of DAND5 was examined by ELISA in 1730 BC patients. Kaplan-Meier and adjusted Cox proportional hazards models were utilized to analyze the prognosis and survival of BC patients. RESULTS: Tissue array results showed that positive DAND5 staining cases displayed a higher likelihood of occurrence of disease events (HR=5.494; 95% CI: 1.008-2.353; P=0.048) in univariate analysis and remained the same trend in multivariate analysis (HR=2.537; 95% CI: 1.056-6.096; P=0.037). DAND5 positive patients exerted generally poor DFS (P=0.041) in the Kaplan-Meier survival analysis. Furthermore, secreted DAND5 promoted tumor growth and angiogenesis in vitro and in vivo. In addition, positive DAND5 in BC patients serum was associated with increased risk of disease events occurrence (univariate: HR=1.58; 95% CI: 1.206-2.070; P=0.001; multivariate: HR=1.4; 95% CI: 1.003-1.954; P=0.048) in univariate and multivariate survival analysis. In the Kaplan-Meier analysis, serum DAND5 positively correlated with poor DFS (P=0.001) and DDFS (P=0.002). CONCLUSIONS: DAND5 was correlated with poor survival and could serve as an easily detectable serum biomarker to predict the survival of breast cancer.

Astrocyte-secreted thrombospondin-1 modulates synapse and spine defects in the fragile X mouse model.

Astrocytes are key participants in various aspects of brain development and function, many of which are executed via secreted proteins. Defects in astrocyte signaling are implicated in neurodevelopmental disorders characterized by abnormal neural circuitry such as Fragile X syndrome (FXS). In animal models of FXS, the loss in expression of the Fragile X mental retardation 1 protein (FMRP) from astrocytes is associated with delayed dendrite maturation and improper synapse formation; however, the effect of astrocyte-derived factors on the development of neurons is not known. Thrombospondin-1 (TSP-1) is an important astrocyte-secreted protein that is involved in the regulation of spine development and synaptogenesis. In this study, we found that cultured astrocytes isolated from an Fmr1 knockout (Fmr1 KO) mouse model of FXS displayed a significant decrease in TSP-1 protein expression compared to the wildtype (WT) astrocytes. Correspondingly, Fmr1 KO hippocampal neurons exhibited morphological deficits in dendritic spines and alterations in excitatory synapse formation following long-term culture. All spine and synaptic abnormalities were prevented in the presence of either astrocyte-conditioned media or a feeder layer derived from FMRP-expressing astrocytes, or following the application of exogenous TSP-1. Importantly, this work demonstrates the integral role of astrocyte-secreted signals in the establishment of neuronal communication and identifies soluble TSP-1 as a potential therapeutic target for Fragile X syndrome.

Importance of the GDF9 signaling pathway on cumulus cell expansion and oocyte competency in sheep.

Acquisition of developmental competency in cumulus oocyte complexes (COCs) is derived from endocrine hormones and oocyte secreted factors. The contribution of these factors in oocyte maturation and development is an active area of research. The objective of this research was to investigate whether growth differentiation factor 9 (GDF9) that is secreted by oocyte affects cumulus expansion and oocyte development in sheep. Immature ovine COCs were cultured in the presence of recombinant human GDF9 (rhGDF9), denuded oocytes, SB-431542, a specific inhibitor of activin-like kinase 4/5/7; or a combination of these factors. Routine in vitro maturation of COCs and denuded oocytes were used as external control samples. Cultured COCs were used for assessment of (1) cumulus expansion; (2) expression of cumulus-related transcripts including pentraxin 3, hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, B-cell lymphoma 2 (BCL2), and Bcl2-associated X (BAX); and (3) yield and quality of embryo development. It was observed that cumulus expansion was not affected by any of these treatments. HAS2 mRNA expression confirmed this observation. In the presence of exogenous GDF9, cleavage rate was reduced, blastocyst rate did not differ from other groups, and trophectoderm cell number significantly increased. This suggests that exogenous GDF9 could improve embryo quality. It was also observed that oocyte secreted factors reduced proapoptotic BAX mRNA, and BCL2 mRNA expression was not significantly different from other groups. This study provides evidence that GDF9 signaling might have a minor influence on ovine cumulus expansion and oocyte development and that other signaling pathway(s) might have a dominant role.

