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Molecular Endocrinology, doi:10.1210/me.2006-0005
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Molecular Endocrinology 21 (2): 499-511
Copyright © 2007 by The Endocrine Society

Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2 Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1

Jian-Mei Li, Masaki Mogi, Kana Tsukuda, Hirokazu Tomochika, Jun Iwanami, Li-Juan Min, Clara Nahmias, Masaru Iwai and Masatsugu Horiuchi

Department of Molecular and Cellular Biology (J.-M.L., M.M., K.T., H.T., J.I., L.-J.M., M.I., M.H.) Division of Medical Biochemistry and Cardiovascular Biology, Ehime University School of Medicine, Tohon, Ehime 791-0295, Japan; and Department of Cell Biology (C.N.), Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 567-Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, 75014 Paris, France

Address all correspondence and requests for reprints to: Masatsugu Horiuchi, M.D., Ph.D., Department of Molecular and Cellular Biology, Division of Medical Biochemistry and Cardiovascular Biology, Ehime University School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. E-mail: horiuchi{at}m.ehime-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development, but also in pathological conditions to protect the brain against stroke; however, the detailed mechanisms are unclear. Here, we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2, one of the ubiquitin-conjugating enzyme variants. The AT2 receptor, MMS2, Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1), and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner, reaching a peak after 4 h of stimulation, and this effect was enhanced with AT1 receptor blocker, valsartan, but inhibited by AT2 receptor blocker PD123319. Moreover, we observed that an AT2 receptor agonist, CGP42112A, alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover, the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor, sodium orthovanadate. After AT2 receptor stimulation, ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore, increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOTENSIN (Ang) II, a potent vasoactive peptide, has a variety of functions in the brain (1). Ang II binds two major receptor subtypes, type 1 (AT1) and type 2 (AT2) receptors; however, AT2 receptor signaling and its possible unique roles in the brain are not well defined. The AT2 receptor is reported to be widely expressed in the fetal-placental unit (2), but is observed at low levels in adult tissues and is reexpressed in some pathological conditions (3), indicating an important role of AT2 receptor activation in tissue regeneration. The AT2 receptor has also been reported to be expressed in the area of the adult brain related to learning and control of motor activity (4, 5) and to promote cell differentiation and regeneration in neural cells and tissue (6). For example, AT2 receptor stimulation induces neurite outgrowth and regulates neurofilaments in neural cell lines, NG108–15 cells and PC12W cells (7, 8). Furthermore, a recent genetic approach indicated that mutations of the AT2 receptor located on the X-chromosome are found in male patients with mental retardation (9), and AT2 receptor-deficient (AT2KO) mice display central neurological anomalies (10), suggesting that the AT2 receptor gene is involved in brain development and neural maturation in humans. However, the detailed mechanism of the effect of AT2 receptor stimulation on neural differentiation is unclear.

From the clinical perspective, the Study on Cognition and Prognosis in the Elderly (SCOPE) study (11) and a clinical double-blind study (12) have proved that AT1 receptor blockers have further therapeutic effects on impaired cognitive function beyond their antihypertensive effects, compared with other antihypertensive drugs. We previously demonstrated that AT2KO mice exhibited a larger ischemic size and greater neurological deficit after subjection to middle cerebral artery occlusion, at least partly due to impairment of cerebral blood flow in the penumbra and an increase in oxidative stress in the ischemic brain (13). Although we have demonstrated indirect effects of AT2 receptor signaling in cerebral protection, little is known about the direct mechanism of AT2 receptor signaling in neural differentiation.

Here, we focused on MMS2 as a target in AT2 receptor signaling-induced neural differentiation. MMS2 is one of the ubiquitin-conjugating enzyme-like proteins and is reported to play an important role in the ubiquitin-proteasome system (UPS) and DNA repair (14). MMS2 has been identified to be distributed widely in the rat brain in the late embryonic developmental stage and appears to have an important role in brain development and neural differentiation (15). Abnormalities of the UPS in the brain contribute to Parkinson disease, Alzheimer disease, prion disease, amyotrophic lateral sclerosis, and polyglutamine disease (16). Moreover, Ang II stimulation is reported to affect the ubiquitin-proteasome pathway, resulting in the degradation of inositol 1,4,5-trisphosphate receptors (17). However, the association between Ang II and MMS2 has never been investigated. Therefore, we examined such an association and demonstrated here that Ang II stimulation increased MMS2 expression via the AT2 receptor-signaling cascade, which could play a pivotal role in neural differentiation.

Recently several interacting proteins or downstream signaling proteins of AT2 receptor signaling, such as AT2 receptor-interacting protein (ATIP) and Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1), have been reported to have unique roles in AT2 receptor-induced signaling (18, 19, 20). We have cloned ATIP as a protein interacting with the C-terminal tail of the AT2 receptor, and it has been recently indicated to cooperate with the AT2 receptor to transinactivate receptor tyrosine kinases (18). ATIP (also known as ATBP, AT2 receptor-binding protein) is also reported to potentially act as a membrane–associated Golgi protein that dictates delivery of the AT2 receptor to the cell surface (19). ATIP was found to be identical to a ubiquitously expressed tumor suppressor protein localized in mitochondria (21). Therefore, ATIP seems to act as a potential novel early component of the growth and/or differentiation-regulating signaling cascade mediated by the AT2 receptor (18). On the other hand, SHP-1 is one of the tyrosine phosphatases activated by AT2 receptor stimulation and differentially phosphorylated and activated by neural growth factor in PC12 cells, resulting in its playing a role in neural growth factor-induced neural differentiation (22). SHP-1 is highly expressed in undifferentiated murine embryonic carcinoma cell line, P19 cells, and is reduced to an undetectable level after their differentiation (23). In contrast, SHP-1 is also expressed in adult mouse brain and especially highly expressed after middle cerebral artery occlusion (24), suggesting that SHP-1 could play important roles in neural repair. Furthermore, Massa et al. (25) demonstrate that SHP-1-deficient mice exhibit decreased myelin formation, suggesting a potential importance for SHP-1 in oligodendrocytes and myelin development. However, the interaction and involvement of ATIP and SHP-1 in neural differentiation by AT2 receptor stimulation have never been studied.

