Paracrine signaling | EPFL Graph Search

In cellular biology, paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance (local action), as opposed to cell signaling by endocrine factors, hormones which travel considerably longer distances via the circulatory system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain. Although paracrine signaling elicits a diverse array of responses in the induced cells, most paracrine factors utilize a relatively streamlined set of receptors and pathways. In fact, different organs in the body - even betw

Characterization of the paracrine stimulation potential of human CAP cells for cardiac repair

Xavier Adam

According to the last WHO numbers, cardiac diseases remain the first cause of death in the world. Cell therapy represents a promising approach to restore heart function. Cardiac derived adherent proliferation (CAP) cells were shown to support the heart regeneration by unexplained mechanisms when injected in a mouse myocarditis model. Not differentiating themselves in cardiomyocytes and expressing cellular markers for angiogenesis, they are now supposed to have a paracrine effect on heart resident cell populations. In the present work, this paracrine effect had to be characterized on a model cell line. A paracrine effect test system based the P19Cl6 teratocarcinoma cell line had to be established and used to carry out a first screening of paracrine interactions on CAP cells and mesenchymal stem cells (MSC) from bone marrow as reference. Initially, critical parameter of the test system had to be adjusted. Specific marker must be identified and their detection had to be proven possible in the designed test system. Suitable serum lots (k,g) have been identified on the base of growth rates and cardiac differentiation potential (beating activity, Gata-4, Myl2, Mef2c gene expression) using standard protocol (ɑMEM, 1% DMSO). Pretreatment of P19Cl6 with 10 [micro]M 5-azacytidine substantially raised the response to known potent cardiomyogenic inductors (DMSO, Oxytocin). Beating activity was remarkedly elevated, as well as Gata4 (5 to 300 fold), Myl2 (2 to 300 fold), and MEF2C (3 to 4 fold) expression induced compared to non-treated culture. Troponin I ELISA-(2 to 5 fold) and Connexin 43 imunnohistochemistry confirmed the reliable induction of cardiac differentiation in pretreated culture. Based on those results, the test system was conducted using 5-azacytidine pretreated P19Cl6 cells and explored their reactions to different direct (cell to cell in coculture) and indirect (secreted factors in preconditioned medium) interactions with CAP cells or MSC. Since no reference setup existed, several coculture compositions with fixed total cell number (10-90%) or fixed P19Cl6 cell number (10-1000%) were tested, as well as exposition to differently conditioned media (24h, 72h) in different dilutions (100%, 50%, 33%). Beating activity was almost never observed. However, CAP cell experiments (n=6) showed a significant increase in P19Cl6 GATA-4 expression compared to control P19Cl6 (3.3 fold). This expression was also increased in MSC analyses (n=4) but in small amount (1.8 fold). Screening of CAP and MSC interactions led to the identification of active/effective compositions and dilutions. In addition, aza pretreatment proved itself enhancing the sensitivity of the test system. To sum up, the results suggested that CAP cells as wells as MSC have a paracrine stimulation potential and gave starting points for deeper analyses.

2011

Immunosuppressive roles of Activin-A in melanoma and characterization of its precursor cleavage

Katarina Pinjusic

Activin-A, a transforming growth factor êµ family member, is a pleiotropic cytokine with diverse functions in development, fertility, adult tissue homeostasis, and aging. Accordingly, deregulation of Activin-A signaling has been associated with many pathological conditions, including cancer and cancer-associated muscle wasting (cachexia). Activin-A was shown to signal in endocrine, but also local autocrine and paracrine manner. Its secretion in many cancer types is associated with a poor prognosis. Previous studies suggest that Activin-A expression in melanoma promotes tumor growth and metastasis indirectly by inhibiting adaptive anti-tumor immunity, leading to a decrease in the intratumoral frequency of cytotoxic CD8+ T cells (CTLs). However, the underlying mechanisms and the relevant direct target cells of Activin-A and their significance for cancer therapies remained unknown. Similar to other TGFêµ-related ligands, proActivin-A is cleaved by proprotein convertases (PCs) to release mature ligands. However, unlike other family members whose signaling is tightly regulated, Activin-A apparently does not have latency.In this thesis, I characterize proActivin-A cleavage and show that one prodomain can be cleaved independently of known PCs. The resulting hemi-cleaved Activin-A can bind activin type II receptors and induce SMAD3 signaling in reporter cells. A protein interactome screen and analysis of cleavage intermediates in plasma revealed that the hemi-cleaved Activin-A likely interacts with extracellular matrix proteins via the remaining prodomain where it promotes local signaling and regulates trafficking. Luciferase reporter assay showed that hemi-cleaved Activin-A can avoid, at least partially, inhibition by endogenous inhibitors FSTL3 and FST, and enable parallel activation of BMP and Activin signaling pathways. Finally, analysis of B16-F1 melanoma models revealed that only fully cleaved Activin-A in source cells increased tumor growth by acting locally in the tumor microenvironment suggesting that the context of Activin-A precursor processing regulates its tumor growth-promoting effect.The second part of this thesis addresses the mechanisms of Activin-induced immunosuppression in melanoma. We found that Activin-A impairs CTL function indirectly and independently of CD4+ T cell, CSF1R+ macrophages, and IL4 signaling. At least in part, anti-tumor CTL responses are impaired by the inhibitory effects of Activin-A on the secretion of critical CTL-recruiting chemokines CXCL9 and CXCL10 by macrophages and dendritic cells (DCs). Furthermore, Activin-A conferred resistance to immune checkpoint blockade therapy. Characterization of Activin-induced changes in the gene expression of tumor-infiltrating cells by Single cell RNA sequencing revealed that INHBA expression induced the most changes in DCs and least in T cells. Interestingly, the most prominently induced hallmark in Activin-secreting tumors across cell types was the IFN signature. In vitro analyses showed that Activin-A increases IFNÎ³-induced activation of STAT1 and confers resistance to cytostatic IFN signaling in cancer cells.Altogether, the findings of this thesis expand our knowledge on the regulation of Activin-A signaling by precursor cleavage and offer insights into mechanisms of Activin-induced immunosuppression in melanoma. Inhibition of Activin-A maturation thus emerges as a potential new strategy to improve responses to immunotherapies in melanoma and likely other cancers

