Ca2+ Release by IP3 Receptors Is Required to Orient the Mitotic Spindle.

The mitotic spindle distributes chromosomes evenly to daughter cells during mitosis. The orientation of the spindle, guided by internal and external cues, determines the axis of cell division and thereby contributes to tissue morphogenesis. Progression through mitosis requires local Ca2+ signals at critical steps, and because store-operated Ca2+ entry is inhibited during mitosis, those signals probably require Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs). In cells without IP3Rs, astral microtubules around the daughter centrosome are shorter than those at the mother centrosome, and the mitotic spindle fails to align with the substratum during metaphase. The misalignment is due to the spindle ineffectively detecting internal cues rather than a failure of cells to recognize the substratum. Expression of type 3 IP3R is sufficient to rescue spindle alignment, but only if the IP3R has a functional pore. We conclude that Ca2+ signals evoked by IP3Rs are required to orient the mitotic spindle.

Connexins in Astrocyte Migration.

Astrocytes have long been considered the supportive cells of the central nervous system, but during the last decades, they have gained much more attention because of their active participation in the modulation of neuronal function. For example, after brain damage, astrocytes become reactive and undergo characteristic morphological and molecular changes, such as hypertrophy and increase in the expression of glial fibrillary acidic protein (GFAP), in a process known as astrogliosis. After severe damage, astrocytes migrate to the lesion site and proliferate, which leads to the formation of a glial scar. At this scar-forming stage, astrocytes secrete many factors, such as extracellular matrix proteins, cytokines, growth factors and chondroitin sulfate proteoglycans, stop migrating, and the process is irreversible. Although reactive gliosis is a normal physiological response that can protect brain cells from further damage, it also has detrimental effects on neuronal survival, by creating a hostile and non-permissive environment for axonal repair. The transformation of astrocytes from reactive to scar-forming astrocytes highlights migration as a relevant regulator of glial scar formation, and further emphasizes the importance of efficient communication between astrocytes in order to orchestrate cell migration. The coordination between astrocytes occurs mainly through Connexin (Cx) channels, in the form of direct cell-cell contact (gap junctions, GJs) or contact between the extracellular matrix and the astrocytes (hemichannels, HCs). Reactive astrocytes increase the expression levels of several proteins involved in astrocyte migration, such as αvß3 Integrin, Syndecan-4 proteoglycan, the purinergic receptor P2X7, Pannexin1, and Cx43 HCs. Evidence has indicated that Cx43 HCs play a role in regulating astrocyte migration through the release of small molecules to the extracellular space, which then activate receptors in the same or adjacent cells to continue the signaling cascades required for astrocyte migration. In this review, we describe the communication of astrocytes through Cxs, the role of Cxs in inflammation and astrocyte migration, and discuss the molecular mechanisms that regulate Cx43 HCs, which may provide a therapeutic window of opportunity to control astrogliosis and the progression of neurodegenerative diseases.

Bisphenol-A and Nonylphenol Induce Apoptosis in Reproductive Tract Cancer Cell Lines by the Activation of ADAM17.

Endocrine-disruptor chemicals (EDCs), such as bisphenol A (BPA) and nonylphenol (NP), have been widely studied due to their negative effects on human and wildlife reproduction. Exposure to BPA or NP is related to cell death, hormonal deregulation, and cancer onset. Our previous studies showed that both compounds induce A Disintegrin And Metalloprotease 17 (ADAM17) activation. Here, we show that BPA and NP induce apoptosis in prostate and ovary cancer cell lines, in a process dependent on ADAM17 activation. ADAM17 knockdown completely prevented apoptosis as well as the shedding of ADAM17 substrates. Both compounds were found to induce an increase in intracellular calcium (Ca2+) only in Ca2+-containing medium, with the NP-treated cells response being more robust than those treated with BPA. Additionally, using a phosphorylated protein microarray, we found that both compounds stimulate common intracellular pathways related to cell growth, differentiation, survival, and apoptosis. These results suggest that BPA and NP could induce apoptosis through ADAM17 by activating different intracellular signaling pathways that may converge in different cellular responses, one of which is apoptosis. These results confirm the capacity of these compounds to induce cell apoptosis in cancer cell lines and uncover ADAM17 as a key regulator of this process in response to EDCs.

Intracellular Ca2+ Increases and Connexin 43 Hemichannel Opening Are Necessary but Not Sufficient for Thy-1-Induced Astrocyte Migration.