Therefore, we examined the potential roles of AT2 receptor signaling in neural differentiation and its molecular mechanism, focusing on MMS2, ATIP, and SHP-1. Here, we demonstrated a new signaling cascade via AT2 receptor stimulation in neural differentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Ang II Receptors, MMS2, and AT2-Receptor Related Proteins in Rat Neural Cells
First, to identify the primary cultured cells prepared from the rat fetal or newborn brain cortex as neurons, we checked the expression of a neural specific marker, MAP2, 3 d after isolation. MAP2 was highly expressed in these cells but was not observed in adult vascular smooth muscle cells as a negative control (Fig. 1AGo). To assess the expression of AT1 and AT2 receptors in these neuronal cells, a radioligand receptor binding assay was performed. As shown in Fig. 1BGo, the AT2 receptor was highly expressed in neuronal cells prepared from 18-d-old rat fetus, and then its expression gradually decreased after birth (6.8 ± 0.38 fmol/105 cells on embryonic day 18, 3.9 ± 0.22 fmol/105 cells at 10 d of age, and 2.1 ± 0.09 fmol/105 cells at 35 d of age). In contrast, no significant development-related change in AT1 receptor expression was found in these neuronal cells (Fig. 1BGo). On the other hand, mRNAs of SHP-1, ATIP, and MMS2 were highly expressed in neuronal cells on embryonic d 18, and decreased in a time-dependent manner after birth as determined by real-time RT-PCR method for SHP-1 and ATIP, and by Northern blot analysis of MMS2 (Fig. 1CGo). These changes in their mRNA expressions were similar to that in AT2 receptor expression. Protein levels of ATIP, SHP-1, and MMS2 determined by immunoblot analysis were also higher in embryonic and neonatal neurons, whereas their expressions declined after birth (Fig. 1DGo). Therefore, we employed neural cells prepared from the cerebral cortex of newborn rat, which express AT1 receptor, AT2 receptor, ATIP, and SHP-1.


Figure 1
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  		Fig. 1. Time Course of Expression of Ang II Receptors, MMS2, ATIP, and SHP-1 in Fetal and Newborn Rat Neurons

A, MAP2 protein level in primary rat neural cells determined by immunoblot. B, AT1 and AT2 receptor expression determined by radioligand-binding assay performed as described in Materials and Methods. C, mRNA expressions of SHP-1, ATIP, and MMS2. SHP-1 and ATIP mRNA expressions were determined by real-time PCR. MMS2 mRNA expressions were determined by Northern blot. D, Protein levels of SHP-1, ATIP, and MMS2 detected by immunoblot. Representative images and histogram of MMS2 expression are shown. Values are expressed as mean ± SEM (n = 3). E18, Embryonic d 18; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P1, postnatal d 1; VSMC, vascular smooth muscle cells.

 
Pivotal Role of MMS2 in Neural Differentiation
First, to address the roles of AT2 receptor-mediated MMS2 expression in neuronal cells, we employed RNA interference (RNAi) in neurons and evaluated neurite outgrowth and synapse formation. Neurons were transiently transfected with lamin A/C small interfering RNA (siRNA) as a control or MMS2-specific siRNA. Treatment with MMS2-RNAi markedly reduced MMS2 protein level (Fig. 2AGo) and inhibited neurite outgrowth (Fig. 2BGo) and synapse formation (Fig. 2CGo) assessed by immunofluorescent assay against a mature neural cell marker, ßIII-tubulin. After failing to exhibit neurite outgrowth, the neurons treated with MMS2-RNAi detached from the dishes and underwent apoptosis (data not shown). In PC12W cells, a rat pheochromocytoma cell line, treatment with MMS2-RNAi also induced detachment and apoptosis (data not shown).


Figure 2
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  		Fig. 2. Effect of MMS2 Expression on Neuronal Cell Differentiation in Rat Neural Cells

A, Comparison of MMS2 protein levels with or without MMS2-siRNA treatment. B, Effect of treatment with MMS2-targeted siRNA (MMS2-siRNA) on neurite outgrowth. Representative images and histogram of mean neurite length are shown. B, Effect of treatment with MMS2-siRNA on synapse formation detected by immunofluorescence against ßIII-tubulin. Representative images and histogram of ßIII-tubulin staining density are shown. Control, Control-siRNA treatment. *, P < 0.05 vs. control.

 
AT2 Receptor Stimulation Increased MMS2 Expression
Next, we examined the effect of Ang II on MMS2 expression in rat neuronal cells. Administration of Ang II (10–7 M) increased MMS2 mRNA expression, reaching a peak after 4 h of stimulation (Fig. 3AGo), in a dose-dependent manner (Fig. 3BGo). Consistent with previous reports that an AT2 receptor agonist, CGP42112A, induced neurite outgrowth in NG108–15 cells (7) and in microexplant cultures of the cerebellum (26), we also observed that treatment with CGP42112A significantly induced MMS2 mRNA expression (Fig. 3CGo), protein levels and neurite outgrowth (data not shown) in rat neuronal cells, indicating that AT2 receptor stimulation enhanced MMS2 expression in neurons. Ang II stimulation (10–7 M) also increased the expression of MMS 2 (Fig. 3DGo), mature neural cell markers, ßIII-tubulin (Fig. 3EGo) and MAP2 (Fig. 3FGo). These Ang II-mediated effects were further enhanced by addition of an AT1 receptor blocker, valsartan (10–5 M), but inhibited by an AT2 receptor antagonist, PD123319 (10–5 M). Moreover, treatment with MMS2-siRNA markedly inhibited the AT2 receptor-induced increase in the expression of these neural cell markers compared with treatment with control-siRNA (Fig. 3Go, E and F).


