ABSTRACT
Secreted protein acidic rich in cysteine (SPARC) is a matricellular protein that modulates the activity of growth factors, cytokines, and extracellular matrix to play multiple roles in tissue development and repair, such as cellular adhesion, migration, and proliferation. Throughout the CNS, SPARC is highly localized in mature ramified microglia, but its role in microglia--in development or during response to disease or injury--is not understood. In the postnatal brain, immature amoeboid myeloid precursors only induce SPARC expression after they cease proliferation and migration, and transform into mature, ramified resting microglia. SPARC null/CX3CR1-GFP reporter mice reveal that SPARC regulates the distribution and branching of mature microglia, with significant differences between cortical gray and white matter in both controls and SPARC nulls. Following ischemic and excitotoxic lesion, reactive, hypertrophic microglia rapidly downregulate and release SPARC at the lesion, concomitant with reactive, hypertrophic perilesion astrocytes upregulating SPARC. After photothrombotic stroke in the forelimb sensorimotor cortex, SPARC nulls demonstrate enhanced microgliosis in and around the lesion site, which accompanies significantly enhanced functional recovery by 32 d after lesion. Microglia from SPARC nulls also intrinsically proliferate at a greater rate in vitro--an enhanced effect that can be rescued by the addition of exogenous SPARC. SPARC is thus a novel regulator of microglial proliferation and structure, and, in addition to regulating glioma progression, may play an important role in differently regulating the gray and white matter microglial responses to CNS lesion--and modulating behavioral recovery--after injury.
Subject(s)
Brain Ischemia/complications , Brain Ischemia/pathology , Cerebral Cortex/pathology , Gliosis/etiology , Glycoproteins/metabolism , Recovery of Function/physiology , Tumor Suppressor Proteins/metabolism , Age Factors , Animals , Animals, Newborn , Brain Infarction/etiology , Brain Infarction/pathology , Brain Ischemia/etiology , CX3C Chemokine Receptor 1 , Calcium-Binding Proteins/metabolism , Cell Count , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Size , Cells, Cultured , Disease Models, Animal , Excitatory Amino Acid Agonists/toxicity , Female , Forelimb/physiopathology , Galectin 3/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Genotype , Glial Fibrillary Acidic Protein/metabolism , Glycoproteins/deficiency , Glycoproteins/pharmacology , Green Fluorescent Proteins/genetics , Intracranial Thrombosis/complications , Lectins/metabolism , Male , Mice , Mice, Transgenic , Microfilament Proteins/metabolism , Microglia/drug effects , Microglia/physiology , Motor Skills/drug effects , Motor Skills/physiology , Mutation/genetics , N-Methylaspartate/toxicity , Olfactory Bulb/injuries , Osteonectin , Proliferating Cell Nuclear Antigen/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Chemokine/genetics , Time Factors , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/pharmacology , NF-kappaB-Inducing KinaseABSTRACT
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) 3-kinases (IP(3)Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P(3) into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca(2+) signaling, and how, is still not fully understood. In this study, we have used a simple Ca(2+) imaging assay to compare the abilities of various Ins (1,4,5)P(3)-metabolizing enzymes to regulate a maximal histamine-stimulated Ca(2+) signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP(3)K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca(2+) release stimulated by 100 microm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP(3)Ks to dampen the Ins (1,4,5)P(3)-mediated Ca(2+) signals. We also compared the effects of the these IP(3)Ks with other enzymes that metabolize Ins(1,4,5)P(3), including the Type I Ins(1,4,5)P(3) 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP(3)K found in Drosophila. All reduce the Ca(2+) signal but to varying degrees. We demonstrate that the activity of only one of two IP(3)K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP(3)Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P(3) signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.
Subject(s)
Calcium Signaling/physiology , Calcium/physiology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Amino Acid Sequence , Animals , COS Cells , Calcium/metabolism , Chlorocebus aethiops , Drosophila melanogaster/enzymology , HeLa Cells , Humans , Isoenzymes/metabolism , Mice , Molecular Sequence Data , RatsABSTRACT
IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.
