STAT3-Ser/Hes3 Signaling: A New Molecular Component of the Neuroendocrine System?

The endocrine system involves communication among different tissues in distinct organs, including the pancreas and components of the Hypothalamic-Pituitary-Adrenal Axis. The molecular mechanisms underlying these complex interactions are a subject of intense study as they may hold clues for the progression and treatment of a variety of metabolic and degenerative diseases. A plethora of signaling pathways, activated by hormones and other endocrine factors have been implicated in this communication. Recent advances in the stem cell field introduce a new level of complexity: adult progenitor cells appear to utilize distinct signaling pathways than the more mature cells in the tissue they co-reside. It is therefore important to elucidate the signal transduction requirements of adult progenitor cells in addition to those of mature cells. Recent evidence suggests that a common non-canonical signaling pathway regulates adult progenitors in several different tissues, rendering it as a potentially valuable starting point to explore their biology. The STAT3-Ser/Hes3 Signaling Axis was first identified as a major regulator of neural stem cells and, subsequently, cancer stem cells. In the endocrine/neuroendocrine system, this pathway operates on several levels, regulating other types of plastic cells: (a) it regulates pancreatic islet cell function and insulin release; (b) insulin in turn activates the pathway in broadly distributed neural progenitors and possibly also hypothalamic tanycytes, cells with important roles in the control of the adrenal gland; (c) adrenal progenitors themselves operate this pathway. The STAT3-Ser/Hes3 Signaling Axis therefore deserves additional research in the context of endocrinology.

The effects of stress on brain and adrenal stem cells.

The brain and adrenal are critical control centers that maintain body homeostasis under basal and stress conditions, and orchestrate the body's response to stress. It is noteworthy that patients with stress-related disorders exhibit increased vulnerability to mental illness, even years after the stress experience, which is able to generate long-term changes in the brain's architecture and function. High levels of glucocorticoids produced by the adrenal cortex of the stressed subject reduce neurogenesis, which contributes to the development of depression. In support of the brain-adrenal connection in stress, many (but not all) depressed patients have alterations in the components of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, with enlarged adrenal cortex and increased glucocorticoid levels. Other psychiatric disorders, such as post-traumatic stress disorder, bipolar disorder and depression, are also associated with abnormalities in hippocampal volume and hippocampal function. In addition, hippocampal lesions impair the regulation of the LHPA axis in stress response. Our knowledge of the functional connection between stress, brain function and adrenal has been further expanded by two recent, independent papers that elucidate the effects of stress on brain and adrenal stem cells, showing similarities in the way that the progenitor populations of these organs behave under stress, and shedding more light into the potential cellular and molecular mechanisms involved in the adaptation of tissues to stress.

Expression profiles of the nuclear receptors and their transcriptional coregulators during differentiation of neural stem cells.

Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0-5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUP-TFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands.

Chromaffin cells: the peripheral brain.

Chromaffin cells probably are the most intensively studied of the neural crest derivates. They are closely related to the nervous system, share with neurons some fundamental mechanisms and thus were the ideal model to study the basic mechanisms of neurobiology for many years. The lessons we have learned from chromaffin cell biology as a peripheral model for the brain and brain diseases pertain more than ever to the cutting edge research in neurobiology. Here, we highlight how studying this cell model can help unravel the basic mechanisms of cell renewal and regeneration both in the central nervous system (CNS) and neuroendocrine tissue and also can help in designing new strategies for regenerative therapies of the CNS.

Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha.

Hypoxia contributes to the progression of a variety of cancers by activating adaptive transcriptional programs that promote cell survival, motility and tumor angiogenesis. Although the importance of hypoxia and subsequent hypoxia-inducible factor-1alpha (HIF-1alpha) activation in tumor angiogenesis is well known, their role in the regulation of glioma-derived stem cells is unclear. In this study, we show that hypoxia (1% oxygen) promotes the self-renewal capacity of CD133-positive human glioma-derived cancer stem cells (CSCs). Propagation of the glioma-derived CSCs in a hypoxic environment also led to the expansion of cells bearing CXCR4 (CD184), CD44(low) and A2B5 surface markers. The enhanced self-renewal activity of the CD133-positive CSCs in hypoxia was preceded by upregulation of HIF-1alpha. Knockdown of HIF-1alpha abrogated the hypoxia-mediated CD133-positive CSC expansion. Inhibition of the phosphatidylinositol 3-kinase(PI3K)-Akt or ERK1/2 pathway reduced the hypoxia-driven CD133 expansion, suggesting that these signaling cascades may modulate the hypoxic response. Finally, CSCs propagated at hypoxia robustly retained the undifferentiated phenotype, whereas CSCs cultured at normoxia did not. These results suggest that response to hypoxia by CSCs involves the activation of HIF-1alpha to enhance the self-renewal activity of CD133-positive cells and to inhibit the induction of CSC differentiation. This study illustrates the importance of the tumor microenvironment in determining cellular behavior.

Signaling pathways controlling neural stem cells slow progressive brain disease.

The identification and characterization of multipotent neural precursors open the possibility of transplant therapies, but this approach is complicated by the widespread pathology of many degenerative diseases. Activation of endogenous precursors that support regenerative mechanisms is a possible alternative. We have previously shown that Notch ligands promote stem cell survival in vitro. Here, we show that there is an intimate interaction between insulin and Notch receptor signaling. Notch ligands also expand stem cell numbers in vivo with correlated benefits in brain ischemia. We now show that insulin promotes recovery of injured dopamine neurons in the adult brain. This response suggests that activating survival mechanisms in neural stem cells will promote recovery from progressive degenerative disease.

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter.

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.

A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.

The depolarisation-induced release of [125I]BDNF from brain tissue.

The pattern of release of radioactive brain-derived neurotrophic factor ([125I]BDNF) from brain tissue was studied. Rat brain slices from cerebral cortex and synaptosomes from cerebral cortex and hippocampus were preloaded with [125I]BDNF. Depolarising stimulation by veratridine (final conc. 50 microM) and high KCl (final conc. 45 mM) caused a short-term, greatly enhanced depolarisation-induced release of [125I]BDNF during superfusion and batch protocol experiments. The results suggested that the evoked release was independent of the presence of extracellular calcium ions, but dependent on intracellular calcium ion stores, since the intracellular calcium ion chelator BAPTA-AM, but not the extracellular chelator EGTA abolished the high-potassium-induced [125I]BDNF release from synaptosomes. The release was blocked by tetrodotoxin (1 microM) when synaptosomes were stimulated by veratridine or potassium chloride. Short time-fraction (30 s) superfusion experiments showed that the [125I]BDNF release from synaptosomes appeared in two temporal phases.