The SH2 domain-containing 5-phosphatase SHIP2 is expressed in the germinal layers of embryo and adult mouse brain: increased expression in N-CAM-deficient mice

doi:10.1016/S0306-4522(01)00240-8

Neuroscience

Volume 105, Issue 4, 22 August 2001, Pages 1019-1030

https://doi.org/10.1016/S0306-4522(01)00240-8 Get rights and content

Abstract

The germinative ventricular zone of embryonic brain contains neural lineage progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. The ability to generate neurons persists at adulthood in restricted brain areas. During development, many growth factors exert their effects by interacting with tyrosine kinase receptors and activate the phosphatidylinositol 3-kinase and the Ras/MAP kinase pathways. By its ability to modulate these pathways, the recently identified Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2, SHIP2, has the potential to regulate neuronal development. Using in situ hybridization technique with multiple synthetic oligonucleotides, we demonstrated that SHIP2 mRNA was highly expressed in the ventricular zone at early embryonic stages and subventricular zones at latter stages of brain and spinal cord and in the sympathetic chain. No significant expression was seen in differentiated fields. This restricted expression was maintained from embryonic day 11.5 to birth. In the periphery, large expression was detected in muscle and kidney and moderate expression in thyroid, pituitary gland, digestive system and bone. In the adult brain, SHIP2 was mainly restricted in structures containing neural stem cells such as the anterior subventricular zone, the rostral migratory stream and the olfactory tubercle. SHIP2 was also detected in the choroid plexuses and the granular layer of the cerebellum. The specificity of SHIP2 expression in neural stem cells was further demonstrated by (i) the dramatic increase in SHIP2 mRNA signal in neural cell adhesion molecule (N-CAM)-deficient mice, which present an accumulation of progenitor cells in the anterior subventricular zone and the rostral migratory stream, (ii) the abundant expression of 160-kDa SHIP2 by western blotting in proliferating neurospheres in culture and its downregulation in non-proliferating differentiated neurospheres.

In conclusion, the close correlation between the pattern of SHIP2 expression in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.

Section snippets

Animals

Female BALB/c and C57BL/6 mice (6–10 weeks old) were purchased from IffaCredo (Bruxelles, Belgium) and maintained in our animal facility. Pregnancies were dated by inspecting females for the presence of a vaginal plug, the day of the plug being embryonic day zero (E0). Embryonic brains were examined at the following stages: E11.5, E16.5 and E18.5. Neural cell adhesion molecule (N-CAM)-deficient mice were generated on a C57BL/6 genetic background by Cremer et al. (1994). Note that all efforts

SHIP2 probe characterization and signal specificity

Alternative spliced forms have been described for SHIP1 (Kavanaugh et al., 1996, Rohrschneider et al., 2000, Wolf et al., 2000) and rat SHIP2 (Ishihara et al., 1999). To test the possibility that the exonic localization of probes may affect the apparent SHIP2 distribution, seven antisense probes widespreadly distributed in the mouse SHIP2 sequence (Schurmans et al., 1999) were prepared (see Experimental procedures). Hybridization with these SHIP2 probes on embryos at E16.5 (data not shown) and

Discussion

We have shown that during mouse embryonic development, SHIP2 5-phosphatase was highly expressed in the germinal layer or neuroepithelium of brain and spinal cord throughout the embryonic development, whereas no significant expression was detected in regions containing migrating or more differentiated cells. In the periphery, SHIP2 mRNA expression was intense in the developing muscular tissues and moderate in several other organs. In the adult brain, SHIP2 expression was mainly detected in

Conclusion

The pattern of SHIP2 expression in the brain germinal epithelium, where it is susceptible to tyrosine phosphorylation by stimulation with exogenous factors, correlates with the proliferative and early differentiative events that occur in the brain. This suggests that this 5-phosphatase may have important roles in neural development and may define a completely new pathway to control the switch between proliferation and differentiation of immature neuroblasts. SHIP2 expression in neural stem

Acknowledgements

We thank Mrs. Michèle Authelet for technical assistance in the initial part of this work. We would like to thank Xavier Pesesse for providing the SH2 antibodies and Colette Moreau for the help in western blot analysis. This work was supported by grants of the Fonds de la Recherche Scientifique Médicale (3.4551.98) (Belgium), Action de Recherche Concertée of the Communauté Française de Belgique (Belgium), Fondation Médicale Reine Elisabeth (Neurobiologie 1999–2001) (Belgium), by the Belgian

