Novel insights into the pharmacological modulation of human periodontal ligament stem cells by the amino-bisphosphonate Alendronate.

Alendronate (ALN) is a second-generation bisphosphonate widely used for osteoporosis and cancer-induced bone lesions. Many studies have confirmed a strong relationship between osteonecrosis of the jaws (ONJ) development and oral bisphosphonates, especially ALN, although the molecular mechanisms underlying this pathology have not yet been elucidated. The reduction in bone turnover and vascularization usually observed in ONJ are the result of ALN action on different cell types harboured in oral microenvironment, such as osteoclasts, endothelial cells, and periodontal ligament stem cells (PDLSCs). In this perspective, the present study aims to investigate the effects of different ALN concentrations (2 µM, 5 µM, 10 µM, 25 µM, 50 µM) on the phenotype and functional properties of human PDLSCs (hPDLSCs). hPDLSCs showed a decrease in cell viability (MTT assay) only when treated with ALN concentration of 10 µM or larger for 48 h and 72 h. Cell cycle analysis revealed a moderate increase in proportion of S-phase cells after exposure to low ALN concentration (2-5 µM), an effect that was reverted after exposure to 10-50 µM ALN. Conversely, cell death was evidenced via Annexin V/PI assay at very high concentration of ALN (50 µM) after 4 days of treatment. In addition, we explored whether the effects of ALN on hPDLSCs growth and survival can be mediated by its ability to modulate oxidative stress. To this, we quantified the intracellular ROS amount and lipid peroxidation by using DCF probe and Bodipy staining, respectively. Flow cytometry analysis showed that ALN induced a dose-dependent reduction of intracellular oxidative stress and lipid peroxidation upon treatment with low concentrations at both 48 h and 72 h. Increased levels of oxidative stress was reported at 50 µM ALN and was also confirmed via TEM analysis. Despite the stability of the cellular immunophenotype, hPDLSCs showed impaired mobility after ALN exposure. Chronic exposure (7-14 days) to ALN in the range of 2-10 µM significantly decreased the expression of the differentiation-related factors ALP, RUNX2, COLI, and OPN as well as the osteogenic ability of hPDLSCs compared with untreated cells. Conversely, higher doses were found to be neutral. Our findings indicated that the effects of ALN on hPDLSCs behavior are dose-dependent and suggest a role for oxidative stress in ALN-induced cell death that may lead to novel therapeutic approaches for ONJ.

A Review of Novel Strategies for Human Periodontal Ligament Stem Cell Ex Vivo Expansion: Are They an Evidence-Based Promise for Regenerative Periodontal Therapy?

Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines' or growth factors' mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes.

Correlation of distinct behaviors to the modified expression of cerebral Shank1,3 and BDNF in two autistic animal models.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder featuring altered neuronal circuitry and consequently impaired social interactions, restrictive interests plus repetitive stereotypic activities. In the present study, differentiated behaviors of valproic (VPA) and propionic (PPA) acid-mediated autism rats were correlated to cerebral scaffolding proteins (Shank1,3) and BDNF expression variations. Sprague-Dawley offspring that received VPA during pregnancy displayed a notably diminished permanence (-78 %, p < 0.01) in the light chamber of light dark (LD) test, reduced exploratory tasks, i.e. grooming (-90 %) and rearing (-65 %). Moreover, they executed extremely greater climbing intervals (+300 %, p < 0.001) in novel cage (NC) test, plus exhibited an extremely reduced (-331 %) discrimination index in novel object recognition (NOR) test when compared to controls. PPA-treated postnatal days (PND) 12-16 rats also displayed anxiety-like behaviors, although in a less evident manner, as indicated by a moderate time (+55 %; p < 0.05) spent in dark chamber along with notable and moderate decreases in digging (-78 %) plus grooming (-52 %), respectively. Contextually, VPA- more than PPA supplied opposite Shank1,3 expression changes in cerebellum (CB; -62 %; +78 %), dorsomedial prefrontal cortex (DM-PFC; +95 % -76 %), respectively, while resulting extremely upregulated in hippocampus (HIP; +125 % - +155 %). Even BDNF resulted to be substantially and notably diminished in HIP (-85 %) and DM-PFC (-72 %), respectively, of VPA rats while it was only moderately reduced (-35 % to -45 %) in these same areas of PPA rats. The early altered brain-specific expression levels accounting for different behavioral performances may provide useful diagnostic indications and constitute valuable therapeutic strategies for autistic patients.

Daidzein Pro-cognitive Effects Coincided with Changes of Brain Neurotensin1 Receptor and Interleukin-10 Expression Levels in Obese Hamsters.