Figure 3
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  		Fig. 3. Ang II Stimulation Increases MMS2 Expression and Neuronal Cell Differentiation in Rat Neural Cells

A–C, Time courses of MMS2 mRNA expression detected by Northern blot after Ang II (10–7 M) stimulation (A), of 4-h Ang II stimulation at various doses (B), or addition of an AT2 receptor agonist, CGP42112A (C). Representative images and histogram of MMS2 expression are shown. Similar results were obtained in three different neuron cultures. D, Protein levels of MMS2. Histograms of MMS2-levels are shown. E and F, Effect of AT2 receptor stimulation on mature neural cell marker, ßIII-tubulin (E), and MAP2 (F) protein levels in neural cells treated with or without MMS2-siRNA. Representative images and histogram of ßIII-tubulin and MAP2 staining density are shown. Similar results were obtained in three different neuron cultures. Values are expressed as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01 vs. control. Control, Control-siRNA treatment; MMS2-siRNA, MMS2-targeted small interfering RNA; Ang II, angiotensin II (10–7 M); Val, valsartan (10–5 M); CGP, CGP42112A; PD, PD123319 (10–5 M); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Enforced Expression of ATIP Affects MMS2 Expression and SHP-1 Activity
To further examine the AT2 receptor-mediated signaling cascade in MMS2 up-regulation, we focused on ATIP and SHP-1. In neurons transfected with mATIP, ATIP was highly expressed (~3-fold), which was detected by immunoblot (data not shown). Interestingly, Ang II (10–7 M) with valsartan (10–5 M) stimulation further enhanced MMS2 mRNA expression in neurons with ATIP overexpression compared with neurons transfected with a control vector, pcDNA3 (4.87 ± 0.22-fold vs. 7.41 ± 0.29-fold, respectively, P < 0.05), and the effect of Ang II was inhibited by PD123319 (10–5 M) (Fig. 4AGo). Even CGP42112A (10–9 M) stimulation alone also increased MMS2 expression to more than that in control neurons (Fig. 4AGo), indicating that ATIP is important for enhancing AT2 receptor signaling-induced MMS2 mRNA expression. Moreover, treatment with Ang II (10–7 M) increased SHP-1 phosphatase activity more in ATIP-transfected neurons, and this Ang II-mediated effect was enhanced by addition of valsartan (10–5 M) but inhibited by PD123319 (10–5 M) (Fig. 4BGo). Moreover, we observed that CGP42112A (10–9 M) also increased SHP-1 phosphatase activity more markedly in ATIP-transfected neurons (Fig. 4BGo), indicating that ATIP could also be involved in the increase in SHP-1 phosphatase activity mediated by AT2 receptor signaling. Therefore, we speculated that a possible association between ATIP and SHP-1 triggered by AT2 receptor stimulation could be involved in AT2 receptor-induced MMS2 up-regulation.


Figure 4
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  		Fig. 4. Effect of Enforced Expression of ATIP on MMS2 Expression and SHP-1 Phosphatase Activity in Rat Neural Cells

A, Effect of overexpression of ATIP on MMS2 expression determined by Northern blot. B, Effect of overexpression of ATIP on SHP-1 phosphatase activity determined as described in Materials and Methods. Image shows representative data from three separate experiments. Values are expressed as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01 vs. no treatment. {dagger}, P < 0.01 vs. Ang II (+). Ang II, angiotensin II (10–7 M); Val, valsartan (10–5 M); PD, PD123319 (10–5 M); CGP, CGP42112A (10–9 M); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Interaction between ATIP and SHP-1 Induced by Ang II Stimulation
To explore the possibility of an interaction of ATIP and SHP-1, we performed immunoblot analysis with separated cell fractions, from the membrane, cytoplasm, and nucleus. Without Ang II stimulation, ATIP was distributed mainly in the plasma membrane in neurons and translocated into the nucleus after treatment with Ang II (10–7 M) for 15 min. SHP-1 was also distributed mainly in the cytoplasm without Ang II stimulation and translocated to the nucleus after Ang II stimulation (Fig. 5AGo). Moreover, immunoprecipitation of SHP-1 and ATIP showed that Ang II (10–7 M) stimulation for 15 min promoted the association of SHP-1 and ATIP, which was further increased by valsartan, but inhibited by PD123319, indicating that AT2 receptor stimulation increases the association of ATIP and SHP-1 and induces them to form and complex (Fig. 5BGo).


Figure 5
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  		Fig. 5. Interaction between ATIP and SHP-1 with AT2 Receptor Stimulation in Rat Neural Cells

A, Expression of ATIP and SHP-1 determined by immunoblotting with cell fractions with or without Ang II stimulation. Representative images and histogram of expression are shown. M, Membrane; C, cytoplasm; N, nucleus. B, Effect of Ang II on interaction of SHP-1 and ATIP analyzed by immunoprecipitation with whole-cell extracts. Representative images and histogram of expression are shown. Similar results were obtained in three different experiments. Values are expressed as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01 vs. no treatment. Ang II, Angiotensin II (10–7 M); Val, valsartan (10–5 M); PD, PD123319 (10–5 M); IB, immunoblot; IP, immunoprecipitation.

 
MMS2 Expression Was Dependent on Interaction between ATIP and SHP-1
Next, to investigate the detailed mechanism of the interaction between ATIP and SHP-1 and the roles of SHP-1, we performed transfection of SHP-1-siRNA into rat cultured neuronal cells. As shown in Fig. 6AGo, SHP-1 siRNA treatment effectively inhibited SHP-1 expression. In neuronal cells, treatment with SHP-1-siRNA inhibited Ang II signaling-mediated MMS2 expression (Fig. 6BGo). To examine the possibility that tyrosine phosphatases are involved in AT2 receptor-mediated MMS2 mRNA expression, we used sodium orthovanadate to inhibit tyrosine phosphatase activity. As shown in Fig. 6CGo, pretreatment with orthovanadate 16 h before Ang II stimulation inhibited AT2 receptor-mediated MMS2 mRNA expression, suggesting that phosphatase activity induced by SHP-1 is at least one of the critical regulators of AT2 receptor-mediated neural differentiation. Moreover, treatment with ATIP-siRNA also attenuated mRNA and protein level of MMS2 induced by AT2 receptor stimulation (Fig. 6Go, D–F). Moreover, transient transfection of catalytically dominant-negative SHP-1 (dnSHP-1), which markedly attenuated the phosphatase activity of the SHP-1 (Fig. 6HGo), significantly inhibited MMS2 expression in neurons, indicating that a phosphatase activity of SHP-1 is necessary to induce MMS2 expression (Fig. 6Go, G and I).


