From the Cover: Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP₃ receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a subcompartment of the ER that forms a quasisynaptic structure with mitochondria. The main function of this membrane is to facilitate the transfer of lipids and calcium between the two organelles. MAM thereby controls mitochondrial physiology and apoptosis (1, 2). MAM also mediates ER homeostasis and lipid biosynthesis by harboring chaperones and several key lipid synthesis enzymes (3–6). In mammalian MAM, the ER and mitochondria are physically tethered to each other by the IP₃ receptor (IP3R)-Grp75-VDAC1 (voltage-dependent anion-selective channel 1) trimeric complex (7) and by dimers of the mitofusin (Mfn) proteins Mfn1 and Mfn2 (8) (Fig. S1H). The σ-1 receptor also stabilizes MAM by interacting with IP3R and VDAC (9). MAM formation is regulated by multiple signaling inputs, including calcium and possibly growth factors (10–12). However, the mechanism(s) that controls MAM formation is largely unknown other than it involves recruitment of MAM components by the MAM resident proteins phosphofurin acidic cluster sorting protein 2 (PACS2) and Rab32 (13–15). Akt, an AGC family kinase that is also found at MAM (16), phosphorylates PACS2 (17), but it remains to be determined whether Akt is involved in mediating MAM integrity.Akt, often up-regulated in cancer, also phosphorylates hexokinase 2 (HK2) to promote association of HK2 with the MAM protein VDAC1 (18, 19). This association, possibly at MAM (20, 21), enables HK2, using ATP exiting mitochondria through VDAC1, to phosphorylate glucose and thereby stimulate glycolysis (22). Conversely, upon inhibition of Akt, HK2 dissociates from VDAC1, causing VDAC1 closure and increased mitochondrial membrane potential (19). This regulation of HK2 by Akt has been proposed to account for enhanced glycolysis in cancer cells, also known as the Warburg effect (23). Furthermore, Akt regulates calcium release from MAM by phosphorylating IP3R, thereby controlling apoptosis (24–26). Thus, MAM is increasingly recognized as a signaling hub controlling cell physiology (15), and is implicated in a wide spectrum of diseases, including cancer, neurodegenerative disorders, inflammation, and infection (27).The target of rapamycin (TOR) pathway is a cellular signaling cascade that, like mitochondria, is present in all eukaryotes (28, 29). TOR integrates and relays signals from both extra- and intracellular sources (e.g., growth factors, nutrients, and cellular energy levels), and thereby instructs the cell to grow. TOR is found in two structurally and functionally distinct protein complexes that in mammalian cells are termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (30). mTORC2 comprises mTOR, rictor, mammalian lethal with SEC13 protein 8 (mLST8), stress-activated protein kinase (SAPK)-interacting protein (Sin1), and protor [also known as proline-rich protein 5 (PRR5)] (31), and phosphorylates AGC kinases, such as Akt, serum/glucocorticoid-regulated kinase 1 (SGK1), and PKC, all of which are linked to cancer and diabetes (32). Growth factors activate mTORC2 by promoting mTORC2-ribosome association in a PI3K-dependent manner (33, 34). mTORC2 is antiapoptotic, presumably via its role in phosphorylating and activating Akt (34–38).Various observations indicate that mTORC2 is linked to both the ER and mitochondria. Recent findings suggest that mTORC2 is at the ER, possibly through interaction with ER-bound ribosomes (34, 39). mTORC2 phosphorylates Akt at the ER (39, 40), and mTORC2 signaling is sensitive to ER stress (41, 42). In Chlamydomonas, TOR associates with membranes from the ER (43). With regard to mitochondria, mTOR has been observed in close proximity to the outer mitochondrial membrane (44), and mTOR and mLST8 interact with the mitochondrial outer-membrane protein VDAC1 (45) and the mitochondria-associated protein Grp75 (46), respectively. mTORC2 regulates the cellular distribution of mitochondria (47), and mTORC2-activated Akt is associated with mitochondria (18, 48, 49). Pink1, a regulator of mitochondrial function, has been implicated in