At present, concerns are pointing to "tasteful" high-fat diets as a cause of conditioning physical-social states that through alterations of some key emotional- and nutritional-related limbic circuits such as hypothalamic and amygdalar areas lead to obesity states. Feeding and energetic homeostatic molecular mechanisms are part of a complex neuronal circuit accounting for this metabolic disorder. In an attempt to exclude conventional drugs for treating obesity, daidzein, a natural glycosidic isoflavone, which mimics estrogenic neuroprotective properties against increased body weight, is beginning to be preferred. In this study, evident anxiolytic-like behaviors were detected following treatment of high-fat diet hamsters with daidzein as shown by extremely evident (p < 0.001) exploration tendencies in novel object recognition test and a notably greater amount of time spent (p < 0.01) in open arms of elevated plus maze. Moreover, the isoflavone promoted a protective role against neurodegeneration processes as shown by few, if any, amino cupric silver granules in amygdalar, hypothalamic and hippocampal neuronal fields when compared with obese hamsters. Interestingly, elevated expression levels of the anorexic neuropeptide receptor neurotensin1 in the above limbic areas of obese hamsters were extremely reduced by daidzein, especially during recovery of cognitive events. Contextually, such effects were strongly paralleled by increased levels of the anti-neuroinflammatory cytokine, interleukin-10. Our results corroborate a neuroprotective ability of this natural glycosidic isoflavone, which through its interaction with the receptor neurotensin1 and interleukin-10 pathways is correlated not only to improved feeding states, and subsequently obesity conditions, but above all to cognitive performances.

The Case of Medication-Related Osteonecrosis of the Jaw Addressed from a Pathogenic Point of View. Innovative Therapeutic Strategies: Focus on the Most Recent Discoveries on Oral Mesenchymal Stem Cell-Derived Exosomes.

Bisphosphonates-related osteonecrosis of the jaw (BRONJ) was firstly reported by Marx in 2003. Since 2014, the term medication-related osteonecrosis of the jaw (MRONJ) is recommended by the American Association of Oral and Maxillofacial Surgeons (AAOMS). Development of MRONJ has been associated to the assumption of bisphosphonates but many MRONJ-promoting factors have been identified. A strong involvement of immunity components has been suggested. Therapeutic intervention includes surgical and non-surgical treatments, as well as regenerative medicine procedures for the replacement of the lost tissues. The literature confirms that the combination of mesenchymal stem cells (MSCs), biomaterials and local biomolecules can support the regeneration/repair of different structures. In this review, we report the major open topics in the pathogenesis of MRONJ. Then, we introduce the oral tissues recognized as sources of MSCs, summing up in functional terms what is known about the exosomes release in physiological and pathological conditions.

Reduced learning and memory performances in high-fat treated hamsters related to brain neurotensin receptor1 expression variations.

Recent indications are suggesting that high fat and sugar-enriched foods do not only evoke harmful physiological conditions, but they also endure evident structural alterations in cerebral regions controlling cognitive and feeding behaviors. Food consumption plus neuronal energy regulatory mechanisms seem to constitute a complex system assuring that food calories do not exceed body requirements. At the same time obesogenic-related properties of limbic feeding stations like the hypothalamus (HTH), hippocampus (HIP) and amygdala (AMY) tend to control eating habits through the interaction of distinct neuropeptides. For this purpose, it was our intention to correlate expression differences of a key anti-obesogenic neuropeptide receptor i.e. neurotensin1 (NTR1) on mnemonic performances in the hibernating hamster (Mesocricetus auratus) exposed to a high fat diet (HFD). Interestingly, these hamsters exhibited a notable enhanced (p < 0.01) body weight from the fifth on to the twelfth week of treatment, which was accompanied by elevated blood lipid cholesterolo and triglycerides and glucose levels. At the same time these hamsters provided diminished locomotor activities such as exploratory bouts, rearing and grooming behaviors. Of greater relevance was their very extreme (p < 0.001) inability of identifying new objects during novel object recognition (NOR) tests along with not having correctly chosen the chamber of the conditioned place preference (CPP) apparatus, which contained the gratifying reward. Surprisingly the altered behavioral plus mnemonic tasks of HFD hamsters were tightly related to elevated NTR1 expression changes in the above limbic sites thus proposing this neuronal system as a highly probable alternative for treating obesity-dependent mnemonic dysfunctions.

Role of Brain Neuroinflammatory Factors on Hypertension in the Spontaneously Hypertensive Rat.