Figure 6
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  		Fig. 6. Effect of SHP-1 and ATIP on MMS2 Expression in Rat Neural Cells

A, SHP-1 expression in rat neurons treated with SHP-1-siRNA. B, Effect of treatment with SHP-1-siRNA on AT2 receptor-mediated MMS2 expression. C, Effect of tyrosine phosphatase inhibitor on AT2 receptor-mediated MMS2 mRNA expression. D, ATIP expression in rat neurons treated with ATIP-siRNA. Effect of treatment with ATIP-siRNA on MMS2 mRNA expression (E) and protein level (F) induced by AT2 receptor stimulation. Histograms of MMS2 expression are shown. *, P < 0.05; **, P < 0.01 vs. no treatment. Effect of treatment with dominant-negative form of SHP-1 on SHP-1 expression (G), SHP-1 phosphatase activity (H), and MMS2 mRNA expression after Ang II stimulation (I). *, P < 0.05 vs. pcDNA3-Ang II (–). #, P < 0.05 vs. pcDNA3-Ang II (+). Ang II, Angiotensin II (10–7 M); Val, valsartan (10–5 M); PD, PD123319 (10–5 M); SHP-1 siRNA, SHP-1-targeted siRNA; ATIP siRNA, ATIP-targeted siRNA; Van, sodium orthovanadate (10–5 M); dnSHP-1, dominant negative SHP-1 (C453/S); GAPDH, GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
MMS2 Promoted the Proteolysis of Inhibitor of DNA Binding Protein 1 (Id1) and the DNA Repair
Finally, we examined the effect of AT2 receptor-induced MMS2 on a differentiation inhibitory factor and DNA repair system in neurospheres. The proteins contribute to the regulation of mammalian nervous system development (27). We observed that Id1 protein level was decreased by AT2 receptor-stimulation (Fig. 7AGo), and this decrease was restored by a proteasome inhibitor, MG132, and MMS2-siRNA treatment, indicating that MMS2 could promote the proteolysis of Id1 by the ubiquitin proteasome system. Moreover, a recent paper described that UBC13 works as two different pathways binding with two different UBC variants, such as MMS2 for DNA repair and Uev1A for nuclear factor-{kappa}B activation (28). Therefore, we also analyzed the effects of MMS2 on the DNA repair pathway after AT2 receptor stimulation and performed quantification of abasic sites in genomic DNA in neurospheres after UV radiation. As shown in Fig. 7BGo, AT2 receptor stimulation reduced damaged DNA. Treatment with MMS2-siRNA attenuated such DNA repair effects, indicating that AT2 receptor induced an increase in DNA repair via MMS2.


Figure 7
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  		Fig. 7. MMS2 Promoted the Proteolysis of Id1 Protein and the DNA Repair

A, Id1 protein levels in mouse neurospheres. Representative images and histogram of Id1 levels are shown. B, Effect of AT2 receptor-stimulation on damaged DNA after UV radiation (254 nm) in mouse neurospheres. Quantification of DNA damage was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01 vs. no treatment. {dagger}, P < 0.05 vs. Ang II (+). Ang II, angiotensin II (10–7 M); Val, valsartan (10–5 M); PD, PD123319 (10–5 M); MMS2-siRNA, MMS2-targeted siRNA; MG132, a proteasome inhibitor (2.5 x 10–5 M). AP sites, Apurinic/apyrimidinic sites.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study demonstrated a new signaling mechanism in AT2 receptor-induced neural differentiation. First, one of the ubiquitin-conjugating enzyme variants, MMS2, was a key molecule in the downstream target of the AT2 receptor. Second, novel AT2 receptor-interacting proteins, ATIP and SHP-1, played a role as transactivating proteins in the AT2 receptor-MMS2 cascade. Third, ATIP and SHP-1 formed a complex and translocated into the nucleus to induce MMS2 expression (Fig. 8Go). Treatment with siRNA targeting against ATIP or SHP-1 indicated that both ATIP and SHP-1 would contribute to the AT2 receptor-mediated increase in MMS2 formation; however, we could not define the detailed mechanism of the transcriptional regulation of MMS2 by the ATIP/SHP-1 complex. We speculate that other factors must act as adaptor or translocation proteins in the ATIP/SHP-1 complex to regulate MMS2 expression. Furthermore, targeted proteins that are degraded after MMS2 induction are also necessary to support the mechanism of MMS2-induced neural differentiation. MMS2 plays an important role in UPS through the ubiquitin-conjugating enzyme complex with UBC13. Trausch-Azar et al. (29) reported that degradation of many short-lived cellular proteins, such as the transcription factor MyoD and Id1, occurs via the ubiquitin-proteasome pathway, indicating that the UPS activated by an increase of MMS2 could possibly affect some neural differentiation-related factors, thereby inducing neural differentiation. Our findings suggest that MMS2 promoted the proteolysis of the Id1 protein by ubiquitin proteasome system, although the detailed effects of MMS2 on Id1 proteolysis need to be clarified, and there may be other neural differentiation-related factors regulated by UPS. Moreover, we also demonstrated that AT2 receptor stimulation enhanced DNA repair pathway through MMS2 induction. Therefore, multiple mechanisms are involved in neural protection induced by MMS2 via the UPS. Antiproliferative effects of AT2 receptor stimulation, including apoptosis induction, are widely reported. Cells with damaged DNA should be removed through apoptosis for preventing malformations, indicating that AT2 receptor-induced apoptosis may be caused through DNA-repair pathway; however, the detailed association between apoptosis and DNA-repair pathway by AT2 receptor-stimulation remains unclear. Moreover, AT2 receptor stimulation is known to exert antiinflammatory and antioxidative stress effects, and these mechanisms could be also involved in neural protection. For example, chemokines and inflammatory response exacerbate cerebral ischemia tissue injury and neurodegenerative disease by increasing leukocyte infiltration and subsequent neuronal damage (30). Therefore, inhibitory effects of AT2 receptor stimulation on neuronal damage may be caused, at least in part, through antiinflammatory and antioxidative stress effects. Further investigations are necessary to explore in more detail the relation among DNA repair, apoptosis, inflammation, and oxidative stress.


