AFT3 as a new therapeutic target for skin field cancerization in antagonism with compromised Notch/CSL signaling

Les interactions épithélio-mésenchymateuses jouent un rôle important dans le contrôle du développement normal de la peau, son homéostasie et sa tumorigenèse. Les fibroblastes dermiques (DFs) représentent la catégorie cellulaire la plus abondante dans le stroma et leur rôle est de plus en plus considéré. En ce qui concerne particulièrement la tumorigenèse, des facteurs diffusibles produits par les fibroblastes entourant les tumeurs épithéliales, appelés 'fibroblastes associés au cancer (CAF)', interagissent au niveau de l'inflammation impliquée directement ou indirectement dans la signalisation paracrine, entre le stroma et les cellules épiéliales cancéreuses. Le risque de cancer de la peau augmente de façon exponentielle avec l'âge. Comme un lien probable entre les deux, la sénescence des fibroblastes résulte de la production du sécrétome favorisant la sénescence (SMS), un groupe de facteurs diffusibles induisant une stimulation paracrine de la croissance, l'inflammation et le remodelage de la matrice. De façon fort intéressante, l'induction de ces gènes est aussi une caractéristique des CAFs. Cependant, le lien entre les deux événements cellulaires sénescence et activation des CAFs reste en grande partie inexploré. L'ATF3 (Activating Transcription Factor 3) est un facteur de transcription induit en réponse au stress, dont les fonctions sont hautement spécifiques du type cellulaire. Bien qu'il ait été découvert dans notre laboratoire en tant que promoteur de tumeurs dans les kératinocytes, ses fonctions biologique et biochimique dans le derme n'ont pas encore été étudiées. Récemment, nous avons constaté que, chez la souris, l'abrogation de la voie de signalisation de Notch/CSL dans les DFs, induisait la formation de tumeurs kératinocytaires multifocales. Ces dernières proviennent de la cancérisation en domaine, un phénomène associé à une atrophie du stroma, des altérations de la matrice et de l'inflammation. D'autres études ont montré que CSL agissait comme un régulateur négatif de gènes impliqués dans sénescence des DFs et dans l'activation des CAFs. Ici, nous montrons que la suppression ou l'atténuation de l'expression de ATF3 dans les DFs induit la sénescence et l'expression des gènes liés aux CAFs, de façon similaire à celle déclenchée par la perte de CSL, tandis que la surexpression de ATF3 supprime ces changements. Nous émettons l'hypothèse que ATF3 joue un rôle suppresseur dans l'activation des CAFs et dans la progression des tumeurs kératinocytaires, en surmontant les conséquences de l'abrogation de la voie de signalisation Notch/CSL. En concordance avec cette hypothèse, nous avons constaté que la perte de ATF3 dans les DFs favorisait la tumorigénicité des kératinocytes via le contrôle négatif de cytokines, des enzymes de la matrice de remodelage et de protéines associées au cancer, peut-être par liaison directe des effecteurs de la voie Notch/CSL : IL6 et les gènes Hes. Enfin, dans les échantillons cliniques humains, le stroma sous-jacent aux lésions précancéreuses de kératoses actiniques montre une diminution significative de l'expression de ATF3 par rapport au stroma jouxtant la peau normale. La restauration de l'expression de ATF3 pourrait être utilisée comme un outil thérapeutique en recherche translationnelle pour prévenir ou réprimer le processus de cancérisation en domaine. - Epithelial-mesenchymal interactions play an important role in control of normal skin development, homeostasis and tumorigenesis. The role of dermal fibroblasts (DFs) as the most abundant cell type in stroma is increasingly appreciated. Especially during tumorigenesis, fibroblasts surrounding epithelial tumors, called Cancer Associated Fibroblasts (CAFs), produce diffusible factors (growth factors, inflammatory cytokines, chemokines and enzymes, and matrix metalloproteinases) that mediate inflammation either directly or indirectly through paracrine signaling between stroma and epithelial cancer cells. The risk of skin cancer increases exponentially with age. As a likely link between the two, senescence of fibroblasts results in production of the senescence-messaging-secretome (SMS), a panel of diffusible factors inducing paracrine growth stimulation, inflammation, and matrix remodeling. Interestingly, induction of these genes is also a characteristic of Cancer Associated Fibroblasts (CAFs). However, the link between the two cellular events, senescence and CAF activation is largely unexplored. ATF3 is a key stress response transcription factor with highly cell type specific functions, which has been discovered as a tumor promoter in keratinocytes in our lab. However, the biological and biochemical function of ATF3 in the dermal compartment of the skin has not been studied yet. Recently, we found that compromised Notch/CSL signaling in dermal fibroblasts (DFs) in mice is a primary cause of multifocal keratinocyte tumors called field cancerization associated with stromal atrophy, matrix alterations and inflammation. Further studies showed that CSL functions as a negative regulator of genes involved in DFs senescence and CAF activation. Here, we show that deletion or silencing of the ATF3 gene in DFs activates senescence and CAF-related gene expression similar to that triggered by loss of CSL, while increased ATF3 suppresses these changes. We hypothesize that ATF3 plays a suppressing role in CAF activation and keratinocyte tumor progression, overcoming the consequences of compromised Notch/CSL signaling. In support of this hypothesis, we found that loss of ATF3 in DFs promotes tumorigenic behavior of keratinocytes via negative control of cytokines, matrix-remodeling enzymes and cancer-associated proteins, possibly through direct binding to Notch/CSL targets, IL6 and Hes genes. On the other hand, in human clinical samples, stromal fields underlying premalignant actinic keratosis lesions showed significantly decreased ATF3 expression relative to stroma of flanking normal skin. Restoration of ATF3, which is lost in cancer development, may be used as a therapeutic tool for translational research to prevent or suppress the field cancerization process.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to β-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Use of an oocyte expression assay to reconstitute inductive signaling.