It is already widely known that the different brain areas involved in blood pressure control, are highly vulnerable to the deleterious effects of this condition. Of particular concern are hypertensive and neuroinflammatory-dependent injuries that by modifying blood flow account for artery structural and functional alterations. It was thus our intention to establish if expression changes of some key brain neuroinflammatory factors like caspase-1,3, NF-kB, IL-1ß and NLRP3, which are known to control blood pressure, are actively involved with inflammation regulatory events in a highly valuable spontaneously hypertensive rat (SHR) model. Indeed, notably increased (p < 0.001) caspase-1, NLRP3 and IL-1ß mRNA levels were detected in amygdalar plus hypothalamic areas of SHR. Contextually, similar up-regulated levels of these factors were also reported in brainstem nuclei with respect to the few hippocampal areas. This trend was supported by moderate increases (p < 0.05) of NLRP3 in amygdalar and brainstem sites, while notably greater expression differences of NF-kB protein were observed in hippocampal and hypothalamic areas of SHR. At the same time, moderately increased levels of iNOS were typical of all of the above brain areas with the exception of the consistently (p < 0.01) increased levels featured in the brainstem. Moreover, even immunohistochemical evaluations supplied notably and moderately increased cleaved caspase-3 cell levels in hippocampus and hypothalamus areas, respectively. Overall, evident hypertensive bouts correlated to neuroinflammatory events, especially in brain areas controlling blood pressure, tend to underlie the value of novel therapeutic approaches designed to improve brain blood flow and subsequently reduce hypertensive-dependent cerebral complications.

Innate immunity in cardiac myxomas and its pathological and clinical correlations.

Cardiac myxomas are the most common benign cardiac tumor. We investigated the immunohistochemical properties of 11 surgically excised cardiac myxomas, in order to analyze the correlation between macrophages and mast cell populations and clinical parameters. CD68+/CD163-/iNOS- (M0) cells represent the most abundant macrophage phenotype; however, CD68+/CD163+ cells (M2) were also frequent. CD68+/iNOS+ (M1) elements were rare. Mast cells, defined as a population of c-kit (CD117)+ and/or tryptase+ cells were also detected. Statistical analysis showed significant correlations between c-kit (CD117)+ and tryptase, CD68 and erythrocyte sedimentation rate (ESR), ESR and red blood cell count (RBC), and prothrombin time and platelet count. The inverse correlation between RBCs in peripheral blood and ESR suggested that anemia associated with chronic inflammatory disease is a noncasual event in patients suffering from cardiac myxoma. Mechanical hemolysis may be only a minor component of anemia, according to the lack of correlation between echographic surface and RBCs. Moreover, tumor size did not correlate with ESR, showing that inflammatory state may depend from both tumor cells population and inflammatory infiltrate. In the future, modulation of macrophage polarization in cardiac myxomas might represent important therapeutic target.

Extravascular type of intravascular papillary endothelial hyperplasia mimicking parotid gland neoplasia and the possible role of ferritin in the pathogenesis: A case report.

Intravascular papillary endothelial hyperplasia (IPEH) is defined as a vascular lesion characterized by extensive proliferation of vascular endothelial cells. This lesion was first described by Pierre Masson in 1923 as intravascular hemangioendothelioma. The most frequent sites of involvement are the skin and subcutis. IPEH comprises ~2% of the vascular tumors of the skin and subcutaneous tissue and it has a predilection for the head, neck, trunk and the extremities. The diagnosis is based on histopathology. We herein present the second case of Masson's tumor of the parotid gland described in literature. The patient was a 70-year-old female. Magnetic resonance imaging revealed an irregular lesion with smooth margins, initially considered to be compatible with pleomorphic adenoma. Immunohistochemical analysis revealed positivity of the tumor cells for ferritin heavy and light chains, vimentin and CD31. The aim of the present study was to emphasize the immunohistochemical characteristics and briefly discuss the potential role of ferritin in the pathogenesis of IPEH.

Unpredictable Chronic Mild Stress Paradigm Established Effects of Pro- and Anti-inflammatory Cytokine on Neurodegeneration-Linked Depressive States in Hamsters with Brain Endothelial Damages.