Figure 8
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  		Fig. 8. New Signaling Mechanism in AT2 Receptor-Induced Neural Differentiation

Schematic presentation of AT2 receptor-induced neural differentiation.

 
The AT2 receptor is one of the seven-transmembrane receptor families and interacts with heterotrimeric G proteins, such as G{alpha}i. However, beyond acting on G proteins, its cellular sorting and targeting to cell components has been highlighted. Several proteins including ATIP have been reported such as AT1 receptor-associated protein, which binds to the C terminus of the AT1 receptor and acts as an AT1 receptor desensitizer (31), and promyelocytic zinc finger protein, which binds to the C terminus of the AT2 receptor specifically in cardiomyocytes and induces cardiac hypertrophy (20). We have demonstrated that AT2 receptor stimulation-induced neural differentiation was closely associated with interaction of ATIP protein and SHP-1, beyond acting as a G protein receptor, suggesting a new intracellular signaling mechanism after AT2 receptor stimulation. Therefore, these proteins interacting with Ang II receptors could be attractive as future therapeutic targets for the blockade of the renin angiotensin system.

SHP-1 activity could also play an important role in the AT2 receptor-induced neural differentiation cascade. Loss of SHP-1 has been reported to lead to many hematopoietic abnormalities, infertility, and low body weight (32). Wishcamper et al. (33) demonstrated that the brains of SHP-1-deficient mice are slightly smaller than age-matched wild-type littermates and show decreases in the number of astrocytes and microglia compared with wild-type mice, indicating that SHP-1 is a positive regulator for the normal differentiation and distribution of nervous system. Moreover, our previous report on nuclear factor-{kappa}B inactivation to improve vascular inflammation via AT2 receptor-mediated SHP-1 activation also provides the effects of SHP-1 as a positive regulator (34). Treatment with SHP-1 siRNA and orthovanadate diminished AT2 receptor-mediated MMS2 expression and neural differentiation. Our findings suggest that the increase in SHP-1 tyrosine phosphatase activity mediated by AT2 receptor stimulation could dephosphorylate some tyrosine-phosphorylated factor(s), which would induce colocalization and translocation of the SHP-1 and ATIP complex and thereby increase transcription of MMS2 and Id1 degradation, resulting in neural differentiation. However, we could not find out any specific substrate protein(s) that could be tyrosine dephosphorylated by SHP-1 phosphatase activity in this cascade. Moreover, SHP-1 could act as adaptor protein, and unidentified proteins could be involved (see scheme in Fig. 8Go). These issues must be addressed in order to understand further the mechanism of AT2 receptor-mediated neural differentiation.

Brain aging, dementia, and neurological deficit after stroke are leading causes of disability and impair quality of life in the aged population. Radical treatment for neural damage is limited, and self-repair by neurogenesis after brain damage is reported to be limited (35). Recently, cell replacement therapy using pluripotent cells, such as embryonic stem cells or mesenchymal stem cells, had been expected to repair injured neurons. However, the plasticity of these cells planted within cortical connections was limited, resulting in failure to repair any of the damaged neurons. Therefore, preventing brain damage and neuronal loss is closely related to the management of lifestyle-related diseases, such as hypertension, diabetes mellitus, and hyperlipidemia. Our unpublished data suggest that after the middle cerebral artery occlusion, expression of the AT2 receptor is significantly increased in the ischemic brain. Moreover, AT2KO mice exhibit impairment of cognitive function after stroke as well as an increase in stroke size compared with wild-type littermates.

In conclusion, we demonstrated that stimulation of AT2 receptor signaling induces MMS2 expression, which prevents neural damage and enhances neural cell differentiation via the interaction between ATIP and SHP-1, resulting in brain protection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Treatment
Neurons were prepared from the cerebral cortex of fetal and newborn Wistar rats (Clea Japan, Inc., Tokyo, Japan) as previously described (36). Primary cortical neurons were cultured at 106 cells/cm2 on poly-D-lysine-coated dishes with serum-free neurobasal medium supplemented with 2% B-27 supplement (Invitrogen, Carlsbad, CA), 1 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. Fetal mouse neurospheres were prepared as described previously (37). Cells were plated at 105 cells/ml in neural stem cell basal medium (NeuroCult, StemCell Technologies, Inc., Vancouver, British Columbia, Canada), with differentiation supplements (NeuroCult Differentiation Supplements, StemCell Technologies, Inc.), basic fibroblast growth factor (20 ng/ml), and 100 U/ml penicillin and 100 µg/ml streptomycin sulfate. Neurospheres formed after 7–10 d in culture.

From 3–7 d of culture, the primary neurons were treated with a specific AT1 receptor blocker, valsartan (donated by Novartis Pharma AG, Basel, Switzerland), and/or a specific AT2 receptor blocker, PD123319 (Research Biochemical International, Natick, MA), and/or an AT2 agonist, CGP42112A (Sigma-Aldrich Corp., St. Louis, MO), and/or sodium orthovanadate (Na3VO4: Wako Pure Chemical Industries, Ltd., Osaka, Japan). Ang II binds with AT2 receptors at several sites, such as Arg 182, Asp 297 and N termini of them as well as AT1 receptors, whereas CGP42112A does not have binding sites in the N terminus of AT2 receptors (38). Moreover, CGP42112A was also used at one hundredth lower concentration as AT2 receptor agonist (39), because it has antagonistic effects at higher concentrations. Therefore, we performed CGP42112A treatment with a low-dose concentration compared with that of Ang II with various stimuli.

AT1 and AT2 Receptor Expression
AT1 receptor and AT2 receptor expression was examined by radioligand binding assay as previously reported (40). AT1 and AT2 receptor binding was measured using subconfluent cells grown in 24-well plates. After washing twice with PBS containing 0.1% BSA, the cells were incubated for 1 h at 37 C with 0.1 nM 125I-[Sar1, Ile8]-AII (NEN Life Science Products, Boston, MA) in the absence (for total count) or presence of valsartan (10–5 M) or PD123319 (10–5 M). The cells were then washed twice with ice-cold PBS containing 0.1% BSA and lysed in 0.5 N NaOH. The raw radioactivity count of the lysate was measured by a counter. AT1 receptor binding was calculated as the difference between the total count and the count from samples incubated with valsartan. AT2 receptor binding was determined by subtracting the count of samples incubated with PD123319 from the total count. The net radioactivity count was converted to a molar value using the specific activity of the ligand and was normalized by the cell number, which was measured at the same time.