We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin-expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural-inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors.

In vitro assessment of migratory behavior of two cell populations in a simple multichannel microdevice

Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver “death-signals” to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards each other. Confluent monolayers of MSC and MCF-7 were established in adjacent chambers separated by an array of 14 microchannels. Initially, culture chambers were isolated by air bubbles (air-valves) contained within each microchannel, and then bubbles were displaced to initiate the assay. The MCF-7 cells migrated preferentially towards MSC, whilst the MSC did not migrate preferentially towards the MCF-7 cells. Our results corroborate previous literature that suggests MSC migration towards cancer cells in vivo is in response to the associated inflammation rather than directly to signals secreted by the cancer cells themselves.

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and-4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.

Hormonal therapy in the senescence: Prostatic microenvironment structure and adhesion molecules

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diferenciação hepatocítica de células-tronco mesenquimais da medula óssea humana

Some recent articles have reported that mesenchymal stem cells (MSCs) can be induced to express hepatocyte markers by transplanting them into animal models of liver damage, or by in vitro culture with growth factors and cytokines. In this study, the aim is to evaluate the behavior of MSCs subjected to induction of hepatocyte differentiation. The MSCs were isolated from the bone marrow of 4 normal donors, characterized and subjected to both in vitro and in vivo induction of hepatocyte differentiation. The in vitro induced cells showed morphological changes, acquiring hepatocyte-like features. However, the immunophenotype of these cells was not modified. The induced cells exhibited no increase in albumin, cytokeratin 18 or cytokeratin 19 transcripts, when analyzed by real-time RT-PCR. The expression of albumin, cytokeratin 18 and alpha fetoprotein was also unchanged, according to immunofluorescence tests. In vivo, the MSC demonstrated a potential to migrate to damaged liver tissue in immunodeficient mice. Taken together, the results suggest that bone marrow MSCs are incapable of in vitro differentiation into hepatocytes by the approach used here, but are capable of homing to damaged hepatic tissue in vivo, suggesting a role for them in the repair of the liver. This contribution to tissue repair could be associated with a paracrine effect exerted by these cells.

Ausência de arteriosclerose na porção intramiocárdica das artérias coronárias: Um mistério a ser resolvido?

Several studies show that portions of intramyocardial coronary arteries are spared of arteriosclerosis, involving morphological, embryological, biochemical and pathophysiological aspects. Endothelial function is significantly affected in the segment of transition, as estimated by the vasoactive response to Ach. These findings suggest that myocardial bridge can provide protection against arteriosclerosis by counteracting the negative effects of endothelial dysfunction. The intramyocardial portion's protection phenomenon deserves further scientific research on all research fronts. Improved morphological, biomechanical and especially physiological and embryological knowledge may be the key to a future window of opportunity for chronic arterial disease therapy and prevention. In addition, this review discusses possible therapeutic approaches for symptomatic coronary ischemia caused by myocardial bridges.

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Connexin and pannexin (hemi) channels in the liver

The liver was among the first organs in which connexin proteins have been identified. Hepatocytes harbor connexin32 and connexin26, while non-parenchymal liver cells typically express connexin43. Connexins give rise to hemichannels, which dock with counterparts on adjacent cells to form gap junctions. Both hemichannels and gap junctions provide pathways for communication, via paracrine signaling or direct intercellular coupling, respectively. Over the years, hepatocellular gap junctions have been shown to regulate a number of liver-specific functions and to drive liver cell growth. In the last few years, it has become clear that connexin hemichannels are involved in liver cell death, particularly in hepatocyte apoptosis. This also holds true for hemichannels composed of pannexin1, a connexin-like protein recently identified in the liver. Moreover, pannexin1 hemichannels are key players in the regulation of hepatic inflammatory processes. The current paper provides a concise overview of the features of connexins, pannexins and their channels in the liver.

Tooth eruption: altered gene expression in the dental follicle of patients with cleidocranial dysplasia

OBJECTIVES The dental follicle plays an important role in tooth eruption by providing key regulators of osteogenesis and bone resorption. Patients with cleidocranial dysplasia (CCD) exhibit delayed tooth eruption in combination with increased bone density in the maxilla and mandible, suggesting disturbances in bone remodeling. The aim of this study was to determine the expression of genes relevant for tooth eruption and bone remodeling in the dental follicles of patients with CCD and normal subjects. MATERIAL AND METHODS Thirteen dental follicles were isolated from five unrelated patients with CCD, and fourteen dental follicles were obtained from 10 healthy individuals. All teeth were in the intraosseous phase of eruption. The expression of RANK, RANKL, OPG, and CSF-1 was determined by quantitative RT-PCR. RESULTS In patients with CCD, the mRNA levels of RANK, OPG, and CSF-1 were significantly elevated compared with the control group. Accordingly, the ratios of RANKL/OPG and RANKL/RANK mRNAs were significantly decreased in patients with CCD. CONCLUSION The observed alterations in the expression and ratios of the aforementioned factors in the dental follicle of CCD individuals suggest a disturbed paracrine signaling for bone remodeling that could be responsible for the impaired tooth eruption seen in these patients.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl− secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl− secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl− secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl− secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl− secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.