The mechanisms by which inflammation affects the different emotional moods are only partially known. Previous works have pointed to stress hormones like glucocorticoids plus the vascular factor endothelin-1 as key factors evoking stressful states especially in relation to endothelial dysfunctions. With this work, it was our intention to establish the role of pro- and anti-inflammatory cytokine expression variations towards depression-like behaviors and consequently the development of neurodegeneration events caused by endothelial damages in the hamster (Mesocricetus auratus). Such a rodent, which is considered a valuable animal model to test depression and anxiety states, exhibited a variety of depression-like behaviors including reduction in sucrose consumption, locomotion, and exploration (p < 0.01) following exposure to unpredictable chronic mild stress. Contextually, a tight correlation between unpredictable chronic mild stress-induced depressive states and expression of the pro-inflammatory cytokines was detected as shown by marked expression levels (p < 0.01) of IL-1ß and NF-kB in the hippocampus, amygdala, and prefrontal cortex. Even the anti-inflammatory cytokine IL-10 supplied notably significant (p < 0.001) expression levels in the same areas of resilient hamsters. Application of hemodynamic and endothelial functional studies pointed to altered arterial endothelial activities in depressed with respect to resilient animals. Moreover, evident damaged neuronal fields in the above areas of depressed hamsters allowed us to correlate such a behavioral phenomenon to the upregulation of IL-1ß and NF-κB. Overall, the differing roles of pro- and anti-inflammatory cytokines on depressive states, especially in view of brain endothelial damages, may provide novel therapeutic measures against mood disorders linked to neurodegenerative diseases.

Aurora Kinase A expression predicts platinum-resistance and adverse outcome in high-grade serous ovarian carcinoma patients.

High-Grade Serous Ovarian Carcinoma (HGSOC) is the predominant histotype of epithelial ovarian cancer (EOC), characterized by advanced stage at diagnosis, frequent TP53 mutation, rapid progression, and high responsiveness to platinum-based-chemotherapy. To date, standard first-line-chemotherapy in advanced EOC includes platinum salts and paclitaxel with or without bevacizumab. The major prognostic factor is the response duration from the end of the platinum-based treatment (platinum-free interval) and about 10-0 % of EOC patients bear a platinum-refractory disease or develop early resistance (platinum-free interval shorter than 6 months). On these bases, a careful selection of patients who could benefit from chemotherapy is recommended to avoid unnecessary side effects and for a better disease outcome. In this retrospective study, an immunohistochemical evaluation of Aurora Kinase A (AURKA) was performed on 41 cases of HGSOC according to platinum-status. Taking into account the number and intensity of AURKA positive cells we built a predictive score able to discriminate with high accuracy platinum-sensitive patients from platinum-resistant patients (p < 0.001). Furthermore, we observed that AURKA overexpression correlates to worse overall survival (p = 0.001; HR 0.14). We here suggest AURKA as new effective tool to predict the biological behavior of HGSOC. Particularly, our results indicate that AURKA has a role both as predictor of platinum-resistance and as prognostic factor, that deserves further investigation in prospective clinical trials. Indeed, in the era of personalized medicine, AURKA could assist the clinicians in selecting the best treatment and represent, at the same time, a promising new therapeutic target in EOC treatment.

A reappraisal of macrophage polarization in glioblastoma: Histopathological and immunohistochemical findings and review of the literature.

The survival rate in glioblastoma multiforme patients has scarcely improved in the last decades; however, many new therapeutic strategies have been theorized or developed for these neoplasias. Recently, the inverse correlation observed between patient prognosis and tumor-associated macrophages (TAMs) density in solid tumors has encouraged the development of anti-tumor strategies aiming to target TAMs. As expected, TAMs polarization is influenced by both macrophage localization and tumor microenvironment signals, resulting in a more complex scenario than the simple M1/M2 activation status. Macrophage polarization in glioblastoma has not yet been fully elucidated, and most results have been obtained in experimental non-human settings, with some apparent contradiction. The authors performed a histopathological and immunohistochemical study of 37 cases of glioblastoma in order to characterize the M1 and M2 macrophage populations within TAMs. A high prevalence of CD163+ M2-polarized macrophages was detected in this cohort, whereas iNOS+ macrophages were rarely found. The down-regulation of CD68 expression in microglia/macrophage infiltrating glioblastomas is also reported for the first time. Such a finding is associated with a specific location of TAMs within the lesion, as confirmed by the fact that CD68 staining was lower than CD163, mainly in perivascular areas. The authors discuss the recent literature about the global scenario of macrophage plasticity and polarization in glioblastoma, and suggest some pivotal points for therapeutic applications.

Circulating non-hematological cells during cardiopulmonary bypass: new findings in cardiac surgery procedures.

