Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway

Background

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4⁺CD105⁺CD90^neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4^negCD105⁺CD90^bright and MSCA-1⁺), in the primary cultures, resulted lower than 2%.

Methodology/Principal Finding

We demonstrate that MPCs differentiate to MSCs through an SSEA-4⁺CD105⁺CD90^bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4^neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ = 0.5 µM, p<0.01), while endothelial differentiation was unaffected.

Conclusion

The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.     

Daunorubicin induces cell death via activation of apoptotic signalling pathway and inactivation of survival pathway in muscle-derived stem cells

Daunorubicin (as well as other anthracyclines) is known to be toxic to heart cells and other cells in organism thus limiting its applicability in human cancer therapy. To investigate possible mechanisms of daunorubicin cytotoxicity, we used stem cell lines derived from adult rabbit skeletal muscle. Recently, we have shown that daunorubicin induces apoptotic cell death in our cell model system and distinctly influences the activity of MAP kinases. Here, we demonstrate that two widely accepted antagonistic signalling pathways namely proapoptotic JNK and prosurvival PI3K/AKT participate in apoptosis. Using the Western blot method, we observed the activation of JNK and phosphorylation of its direct target c-Jun along with inactivation of AKT and its direct target GSK in the course of programmed cell death. By means of small-molecule kinase inhibitors and transfection of cells with the genes of the components of these pathways, c-Jun and AKT, we confirm that JNK signalling pathway is proapoptotic, whereas AKT is antiapoptotic in daunorubicin-induced muscle cells. These findings could contribute to new approaches which will result in less toxicity and fewer side effects that are currently associated with the use of daunorubicin in cancer therapies.     

Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain, and oedema. A role in the modulation or induction of healthy breast tissue growth has been postulated for tissue kallikrein present in human milk. Moreover, tissue kallikrein was found in malignant human breast tissue and bradykinin (BK) stimulates the proliferation of immortalised breast cancer cells. Aim of the present article was to investigate whether BK also exerts mitogenic activity in normal breast epithelial cells and partially characterise the signalling machinery involved. Results show that BK increased up to 2-fold the 24 h proliferation of breast epithelial cells in primary culture, and that the BK B2 receptor (not B1) inhibitor alone fully blocked the BK response. Intracellular effects of B2 stimulation were the following: (a) the increase of free intracellular Ca(2+) concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol-to-membrane translocation of conventional (PKC)-alpha and -beta isozymes, novel PKC-delta, -epsilon, and -eta isozymes; (c) the phosphorylation of the extracellular-regulated kinase 1 and 2 (ERK1/2); and (d) the stimulation of the expression of c-Fos protein. EGF, a well known stimulator of cell proliferation, regulated the proliferative response in human epithelial breast cells to the same extent of BK. The effects of BK on proliferation, ERK1/2 phosphorylation, and c-Fos expression were abolished by GF109203X, which inhibits PKC-delta isozyme. Conversely, G?6976, an inhibitor of PKC-alpha and -beta isozymes, and the 18-h treatment of cells with PMA, that led to the complete down-regulation of PKC-alpha, -beta, -epsilon, and -eta, but not of PKC-delta, did not have any effect, thereby indicating that the PKC-delta mediates the mitogenic signalling of BK. Phosphoinositide 3-kinase (PI3K), tyrosine kinase of the epidermal growth factor receptor (EGFR), and mitogen activated protein kinase kinases (MEK) inhibitors were also tested. The results suggest that EGFR, PI3K, and ERK are required for the proliferative effects of BK. In addition, the BK induced cytosol-to-membrane translocation of PKC-delta was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC-delta. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC-delta through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation.     

Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src-overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c-src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain.     

Glycoprotein Ib-mediated platelet activation. A signalling pathway triggered by thrombin.

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin-GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active-site-blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin alphaIIbbeta3-dependent platelet-platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet-platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin.     

An isopentenyl-substituted flavonoid norartocarpin activates Nrf2 signalling pathway and prevents oxidative insults in human lung epithelial cells

The nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in regulating the intracellular oxidative stress, and thus activation of Nrf2 by nature-derived molecules effectively alleviates the pathological process of oxidative stress-induced chronic diseases. The isopentenyl-substituted flavonoid norartocarpin (NOR) induced the activity of NAD(P)H: quinone reductase (QR), implying that it might be a potential Nrf2 activator. Further studies indicated that NOR upregulated the protein levels of Nrf2 and its downstream genes, NAD(P)H quinone oxidoreductase 1 (NQO1), and γ-glutamyl cysteine synthetase (GCLM) through facilitating the nuclear translocation of Nrf2 and enhancing Nrf2 protein stability. NOR-induced activation of Nrf2 pathway was associated with multiple upstream kinases, including mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase C (PKC), and protein kinase R-like endoplasmic reticulum kinase (PERK). Moreover, NOR protected human lung epithelial Beas-2B cells against sodium arsenite [As(III)]-induced cytotoxicity in an Nrf2-dependent manner. Collectively, NOR was firstly identified to be an Nrf2 activator, which demonstrated the capability of preventing oxidative insults in human lung epithelial cells.     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.     