Expressions and significances of LIF and RANTES in mouse model of bloodstream infection with single pathogen / 临床检验杂志

Objective To investigate the expressions of leukaemia inhibitory factor (LIF) and regulated upon activation,normal T cell expressed and secreted factor (RANTES) in mice with bloodstream infection by 4 different single pathogen and provide research basis for the early diagnosis of bacteriogenous bloodstream infection.Methods CD-1 (ICR,Institute of Cancer Research) mouse models of bloodstream infection with the standard strains of Staphylococcus aureus (S.aureus),Enterococcus faecalis (E.faecalis),Escherichia coli (E.coli) and Klebsiella pneumonia(K,pneumoniae) were established.The serum samples were collected at the 0.5,1,3,6,12,24 and 48 hours after infection and the concentrations of LIF and RANTES in mouse serum of experimental groups and control were detected by Luminex liquid chip system.Results The median lethal dose (LD50) of S.aureus,E.faecalis,E.coli and K.pneumoniae were 8.1 × 108/mL,9.6 × 108/mL,8.1 × 108/mL and 1.1 × 109/mL,respectively.The concentration of serum LIF was significantly increased in 1 hour after infection.The peak concentrations of LIF in the four groups were (51.6±5.0),(73.2±20.8),(7.3 ±0.9)and (6.1 ± 1.2) pg/mL respectively,and the differences were statistically significant compared with the control group (P ＜ 0.01).The concentrations of RANTES in E.faecalis group,E.coli group and K.pneumoniae group were increased after infection for 1 hour and increased significantly after infection for 3 hours.The increased concentrations of RANTES in E.coli group and K.pneumoniae group were more than those in S.aureus group and E.faecalis group.The peak concentrations of RANTES in S.aureus group,E.faecalis group,E.coli group and K.pneumoniae group were (1 929.0-± 25.2),(1 218.1 ± 227.4),(55.7 ± 10.0) and (179.2 ± 9.2)pg/mL,and the differences were statistically significant compared with the control group (P ＜ 0.01).Conclusion The concentrations of LIF and RANTES increased obviously in 1 h after the bacteria entered bloodstream.After 2 days of infections,the levels of LIF and RANTES in E.coli group and K.pneumoniae group were significantly higher than those in S.aureus group and the E.faecalis group.Combined detections of LIF and RANTES may be of certain values to differentiate the infections caused by the pathogens between gram positive and gram negative bacteria.

Downregulated expression of the secreted glycoprotein follistatin-like 1 (Fstl1) is a robust hallmark of preadipocyte to adipocyte conversion.

Obesity is a public health crisis in the United States. Targeting preadipocyte to adipocyte conversion may be an effective approach to regulate adipose mass. Using differential screening we identified Fstl1, a secreted glycoprotein with roles in immunomodulation, cell growth, cardioprotection, and vascularization, as a "preadipokine". Fstl1 is highly expressed in 3T3-L1 preadipocytes and dramatically downregulated early in their differentiation to adipocytes. Northern blot analysis of murine tissues reveals white adipose tissue (WAT), lung and heart as primary sites of Fstl1 transcript expression. In WAT, Fstl1 transcript is restricted to the preadipocyte-containing stromal-vascular cell population. Time course studies in multiple adipogenesis models reveal downregulation of Fstl1 is a hallmark of white and brown adipocyte conversion. By Western blot, we show culture media of 3T3-L1 preadipocytes contains high levels of Fstl1 protein that rapidly decline in adipocyte conversion. Moreover, we observe a correlation between preadipocyte phenotype and Fstl1 expression in that TNFalpha-mediated de-differentiation of 3T3-L1 adipocytes is accompanied by re-expression of Fstl1 transcript and protein. Treatment of 3T3-L1 preadipocytes with a panel of 18 hormones and other agents revealed the demethylating agent 5-aza-cytidine decreases Fstl1 transcript and protein levels by approximately 90%. Furthermore, of 10 additional preadipocyte-expressed genes analyzed we find Pref-1, Col1A1, Sca-1/Ly6a, Lox and Thbs2, are also downregulated by 5-aza-cytidine. Using luciferase reporter constructs containing 791 or 3922 bp of the Fstl1 5' flanking region, we determine negative transcriptional regulation by Kruppel-like factor 15. Together, our data suggest downregulation of Fstl1 expression may be an important feature of preadipocyte to adipocyte conversion.