BACKGROUND: Several factors have been historically advocated to explain the coagulative and inflammatory disorders following cardiopulmonary bypass (CPB). In this paper, we describe the presence of circulating non-hematological cells, introduced within the bloodstream during CPB. We defined the origin of the cells and tested their impact on coagulation. METHODS: We collected peripheral arterial blood samples in twenty consecutive coronary artery bypass graft cases at four different surgical moments and assessed the presence and nature of circulating cells with the use of the CELLSEARCH® Test, immunocytochemistry and immunofluorescence, evaluating the expression of cytokeratin and calretinin. The effect of the circulating non-hematological cells on coagulation was tested in vitro, using the ROTEM assay. RESULTS: A mean of 263.85 ± 57.5 (median 258.5) cells were present in the samples following the suction of blood from the surgical field while all the other samples were negative (zero cells) (p<0.00001). Immunologic tests confirmed the mesothelial origin of the cells. The ROTEM® assay of the blood samples contaminated by the mesothelial cells presented longer clotting times (53.4 ± 8.2 secs 48.3 ± 8.9 sec, p=0.05), longer clot formation times (137.1 ± 31.5 sec vs 111.9 ± 25.2 sec, p=0.009), smaller alfa angle amplitudes (66.7 ± 9.1° vs 71.1 ± 5.1°, p=0.04) and maximum clot firmness times (59.0 ± 5.4 sec vs 61.9 ±4.6 sec, p=0.004) than the controls. CONCLUSION: The presence of circulating non-hematological cells during CPB with a mesothelial immunophenotype alters in vitro coagulation assays. This finding can help to further understand the pathophysiology of CPB.

Overstimulation of glutamate signals leads to hippocampal transcriptional plasticity in hamsters.

It's known that neurons in mammalian hibernators are more tolerant to hypoxia than those in non-hibernating species and as a consequence animals are capable of awakening from the arousal state without exhibiting cerebral damages. In addition, evidences have suggested that euthermic hamster neurons display protective adaptations against hypoxia, while those of rats are not capable, even though molecular mechanisms involved in similar neuroprotective strategies have not been yet fully studied. In the present work, overstimulation of glutamatergic receptors NMDA recognized as one of the major death-promoting element in hypoxia, accounted for altered network complexity consistent with a moderate reduction of hippocampal neuronal survival (p < 0.05) in hamsters. These alterations appeared to be featured concomitantly with altered glutamatergic signaling as indicated by significant down-regulation (p < 0.01) of NMDAergic (NR2A) and AMPAergic (GluR1, R2) receptor subtypes together with the metabotropic mGluR5 subtype. Diminished mRNA levels were also reported for NMDA receptor binding factors and namely PSD95 plus DREAM, which exert positive and negative regulatory properties, respectively, on receptor trafficking events. Conversely, involvement of glutamatergic signaling systems on neuronal excitotoxicity was strengthened by the co-activation of GABAAR-mediated effects as indicated by toxic morphological effects being notably reduced along with up-regulated GluR1, GluR2, mGluR5, DREAM, and Homer1c scaffold proteins when muscimol was added. Overall, these results point to a neuroprotective role of the GABAergic system against excitotoxicity episodes via DREAM-dependent inhibition of NMDA receptor and activation of AMPA receptor plus mGluR5, respectively, thus proposing them as novel therapeutic targets against cerebral ischemic damages in humans.

Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling.

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells. In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; and iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; and c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin. Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.

H ferritin gene silencing in a human metastatic melanoma cell line: a proteomic analysis.

Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.

Role of endothelin-1 in the migration of human olfactory gonadotropin-releasing hormone-secreting neuroblasts.

FNC-B4 neuroblasts that express both neuronal and olfactory markers have been established and cloned. These cells express GnRH and both the endothelin-1 (ET-1) gene and protein and respond in a migratory manner to GnRH in a dose-dependent manner. Previous research has shown that FNC-B4 cells produce and respond to ET-1 by regulating the secretion of GnRH through endothelin type A receptors and by stimulating their proliferation through endothelin type B (ETB) receptors. In this study, we found that FNC-B4 cells are able to migrate in response to ET-1 through the involvement of ETB receptors. Combined immunohistochemical and biochemical analyses showed that ET-1 triggered actin cytoskeletal remodeling and a dose-dependent increase in migration (up to 6-fold). Whereas the ETB receptor antagonist (B-BQ788) blunted the ET-1-induced effects, the ETA receptor antagonist (A-BQ123) did not. Moreover, we observed that FNC-B4 cells were independently and selectively stimulated by ET-1 and GnRH. We suggest that ET-1, through ETB receptor activation, may be required to maintain an adequate proliferative stem cell pool in the developing olfactory epithelium and the subsequent commitment to GnRH neuronal migratory pattern. The coordinate interaction between ET receptors and GnRH receptor participates in the fully expressed GnRH-secreting neuron phenotype.

Expression and function of gonadotropin-releasing hormone (GnRH) receptor in human olfactory GnRH-secreting neurons: an autocrine GnRH loop underlies neuronal migration.

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.