Real-Time RT-PCR
Real-time quantitative RT-PCR was performed with a SYBR kit (MJ Research, Inc., Waltham, MA). PCR primers for ATIP were 5'-AAA CTG CAC AAC GGA GAC CT-3' (forward) and 5'-TTC CCA TGA GAG GGT CAG TC-3' (reverse).

RNA Interference Assay
Neurons were transiently transfected with lamin A/C siRNA as a control or MMS2-specific siRNA or SHP-1-specific siRNA. These siRNAs were cocktails of three siRNAs designed by B-Bridge (Sunnyvale, CA) and were transfected using Lipofectamine PLUS (Invitrogen). Neurons were treated, 36 h after transfection, with or without Ang II and/or valsartan and/or PD123319.

Quantification of Neurite Outgrowth in Neurons
Images of randomly chosen fields of neuron cultures were obtained at x40 magnification with a Leica DMI 6000 B microscope (Leica Microsystems, Wetzlar, Germany) and Nikon DS-5M camera units (Nikon, Tokyo, Japan). Quantification of neurite length was done on exported TIFF file using the NIH image analysis software.

Immunofluorescent Staining
Cells were fixed with 10% formalin and permeabilized with 0.1% saponin before incubation with mouse monoclonal anti-ßIII tubulin antibody (Promega Corp., Madison WI). The secondary antibody was goat antimouse antibody (Molecular Probes, Inc., Eugene, OR). Cells were counterstained with 4',6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Inc., Burlingame, CA). Images were viewed at x10 magnification with an Axioskop microscope (Carl Zeiss, Oberkochen, Germany), using image analysis software (Atto Densitograph, Tokyo, Japan).

ATIP Transfection
The full-length mATIP cDNA fused to the Myc or AcGFP epitope and was transiently transfected in neurons with the Lipofectamine PLUS reagents (Life Science Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. The transfected cells were fed with complete growth medium for 48 h and were then made quiescent by incubation with serum-free DMEM overnight before appropriate treatment.

Northern Blot Analysis
Fetal rat brain cortical neurons were kept in a supplement-free condition for 24 h to reach a quiescent state. Neurons were treated with vehicle or Ang II (10–7 M) with or without valsartan (10–5 M) or PD123319 (10–5 M). An AT2 receptor agonist, CGP42112A, was also used for stimulation. RNA was prepared from the cells using TRIzol reagent (Invitrogen). To prepare the hybridization probe for MMS2, RT-PCR was performed as previously described (29). PCR primers for MMS2 were 5'-AAG GAG TAG GTG ATG GTA CGG T-3' (forward) and 5'-TGC TAA TAC TGG TAT GCT CCG T-3' (reverse). To verify the identities of the PCR products, we sequenced the PCR products and confirmed that the sequences of PCR products matched the predicted sequences. PCR-amplified DNA of MMS2 was subcloned into pGEM-T Easy Vector (Promega Corp.). After size fractionation on denaturing agarose-formaldehyde gel, total RNA (30 µg) was transferred to a nylon membrane (Hybond-N +, Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization was carried out with 32P-labeled probes of rat MMS2 cDNA and/or the 0.78-kb PstI-XbaI fragment of human glyceraldehyde 3-phosphate dehydrogenase in Parid-Hyb buffer (Amersham Pharmacia Biotech). Densitometric analysis was performed using an image scanner (EPSON GT-8000; Ricoh System Kaihatu Co. Ltd., Tokyo, Japan) and NIH imaging software.

Preparation of Neural Cell Fractions
To measure the changes in distribution of ATIP and SHP-1 in the plasma membrane, cytoplasm, and nucleus after Ang II stimulation, the membrane, cytoplasmic, and nuclear fractions of neural cells were isolated as described previously, with minor modification (41, 42). Neural cells with or without Ang II (10–7 M) stimulation cultured in 100-mm dishes were washed and resuspended in PBS followed by centrifugation at 14,000 x g at 4 C for 20 min. The cell pellet was thawed and sonicated in 100 µl ice-cold homogenization buffer (20 mM Tris, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). After centrifugation at 1000 x g for 10 min, the supernatant was centrifuged at 100,000 x g at 4 C for 60 min to separate the plasma membrane and cytoplasmic fractions, the cell pellet was washed in 40 µl ice-cold high-salt buffer [20 mM Tris (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol] and centrifuged at 13,000 x g at 4 C for 5 min to separate the nucleus. After separation of the plasma membrane and cytoplasmic fractions, the supernatant representing the cytoplasmic fraction was removed and subjected to additional centrifugation as described above to sediment any remaining particulate matter, and the pellet representing the membrane fraction was washed once with homogenization buffer by centrifugation as described above and resuspended in receptor-binding buffer (20 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). After separation of the nucleus, the pellet representing the nuclear fraction was saved for later use.

Immunoprecipitation and Immunoblot Analysis
Neurons were kept in a supplement-free condition for 24 h and then treated as indicated in the figure legends. Total protein was prepared from the cultured neurons, and Western blotting was performed as previously described (43). Immunoprecipitation was performed using anti-SHP-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and rabbit anti-ATIP polyclonal antibodies previously reported (18). Immunoblotting was performed using anti-MAP2, anti-ßIII tubulin (Promega Corp.), anti-ß-tubulin (Sigma-Aldrich Corp.), anti-MMS2, anti-SHP-1, anti-ATIP, and anti-Id1 (Santa Cruz Biotechnology, Inc.) antibodies. The cell lysate (20 µg) was run on 10% SDS-PAGE, and the separated proteins were electrophoretically transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech). Blots were incubated with specific antibodies as indicated. The bands were visualized with an ECL system (Amersham Pharmacia Biotech). Densitometric analysis was performed using an image scanner (EPSON GT-8000) and NIH imaging software.

