Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Adipocytes promote prostate cancer stem cell self-renewal through amplification of the cholecystokinin autocrine loop

Obesity has long been linked with prostate cancer progression, although the underlying mechanism is still largely unknown. Here, we report that adipocytes promote the enrichment of prostate cancer stem cells (CSCs) through a vicious cycle of autocrine amplification. In the presence of adipocytes, prostate cancer cells actively secrete the peptide hormone cholecystokinin (CCK), which not only stimulates prostate CSC self-renewal, but also induces cathepsin B (CTSB) production of the adipocytes. In return, CTSB facilitates further CCK secretion by the cancer cells. More importantly, inactivation of CCK receptor not only suppresses CTSB secretion by the adipocytes, but also synergizes the inhibitory effect of CTSB inhibitor on adipocyte-promoted prostate CSC self-renewal. In summary, we have uncovered a novel mechanism underlying the mutual interplay between adipocytes and prostate CSCs, which may help explaining the role of adipocytes in prostate cancer progression and provide opportunities for effective intervention.

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Identification of the semaphorin receptor PLXNA2 as a candidate for susceptibility to schizophrenia

The discovery of genetic factors that contribute to schizophrenia susceptibility is a key challenge in understanding the etiology of this disease. Here, we report the identification of a novel schizophrenia candidate gene on chromosome 1q32, plexin A2 (PLXNA2), in a genome-wide association study using 320 patients with schizophrenia of European descent and 325 matched controls. Over 25,000 single-nucleotide polymorphisms (SNPs) located within approximately 14,000 genes were tested. Out of 62 markers found to be associated with disease status, the most consistent finding was observed for a candidate locus on chromosome 1q32. The marker SNP rs752016 showed suggestive association with schizophrenia (odds ratio (OR) = 1.49, P = 0.006). This result was confirmed in an independent case-control sample of European Americans (combined OR = 1.38, P = 0.035) and similar genetic effects were observed in smaller subsets of Latin Americans (OR = 1.26) and Asian Americans (OR = 1.37). Supporting evidence was also obtained from two family-based collections, one of which reached statistical significance (OR = 2.2, P = 0.02). High-density SNP mapping showed that the region of association spans approximately 60 kb of the PLXNA2 gene. Eight out of 14 SNPs genotyped showed statistically significant differences between cases and controls. These results are in accordance with previous genetic findings that identified chromosome 1q32 as a candidate region for schizophrenia. PLXNA2 is a member of the transmembrane semaphorin receptor family that is involved in axonal guidance during development and may modulate neuronal plasticity and regeneration. The PLXNA2 ligand semaphorin 3A has been shown to be upregulated in the cerebellum of individuals with schizophrenia. These observations, together with the genetic results, make PLXNA2 a likely candidate for the 1q32 schizophrenia susceptibility locus.

Genetics of T cell co-stimulatory receptors -CD28, CTLA4, ICOS and PDCD1 in immunity and transplantation

Co-stimulatory signals are essential for the activation of naïve T cells and productive immune response. Naïve T cells receive first, antigen-specific signal through T cell receptor. Co-stimulatory receptors provide the second signal which can be either activating or inhibitory. The balance between signals determines the outcome of an immune response. CD28 is crucial for T cell activation; whereas cytotoxic T lymphocyte associated antigen 4 (CTLA4) mediates critical inhibitory signal. Inducible co-stimulator (ICOS) augments cytokine expression and plays role in immunoglobulin class switching. Programmed cell death 1 (PDCD1) acts as negative regulator of T cell proliferation and cytokine responses. The co-stimulatory receptor pathways are potentially involved in self-tolerance and thus, they provide a promising therapeutic strategy for autoimmune diseases and transplantation. The genes encoding CD28, CTLA4 and ICOS are located adjacently in the chromosome region 2q33. The PDCD1 gene maps further, to the region 2q37. CTLA4 and PDCD1 are associated with the risk of a few autoimmune diseases. There is strong linkage disequilibrium (LD) on the 2q33 region; the whole gene of CD28 exists in its own LD block but CTLA4 and the 5' part of ICOS are within a same LD block. The 3' part of ICOS and PDCD1 are in their own separate LD blocks. Extended haplotypes covering the 2q33 region can be identified. This study focuses on immune related conditions like coeliac disease (CD) which is a chronic inflammatory disease with autoimmune features. Immunoglobulin A deficiency (IgAD) belongs to the group of primary antibody deficiencies characterised by reduced levels of immunoglobulins. IgAD co-occurs often with coeliac disease. Renal transplantation is needed in the end stage kidney diseases. Transplantation causes strong immune response which is tried to suppress with drugs. All these conditions are multifactorial with complex genetic background and multiple environmental factors affecting the outcome. We have screened ICOS for polymorphisms by sequencing the exon regions. We detected 11 new variants and determined their frequencies in Finnish population. We have measured linkage disequilibrium on the 2q33 region in Finnish as well as other European populations and observed conserved haplotypes. We analysed genetic association and linkage of the co-stimulatory receptor gene region aiming to study if it is a common risk locus for immune diseases. The 2q33 region was replicated to be linked to coeliac disease in Finnish population and CTLA4-ICOS haplotypes were found to be associated with CD and IgAD being the first non-HLA risk locus common for CD and immunodeficiencies. We also showed association between ICOS and the outcome of kidney transplantation. Our results suggest new evidence for CTLA4-ICOS gene region to be involved in susceptibility of coeliac disease. The earlier published contradictory association results can be explained by involvement of both CTLA4 and ICOS in disease susceptibility. The pattern of variants acting together rather than a single polymorphism may confer the disease risk. These genes may predispose also to immunodeficiencies as well as decreased graft survival and delayed graft function. Consequently, the present study indicates that like the well established HLA locus, the co-stimulatory receptor genes predispose to variety of immune disorders.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

FGFR1 regulated gene-expression, cell proliferation and differentiation in the developing midbrain and hindbrain

The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to Vα24+ NKT cell-mediated cytotoxicity

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human Vα24 +/Vβ11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.