mTORC2 activation (50). mTORC2-addicted cancer cells exhibit enhanced dependence on mitochondria, Rab32 and HK2 (51). Finally, Barquilla et al. reported that TORC2 in trypanosomes is localized to both ER and mitochondria (52). Thus, mTORC2 has been physically and functionally linked to both the ER and mitochondria.Here we investigate the localization of mTORC2. We show that ribosome-bound mTORC2 is at MAM. Localization to MAM is growth factor-dependent. MAM-associated mTORC2 activates Akt and thereby controls MAM integrity, mitochondrial metabolism, and cell survival. Thus, our findings describe a critical role for mTORC2 in a MAM signaling hub.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Sec16B is involved in the endoplasmic reticulum export of the peroxisomal membrane biogenesis factor peroxin 16 (Pex16) in mammalian cells

Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.Most eukaryotic cells contain peroxisomes, which are single membrane-bound organelles that function in various metabolic pathways, including the β-oxidation of fatty acids, biosynthesis of plasmalogens and bile acids, and hydrogen peroxide metabolism (1). To perform this variety of functions, peroxisomes are highly dynamic; their number, size, and function change in response to cellular conditions. In addition, unlike mitochondria, peroxisomes can be formed through de novo synthesis as well as through the growth and division of preexisting peroxisomes (2, 3).Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported to peroxisomes (4). This import pathway includes the recognition of two distinct peroxisomal targeting signals (PTS1 and PTS2) by peroxin 5 (Pex5) and Pex7, respectively, followed by translocation across the membrane through the import machinery, including Pex14 and Really Interesting New Gene peroxins (5, 6). The import pathway for peroxisomal membrane proteins (PMPs), on the other hand, is believed to be independent of that used by matrix proteins. Genetic phenotype complementation analysis of yeast and mammalian mutants devoid of peroxisome membranes revealed that Pex3, Pex16, and Pex19 are essential for PMP import (references in ref. 7). Pex3 is a PMP import receptor (8), and Pex19 is a chaperone and import receptor for most PMPs (9). Pex16 appears to function as a Pex3-Pex19 receptor in mammals (7) and as a negative regulator of peroxisome fission in yeast Yarrowia lipolytica (10) but is absent in Saccharomyces cerevisiae (11).Although compelling evidence suggests that PMPs are transported directly from the cytosol to peroxisomes (7–9, 12), recent work has suggested that some PMPs, including the PMP import receptors Pex3 and Pex16, seem to be, at least partly, transported from the endoplasmic reticulum (ER) en route to peroxisomes (13). In addition, several lines of evidence suggest that the ER participates in the de novo formation of peroxisomes (13–20). A very recent study involving a yeast cell-free system revealed that ER-peroxisome carriers are formed in a Pex19-dependent manner (21).In this report, we show that Sec16B plays an important role in the transport of Pex16 from the ER to peroxisomes in mammalian cells. Sec16 was first characterized in yeast S. cerevisiae as a 240-kDa peripheral membrane protein that interacts with coat protein II (COPII) coat components and facilitates their assembly and vesicle budding (22–25). In yeast Pichia pastoris, Sec16 defines ER exit sites (ERESs) (26), special domains where COPII-coated vesicles are formed (27). There are two mammalian orthologs, Sec16A (250 kDa) and Sec16B (117 kDa) (also referred to as Sec16L and Sec16S, respectively) (28–30). Sec16A, which is localized in cup-like structures in ERESs (31), appears to be the primary Sec16 ortholog because its molecular mass is similar to that of Sec16 in yeast (22) and Drosophila (32). Sec16B, which appears to be conserved in vertebrates, is also localized in ERESs, but its function has not been fully examined in the context of membrane trafficking. Our results suggest that Sec16B may participate in the formation of new peroxisomes derived from the ER.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.