Measurement of Tyrosine Phosphatase Activity of SHP-1 and Dominant-Negative SHP-1 Transfection
Activity of SHP-1 was determined as previously described (44). Cell lysates (1 mg protein) were immunoprecipitated with anti-SHP-1 antibody and protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech), as described above. The immunoprecipitates were washed three times with lysis buffer lacking Na3VO4 and NaF, and once with phosphatase assay buffer (50 mM Tris-HCl, pH 7.0; 1 mM EDTA; 5 mM dithiothreitol; and 0.01% Brij35). Protein tyrosine phosphatase activity was measured as the release of [32P]orthophosphate from [32P]ATP-radiolabed myelin basic protein. For further investigation, we used a catalytically inactive SHP-1 (dnSHP-1) (C453/S), which was constructed with the oligonucleotide primer 5-GAT GCC AGC GCT GGA ATG CAC AAT-3, as described previously (45). This mutation completely abolished the phosphatase activity of the SHP-1 mutant transiently expressed in COS-7 cells (46). Overexpression of dnSHP-1 was performed by transient transfection with Lipofectamine PLUS (Invitrogen) as described previously. Cells were treated, 48 h after transfection, with Ang II for 4 h, and then cell lysates were collected to perform the tyrosine phosphatase activity of SHP-1.

Quantification of Damaged DNA
DNA damage was quantified by DNA Damage Quantification Kit (Dojindo, Kumamoto, Japan) based on the detection of abasic sites in genomic DNA with aldehyde reactive probe reagent, which reacts specifically with the open ring form of the abasic sites. We performed this assay according to the manufacturer’s instructions.

Statistical Analysis
Values are expressed as mean ± SEM in the text and figures. The data were analyzed using ANOVA followed by Newman-Keuls’ test for multiple comparisons. If a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. Values of P < 0.05 were considered to be statistically significant.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Takeda Science Foundation, and the Novartis Foundation for Gerontological Research.


   FOOTNOTES
 
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Takeda Science Foundation, and the Novartis Foundation for Gerontological Research (to M.H.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: Ang II, Angiotensin II; AT1 receptor, angiotensin II type 1 receptor; AT2 receptor, angiotensin II type 2 receptor; AT2KO mice, AT2 receptor-deficient mice; ATIP, AT2 receptor-interacting protein; dnSHP-1, dominant-negative SHP-1; Id, the inhibitor of DNA binding; SHP-1, Src homology 2 domain-containing protein-tyrosine phosphatase 1; siRNA, small interfering RNA; UBC, to be defined; UPS, ubiquitin-proteasome system.