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aims

Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM.

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Notch signalling pathway in tooth development and adult dental cells

Notch signalling is a highly conserved intercellular signal transfer mechanism that includes canonical and non-canonical pathways. It regulates differentiation and proliferation of stem/progenitor cells by means of para-inducing effects. Expression and activation of Notch signalling factors (receptors and ligands) are critical not only for development of the dental germ but also for regeneration of injured tissue associated with mature teeth. Notch signalling plays key roles in differentiation of odontoblasts and osteoblasts, calcification of tooth hard tissue, formation of cusp patterns and generation of tooth roots. After tooth eruption, Notch signalling can also be triggered in dental stem cells of the pulp, where it induces them to differentiate into odontoblasts, thus generating fresh dentine tissue. Other signalling pathways, such as TGFβ, NF-κB, Wnt, Fgf and Shh also interact with Notch signalling during tooth development.     

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Background

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.

Methods

In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.

Results

We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.

Conclusions

Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

     

Ectodysplasin A regulates epithelial barrier function through sonic hedgehog signalling pathway

Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X‐linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive. In this study, we found tight junctional proteins ZO‐1 and claudin‐1 expression is largely reduced in epidermal, corneal and lung epithelia in Eda mutant Tabby mice at different postnatal ages. These declines are associated with tail ulceration, corneal pannus formation and lung infection. Furthermore, topical application of recombinant Eda protein markedly mitigated corneal barrier dysfunction. Using cultures of a human corneal epithelial cell line and Tabby mouse skin tissue explants, Eda up‐regulated expression of ZO‐1 and claudin‐1 through activation of the sonic hedgehog signalling pathway. We conclude that EDA gene expression contributes to the maintenance of epithelial barrier function. Such insight may help efforts to identify novel strategies for improving management of XLHED disease manifestations in a clinical setting.     

Rapid effects of androgens in macrophages

We investigated the existence of membrane receptors for testosterone (mAR) in mouse macrophages of the cell lines IC-21 and RAW 264.7 as well as their roles in nongenomic pathways, gene expression and cell functioning. Both cell lines lack intracellular androgen receptors (iARs) and respond to testosterone with rapid rises in [Ca2+]i. These rises in [Ca2+]i can neither be inhibited by iAR- nor by iER blockers, but are rather mediated through mAR. Pharmacological approaches suggest that the mAR belongs to the class of membrane receptors which are coupled to phospholipase C via pertussis toxin (PTX) sensitive G-proteins. The mAR can be localized as specific surface binding sites for testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM)and flow cytometry, and are characterized by their agonist-sequestrability. In order to examine a possible role of the testosterone-induced rise in [Ca2+]i on gene expression, a c-fos promoter reporter gene construct was transfected into RAW 264.7 macrophages. The increase in [Ca2+]i induced by testosterone cannot significantly activate the c-fos promoter directly. Also, no significant activation of ERK1/2, JNK/SAPK and p38 can be observed following testosterone-stimulation alone. However, testosterone-induced rises in [Ca2+]i do have specific effects on gene expression in context with lipopolysaccharide (LPS)-induced genotropic signaling: testosterone specifically down-regulates LPS-induced activation of c-fos promoter, p38 MAPK and NO production. In fetal calf serum (FCS)-induced genotropic signaling, the situation is reversed, i.e. testosterone augments the activation of c-fos promoter and ERK1/2. Our studies demonstrate a cross-talk between the testosterone-induced nongenomic Ca2+ signaling and the genotropic signaling induced by LPS and FCS in macrophages.     

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (approximately 3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E2) and estrone (E1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E2 and E1 thus demonstrating the presence of similar oxidative and reductive activities for 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) [0.45 and 0.40 pmol/3 h/microgram DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E2 to E1 (0.08 pmol/3 h/microgram DNA) but exhibited an approx 5-fold higher rate of reduction of E1 to E2 (1.81 pmol/3 h/microgram DNA). Estriol (E3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.