ABSTRACT
Reversine is a synthetic molecule capable of inducing dedifferentiation of C2C12, a murine myoblast cell line, into multipotent progenitor cells, which can be redirected to differentiate in nonmuscle cell types under appropriate conditions. Reversine is also a potent inhibitor of Aurora B, a protein kinase required for mitotic chromosome segregation, spindle checkpoint function, cytokinesis and histone H3 phosphorylation, raising the possibility that the dedifferentiation capability of reversine is mediated through the inhibition of Aurora B. Indeed, here we show that several other well-characterized Aurora B inhibitors are capable of dedifferentiating C2C12 myoblasts. Significantly, expressing drug-resistant Aurora B mutants, which are insensitive to reversine block the dedifferentiation process, indicating that Aurora B kinase activity is required to maintain the differentiated state. We show that the inhibition of the spindle checkpoint or cytokinesis per se is not sufficient for dedifferentiation. Rather, our data support a model whereby changes in histone H3 phosphorylation result in chromatin remodeling, which in turn restores the multipotent state.
Subject(s)
Myoblasts/cytology , Myoblasts/enzymology , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinase B , Aurora Kinases , Cell Differentiation , Cell Line , Chromatin/metabolism , Cytokinesis/drug effects , Histones/metabolism , Humans , Mice , Models, Biological , Morpholines/pharmacology , Mutant Proteins/metabolism , Myoblasts/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Purines/pharmacologyABSTRACT
Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.
Subject(s)
Blood Platelets/metabolism , Norepinephrine/pharmacology , Receptors, Serotonin, 5-HT1/blood , Adenylyl Cyclases/metabolism , Adrenergic alpha-Antagonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Blood Platelets/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/physiology , Data Interpretation, Statistical , Enzyme Inhibitors/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , In Vitro Techniques , Kinetics , Phentolamine/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Propranolol/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/physiology , Receptors, G-Protein-Coupled/metabolism , Receptors, Serotonin, 5-HT1/drug effectsABSTRACT
Malformations of cortical development (MCD) result from abnormal neuronal positioning during corticogenesis. MCD are believed to be the morphological and perhaps physiological bases of several neurological diseases, spanning from mental retardation to autism and epilepsy. In view of the fact that during development, an appropriate blood supply is necessary to drive organogenesis in other organs, we hypothesized that vasculogenesis plays an important role in brain development and that E15 exposure in rats to the angiogenesis inhibitor thalidomide would cause postnatal MCD. Our results demonstrate that thalidomide inhibits angiogenesis in vitro at concentrations that result in significant morphological alterations in cortical and hippocampal regions of rats prenatally exposed to this vasculotoxin. Abnormal neuronal development was associated with vascular malformations and a leaky blood-brain barrier. Protein extravasation and uptake of fluorescent albumin by neurons, but not glia, was commonly associated with abnormal cortical development. Neuronal hyperexcitability was also a hallmark of these abnormal cortical regions. Our results suggest that prenatal vasculogenesis is required to support normal neuronal migration and maturation. Altering this process leads to failure of normal cerebrovascular development and may have a profound implication for CNS maturation.