Received for publication January 5, 2006. Accepted for publication October 17, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pelegrini-da-Silva A, Martins AR, Prado WA 2005 A new role for the renin-angiotensin system in the rat periaqueductal gray matter: angiotensin receptor-mediated modulation of nociception. Neuroscience 132:453–463[CrossRef][Medline]
  2. Schutz S, Le Moullec JM, Corvol P, Gasc JM 1996 Early expression of all the components of the renin-angiotensin-system in human development. Am J Pathol 149:2067–2079[Abstract]
  3. Matsubara H 1998 Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res 83:1182–1191[Abstract/Free Full Text]
  4. Wright JW, Harding JW 1995 Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their functions. Regul Pept 59:269–295[CrossRef][Medline]
  5. Tsutsumi K, Saavedra JM 1991 Characterization and development of angiotensin II receptor subtypes (AT1 and AT2) in rat brain. Am J Physiol 261:R209–R216
  6. Stroth U, Meffert S, Gallinat S, Unger T 1998 Angiotensin II and NGF differentially influence microtubule proteins in PC12W cells: role of the AT2 receptor. Brain Res Mol Brain Res 53:187–195[Medline]
  7. Laflamme L, Gasparo M, Gallo JM, Payet MD, Gallo-Payet N 1996 Angiotensin II induction of neurite outgrowth by AT2 receptors in NG108–15 cells. Effect counteracted by the AT1 receptors. J Biol Chem 271:22729–22735[Abstract/Free Full Text]
  8. Meffert S, Stoll M, Steckelings UM, Bottari SP, Unger T 1996 The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol Cell Endocrinol 122:59–67[CrossRef][Medline]
  9. Vervoort VS, Beachem MA, Edwards PS, Ladd S, Miller KE, de Mollerat X, Clarkson K, DuPont B, Schwartz CE, Stevenson RE, Boyd E, Srivastava AK 2002 AGTR2 mutations in X-linked mental retardation. Science 296:2401–2403[Abstract/Free Full Text]
  10. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BL, Inagami T 1995 Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377:748–750[CrossRef][Medline]
  11. Lithell H, Hansson L, Skoog I, Elmfeldt D, Hofman A, Olofsson B, Trenkwalder P, Zanchetti A 2003 The Study on Cognition and Prognosis in the Elderly (SCOPE): principal results of a randomized double-blind intervention trial. J Hypertens 21:875–886[CrossRef][Medline]
  12. Tedesco MA, Ratti G, Mennella S, Manzo G, Grieco M, Rainone AC, Iarussi D, Iacono A 1999 Comparison of losartan and hydrochlorothiazide on cognitive function and quality of life in hypertensive patients. Am J Hypertens 12:1130–1134[CrossRef][Medline]
  13. Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T, Horiuchi M 2004 Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation. Circulation 110:843–848
  14. Broomfield S, Chow BL, Xiao W 1998 MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc Natl Acad Sci USA 95:5678–5683[Abstract/Free Full Text]
  15. Hofsaess U, Kapfhammer JP 2003 Identification of numerous genes differentially expressed in rat brain during postnatal development by suppression subtractive hybridization and expression analysis of the novel rat gene rMMS2. Brain Res Mol Brain Res 113:13–27[Medline]
  16. Ciechanover A, Brundin P 2003 The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron 40:427–446[CrossRef][Medline]
  17. Bokkala S, Joseph SK 1997 Angiotensin II-induced down-regulation of inositol trisphosphate receptors in WB rat liver epithelial cells. Evidence for involvement of the proteasome pathway. J Biol Chem 272:12454–12461[Abstract/Free Full Text]
  18. Nouet S, Amzallag N, Li JM, Louis S, Seitz I, Cui TX, Alleaume AM, Di Benedetto M, Boden C, Masson M, Strosberg AD, Horiuchi M, Couraud PO, Nahmias C 2004 Trans-inactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP. J Biol Chem 279:28989–28997[Abstract/Free Full Text]
  19. Wruck CJ, Funke-Kaiser H, Pufe T, Kusserow H, Menk M, Schefe JH, Kruse ML, Stoll M, Unger T 2005 Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein. Arterioscler Thromb Vasc Biol 25:57–64[Abstract/Free Full Text]
  20. Senbonmatsu T, Saito T, Landon EJ, Watanabe O, Price Jr E, Roberts RL, Imboden H, Fitzgerald TG, Gaffney FA, Inagami T 2003 A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy. EMBO J 22:6471–6482[CrossRef][Medline]
  21. Seibold S, Rudroff C, Weber M, Galle J, Wanner C, Marx M 2003 Identification of a new tumor suppressor gene located at chromosome 8p21.3–22. FASEB J 17:1180–1182[Abstract/Free Full Text]
  22. Vambutas V, Kaplan DR, Sells MA, Chernoff J 1995 Nerve growth factor stimulates tyrosine phosphorylation and activation of Src homology-containing protein-tyrosine phosphatase 1 in PC12 cells. J Biol Chem 270:25629–25633[Abstract/Free Full Text]
  23. Mizuno K, Katagiri T, Maruyama E, Hasegawa K, Ogimoto M, Yakura H 1997 SHP-1 is involved in neuronal differentiation of P19 embryonic carcinoma cells. FEBS Lett 417:6–12[CrossRef][Medline]
  24. Wishcamper CA, Brooks DM, Douglas Coffin J, Lurie DI 2003 Focal cerebral ischemia upregulates SHP-1 in reactive astrocytes in juvenile mice. Brain Res 974:88–98[CrossRef][Medline]
  25. Massa PT, Wu C, Fecenko-Tacka K 2004 Dysmyelination and reduced myelin basic protein gene expression by oligodendrocytes of SHP-1-deficient mice. J Neurosci Res 77:15–25[CrossRef][Medline]
  26. Cote F, Do TH, Laflamme L, Gallo JM, Gallo-Payet N 1999 Activation of the AT(2) receptor of angiotensin II induces neurite outgrowth and cell migration in microexplant cultures of the cerebellum. J Biol Chem 274:31686–31692[Abstract/Free Full Text]
  27. Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K, Benezra R 1999 Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401:670–677[CrossRef][Medline]
  28. Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W 2005 Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J Cell Biol 170:745–755[Abstract/Free Full Text]
  29. Trausch-Azar JS, Lingbeck J, Ciechanover A, Schwartz AL 2004 Ubiquitin-proteasome-mediated degradation of Id1 is modulated by MyoD. J Biol Chem 279:32614–32619[Abstract/Free Full Text]
  30. Cartier L, Hartley O, Dubois-Dauphin M, Krause KH 2005 Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases. Brain Res Brain Res Rev 48:16–42[CrossRef][Medline]
  31. Daviet L, Lehtonen JY, Tamura K, Griese DP, Horiuchi M, Dzau VJ 1999 Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor. J Biol Chem 274:17058–17062[Abstract/Free Full Text]
  32. Schweitzer PA, Shultz LD 1988 Characterization of Mott cell hybridomas from autoimmune "viable motheaten" mutant mice. Curr Top Microbiol Immunol 137:223–226[Medline]
  33. Wishcamper CA, Coffin JD, Lurie DI 2001 Lack of the protein tyrosine phosphatase SHP-1 results in decreased numbers of glia within the motheaten (me/me) mouse brain. J Comp Neurol 441:118–133[CrossRef][Medline]
  34. Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M 2004 Regulation of inhibitory protein-{kappa}B and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor-activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells. Mol Endocrinol 18:666–678[Abstract/Free Full Text]
  35. Kornack DR, Rakic P 2001 Cell proliferation without neurogenesis in adult primate neocortex. Science 294:2127–2130[Abstract/Free Full Text]
  36. Bock HH, Jossin Y, May P, Bergner O, Herz J 2004 Apolipoprotein E receptors are required for reelin-induced proteasomal degradation of the neuronal adaptor protein Disabled-1. J Biol Chem 279:33471–33479[Abstract/Free Full Text]
  37. Reynolds BA, Tetzlaff W, Weiss S 1992 A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:4565–4574[Abstract]
  38. Yee DK, Heerding JN, Krichavsky MZ, Fluharty SJ 1998 Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor. Brain Res Mol Brain Res 57:325–329[Medline]
  39. Huang XC, Richards EM, Sumners C 1996 Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors. J Biol Chem 271:15635–15641[Abstract/Free Full Text]
  40. Li G, Chen YF, Greene GL, Oparil S, Thompson JA 1999 Estrogen inhibits vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro. Circulation 100:1639–1645
  41. Tang H, Guo DF, Porter JP, Wanaka Y, Inagami T 1998 Role of cytoplasmic tail of the type 1A angiotensin II receptor in agonist- and phorbol ester-induced desensitization. Circ Res 82:523–531[Abstract/Free Full Text]
  42. Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER{alpha} and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319[Abstract/Free Full Text]
  43. Cui TX, Nakagami H, Nahmias C, Shiuchi T, Takeda-Matsubara Y, Li JM, Wu L, Iwai M, Horiuchi M 2002 Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells. Mol Endocrinol 16:2113–2123[Abstract]
  44. Lehtonen JY, Daviet L, Nahmias C, Horiuchi M, Dzau VJ 1999 Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis. Mol Endocrinol 13:1051–1060[Abstract/Free Full Text]
  45. Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent N, Theveniau M, Nahmias C, Vaysse N, Susini C 1997 The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling. J Biol Chem 272:24448–24454[Abstract/Free Full Text]
  46. Lewin MJ 1992 The somatostatin receptor in the GI tract. Annu Rev Physiol 54:455–468[CrossRef][Medline]



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