Under pro-inflammatory conditions, astrocytes become reactive and acquire a migratory phenotype. Our results show that hemichannels formed by connexin 43 (Cx43) play an important role in Thy-1-induced astrocyte migration. The neuronal protein Thy-1 binds to αvß3 integrin in astrocytes, thereby leading to intricate signaling pathways that include calcium (Ca2+) release from intracellular stores, opening of Cx43 hemichannels, release of ATP, activation of P2X7 receptor, and Ca2+ influx. However, because these Thy-1 effects occur exclusively in reactive astrocytes, we wondered whether by elevating calcium levels and promoting hemichannel opening we could prompt non-reactive astrocytes to respond to Thy-1. Cx43 immunoreactivity increased at juxta-membrane sites, where hemichannels (not gap junctions) participate in astrocyte polarization and migration stimulated by Thy-1. Also, intracellular Ca2+ increase, due to ionomycin treatment, induced hemichannel opening, but activated astrocyte migration only partially, and this limitation was overcome by pre-treatment with tumor necrosis factor (TNF) and Thy-1. Finally, αvß3 integrin formed membrane clusters after TNF stimulation or overexpression of ß3 integrin. We suggest that these microclusters are required for cells to respond to Thy-1 stimulation. Therefore, the large increase in intracellular Ca2+ and hemichannel opening induced by ionomycin are required, but not sufficient, to permit Thy-1-induced astrocyte migration. Thus, we suggest that proinflammatory stimuli prompt astrocytes to respond to migratory signals of neuronal cells.

αVß3 Integrin regulates astrocyte reactivity.

BACKGROUND: Neuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVß3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses. METHODS: Wild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers. RESULTS: Thy-1 induced astrocyte migration only after TNF priming. Increased expression of αVß3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of ß3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while ß3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1. CONCLUSIONS: Therefore, inflammation induces expression of αVß3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of ß3 Integrin levels modulates these responses regardless of inflammation.

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration.

Our previous reports indicate that ligand-induced αVß3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca(2+) to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVß3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVß3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca(2+), hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVß3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca(2+) entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

A mechanism of male germ cell apoptosis induced by bisphenol-A and nonylphenol involving ADAM17 and p38 MAPK activation.

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation.

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers.

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.

Contribution of environmental pollutants to male infertily: A working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.

The emerging role of matrix metalloproteases of the ADAM family in male germ cell apoptosis.

Constitutive germ cell apoptosis during mammalian spermatogenesis is a key process for controlling sperm output and to eliminate damaged or unwanted cells. An increase or decrease in the apoptosis rate has deleterious consequences and leads to low sperm production. Apoptosis in spermatogenesis has been widely studied, but the mechanism by which it is induced under physiological or pathological conditions has not been clarified. We have recently identified the metalloprotease ADAM17 (TACE) as a putative physiological inducer of germ cell apoptosis. The mechanisms involved in regulating the shedding of the ADAM17 extracellular domain are still far from being understood, although they are important in order to understand cell-cell communications. Here, we review the available data regarding apoptosis during mammalian spermatogenesis and the localization of ADAM proteins in the male reproductive tract. We propose an integrative working model where ADAM17, p38 MAPK, protein kinase C (PKC) and the tyrosine kinase c-Abl participate in the physiological signalling cascade inducing apoptosis in germ cells. In our model, we also propose a role for the Sertoli cell in regulating the Fas/FasL system in order to induce the extrinsic pathway of apoptosis in germ cells. This working model could be applied to further understand constitutive apoptosis in spermatogenesis and in pathological conditions (e.g., varicocele) or following environmental toxicants exposure (e.g., genotoxicity or xenoestrogens).

Calpain inhibitors prevent p38 MAPK activation and germ cell apoptosis after heat stress in pubertal rat testes.

Testicular injuries like torsion or cryptorchidism can cause massive germ cell death, which could have great impact on male reproductive health. In addition, it has been proposed that modern life style, in the form of underwear or sedentary work position, could increase the testicular temperature, induce germ cell apoptosis, reduce spermatozoa quality and promote male infertility. In this work we showed that a heat stress stimulus induced massive germ cells apoptosis, which was associated with p38 mitogen-activated protein kinase (MAPK) phosphorylation along with an increase in the levels of mRNA encoding calpain 2. Synthetic calpain inhibitors prevented heat stress-induced germ cell apoptosis through inhibition of p38 MAPK phosphorylation. Thus, our results indicate that the blockage of calpains suppresses p38 MAPK phosphorylation, and identifies calpain activation (most likely calpain 2) as an early event in heat stress-induced male germ cell apoptosis.

Mitotic, but not meiotic, oriented cell divisions in rat spermatogenesis.

The process of mammalian spermatogenesis involves both mitosis and meiosis at the same developmental age. Most previous studies have focused on mitotic spindle orientation during development, but not during meiotic division. Therefore, we asked whether there is a difference between mitotic and meiotic germ cell spindle orientation during rat spermatogenesis. Our results showed that mitotic spindles of spermatogonia were mainly oriented with angles ranging from 60 to 90 degrees, perpendicular in relation to the basement membrane of the seminiferous tubules. On the other hand, meiotic spindles showed a random orientation. Nocodazole treatment (at a concentration that depolymerizes only astral microtubules) induced a significant increase in cells with an angle between 0 and 30 degrees (parallel) in relation to the basement membrane. Meiotic spindles did not show a significant change in their orientation after the Nocodazole treatment. Therefore, our results suggest differences between the mechanisms controlling positioning and orientation of mitotic and meiotic spindles during rat spermatogenesis. It seems that a phylogenetically conserved programme controls the mitotic spindle orientation in organisms ranging from worms to mammals.