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In addition to its central catalytic domain, the 1258 amino acids SHIP2 protein contains several motifs known to be involved in protein-protein interactions, meaning that SHIP2 is also a docking protein for specific intracellular molecules. The SHIP2 protein is largely expressed in hematopoietic and non-hematopoietic cells during embryonic and adult life (Schurmans et al., 1999; Muraille et al., 2001). Functionally, SHIP2 is important for the control of many cellular processes, including cell migration, proliferation and adhesion, endocytosis, as well as for insulin and growth factors signaling (Suwa et al., 2010; Hakim et al., 2012; Dyson et al., 2012; Elong Edimo et al., 2014; Thomas et al., 2017; Ramos et al., 2019).
Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2^Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2^Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition.
Altogether, our results indicate that Ship2^Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.
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Ship2 mRNA has been previously detected in mouse embryonic and adult kidney by in situ hybridization and semiquantitative reverse transcriptase polymerase chain reaction, but no data are currently available on SHIP2 protein expression in the mouse renal PT.4,5
The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule–specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.
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It is for example highly expressed in mouse heart and brain (Muraille et al., 1999). During mouse embryonic development, SHIP2 is very much expressed in the germinal layer or the neuroepithelium of the brain and spinal cord throughout the embryonic development (Muraille et al., 2001). No significant expression was detected in regions containing migrating or more differentiated cells.
Phosphoinositide 5-phosphatases are critical enzymes in modulating the concentrations of PI(3,4,5)P₃, PI(4,5)P₂ and PI(3,5)P₂. The SH2 domain containing inositol 5-phosphatases SHIP1 and SHIP2 belong to this family of enzymes very much involved in physiopathology and development. Therefore activity and localization of the enzymes are particularly important taking into account both catalytic and non-catalytic mechanisms of the SHIP phosphatases. Several different mechanisms have been reported for SHIP2 targeting that often result from specific protein:protein interactions. In unstimulated astrocytoma cells, SHIP2 has a perinuclear and cytoplasmic localization. In serum-stimulated cells, SHIP2 can be localized at the plasma membrane and at focal contacts in polarized cells. A phosphorylated form of SHIP2 on S132 can be found in the nucleus and nuclear speckles. When present at the plasma membrane, SHIP2 may control the intracellular level of PI(3,4,5)P₃ thereby producing PI(3,4)P₂. When present in the nucleus, SHIP2 probably associates to other nuclear proteins such as lamin A/C and could potentially control nuclear PI(4,5)P₂. Finally, its presence at focal adhesions and lamellipodia could suggest a role in cell adhesion and migration. It is proposed that the complex phenotype observed in SHIP2 mutant mice in tissue development and growth could result from the addition of plasma membrane and nuclear effects consecutive to SHIP2 alteration.
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Phosphatidyl inositol 3-kinase (PI3-kinase) functions as a lipid kinase to produce PI(3,4,5)P₃ from PI(4,5)P₂ in vivo. PI(3,4,5)P₃ is crucial as a lipid second messenger in various metabolic effects of insulin. Lipid phosphatases, src homology 2 domain containing inositol 5′-phosphatase 2 (SHIP2) and skeletal muscle and kidney-enriched inositol phosphatase (SKIP) hydrolyze PI(3,4,5)P₃ to PI(3,4)P₂ and phosphatase and tensin homolog deleted on chromosome ten (PTEN) hydrolyzes PI(3,4,5)P₃ to PI(4,5)P₂. SHIP2 negatively regulates insulin signaling relatively specifically via its 5′-phosphatase activity. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity and conferred protection from obesity induced by a high-fat diet. Polymorphisms in the human SHIP2 gene are associated, at least in part, with the insulin resistance of type 2 diabetes. Importantly, inhibition of endogenous SHIP2 through the liver-specific expression of a dominant-negative SHIP2 improves glucose metabolism and insulin resistance in diabetic db/db mice. Overexpression of PTEN and SKIP also inhibited insulin-induced phosphorylation of Akt and the uptake of glucose in cultured cells. Although a homozygous disruption of the PTEN gene in mice results in embryonic lethality, either skeletal muscle or adipose tissue-specific disruption of PTEN ameliorated glucose metabolism without formation of tumors in animal models of diabetes. The role of SKIP in glucose metabolism remains to be further clarified in vivo. Taken together, inhibition of endogenous SHIP2 in the whole body appears to be effective at improving the insulin resistance associated with type 2 diabetes and/or obesity. Inhibition of PTEN in the tissues specifically targeted, including skeletal muscle and fat, may result in an amelioration of insulin resistance in type 2 diabetes, although caution against the formation of tumors is needed.
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The SH2 domain-containing 5-phosphatase SHIP2 is expressed in the germinal layers of embryo and adult mouse brain: increased expression in N-CAM-deficient mice

Abstract

Section snippets

Animals

SHIP2 probe characterization and signal specificity

Discussion

Conclusion

Acknowledgements

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Curr. Biol.

Mol. Brain Res.

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Blood

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FEBS Lett.

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Exp. Cell Res.

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