ABSTRACT
OBJECTIVE: Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. METHODS: Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. RESULTS: Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. CONCLUSION: Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.
ABSTRACT
This study provides a novel view on the interactions between the MS-KIF18A, a kinesin protein, and estrogen receptor alpha (ERalpha) which were studied in vivo and in vitro. Additionally, the regulation of MS-KIF18A expression by estrogen was investigated at the gene and protein levels. An association between recombinant proteins; ERalpha and MS-KIF18A was demonstrated in vitro in a pull down assay. Such interactions were proven also for endogenous proteins in MBA-15 cells were detected prominently in the cytoplasm and are up-regulated by estrogen. Additionally, an association between these proteins and the transcription factor NF-kappaB was identified. MS-KIF18A mRNA expression was measured in vivo in relation to age and estrogen level in mice and rats models. A decrease in MS-KIF18A mRNA level was measured in old and in OVX-estrogen depleted rats as compared to young animals. The low MS-KIF18A mRNA expression in OVX rats was restored by estrogen treatment. We studied the regulation of MS-KIF18A transcription by estrogen using the luciferase reporter gene and chromatin immuno-precipitation (ChIP) assays. The luciferase reporter gene assay demonstrated an increase in MS-KIF18A promoter activity in response to 10(-8) M estrogen and 10(-7)M ICI-182,780. Complimentary, the ChIP assay quantified the binding of ERalpha and pcJun to the MS-KIF18A promoter that was enhanced in cells treated by estrogen and ICI-182,780. In addition, cells treated by estrogen expressed higher levels of MS-KIF18A mRNA and protein and the protein turnover in MBA-15 cells was accelerated. Presented data demonstrated that ERalpha is a defined cargo of MS-KIF18A and added novel insight on the role of estrogen in regulation of MS-KIF18A expression both in vivo and in vitro.
Subject(s)
Gene Expression Regulation , Kinesins/metabolism , Receptor Cross-Talk , Receptors, Estrogen/metabolism , Animals , Base Sequence , Cell Line , Chromatin Immunoprecipitation , DNA Primers , Humans , Kinesins/genetics , Mice , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/genetics , RatsABSTRACT
The present study highlights on the biochemical and immunological analysis of MS-KIF18A in pre-osteogenic MBA-15 cells. The protein distribution in various cellular compartments was demonstrated by imaging and Western blot (WB) analysis. MS-KIF18A interactions with cytoskeletal proteins were confirmed for tubulin and actin. The complex between MS-KIF18A and microtubules (MT) was demonstrated in cellular system for endogenous proteins and also between recombinant proteins in pull down and immunoprecipitation (IP) assays. Multiple assays including metabolic labeling, cell fractionation and IP with anti-MS-KIF18A antibody demonstrated an association with actin that was prominent in the cell cytoplasm. Sub-cellular fractionation identified diverse forms of MS-KIF18A in cytoplasm and membrane/nucleus compartments which are suggested to represent the result of post-transcriptional modifications, such as phosphorylation and glycosylation. These modifications on MS-KIF18A were analyzed by bioinformatics and immunological assays. Furthermore, we studied the role of ubiquitin-proteasome system in the MS-KIF18A degradation. Taken together, the current study sheds light on MS-KIF18A a MT-dependent kinesin and adds insights on the post-translational modifications that potentially control the protein cellular distribution and its co-association with cytoskeletal proteins.
Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Protein Processing, Post-Translational , Actins/metabolism , Cell Line , Glycosylation/drug effects , Humans , Microtubules/drug effects , Nocodazole/pharmacology , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Binding/drug effects , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , Serum , Tubulin/metabolismABSTRACT
Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.