EPFL2022

CO-REGULATION OF ENDOTHELIAL AND MYOGENIC CELL FATES THROUGH INTERCELLULAR CROSSTALK IN SKELETAL MUSCLE

Umji Lee

Skeletal muscle is one of the most dynamic organs in the human body, which has a vigorous potential to regenerate and adapt to environmental changes. Depending on environments, skeletal muscle enables essential life activities to survive by controlling movement and metabolism. The functional and structural adaptations of skeletal muscle crosstalk with other physiological systems enabling nutrition and oxygen delivery, and the elimination of metabolic waste products. The work in this dissertation focuses on the intercellular signals produced by myogenic cells to dynamically remodel skeletal muscle during exercise or muscle repair, and the reciprocal signals from the local niche and the systemic environment that regulate myogenic cell fate and metabolism according to physiological needs. This complex crosstalk is dissected in three specific chapters where we study the crosstalk of myogenic cells with endothelial cells and the vasculature to coordinate local niche interactions and systemic crosstalks. First, we demonstrate that an exercise-induced myokine called apelin is produced by muscle fiber and mediates intercellular signaling to endothelial cells through the apelin receptor. In addition, through a yeast one-hybrid screen of transcription factor binding to the apelin promoter, we identified the myogenic transcription factor TEA domain family member 1 (Tead1) as a regulator of Apln transcription. We observed via single-cell transcriptomic analysis of regenerating skeletal muscle that Aplnr (Apelin receptor) is enriched in muscle endothelial cells, whereas Tead1 is enriched in myogenic cells. Myofiber-specific over-expression of Tead1 suppresses apelin secretion at the whole-body level, and apelin secretion via Tead1 knock-down in muscle cells stimulates endothelial cell proliferation in co-cultures. By showing that apelin peptide supplementation in vivo enhances endothelial cell expansion following muscle injury, we conclude that paracrine crosstalk in which apelin secretion controlled by Tead1 in myogenic cells influences endothelial remodeling during skeletal muscle repair. Secondly, we show how tissue-resident support cells affect muscle progenitor activation in different metabolic environments. Skeletal muscle progenitors (SKMP) reside in close proximity to supportive cell types in the stem cell niche and dynamically interact with each other to adapt to environmental changes. Here, we studied how different niche cell types affect SKMPs in response to glycemic levels. Using co-cultures of SKMPs and niche cells, we discovered that endothelial cells synergistically enhance SKMP proliferation in a low glycemic environment, while fibroblasts and macrophages had no effect. We observed that the crosstalk between SKMPs and endothelial cells was mediated by direct cell-cell contacts independent of soluble paracrine signals. Transcriptomic analysis revealed that the endothelial alpha/beta hydrolase N-Myc Downstream Regulated1 (NDRG1) is induced by a low glycemic environment and is associated with biological adhesion. SKMPs co-cultured with Ndrg1 knock-down endothelial cells lose their synergistic functional relationship in response to glucose levels. Therefore, our findings suggest that Ndrg1 is a key mediator of endothelial cell-mediated glycemic control of SKMPs and provides a link between systemic energy levels and the skeletal muscle stem cell niche. Lastly, I developed intravital imaging to monitor muscle stem cells and vasculature.

EPFL2021