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Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF.

Alsina FC, Irala D, Fontanet PA, Hita FJ, Ledda F, Paratcha G - PLoS ONE (2012)

Bottom Line: Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA.In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF.Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof Dr E De Robertis-CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

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Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.
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pone-0032087-g005: Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.

Mentions: Next, we also examined the role of Spry4 on neurite outgrowth induced by NGF in primary DRG neurons. To this end, dissociated DRG (E14.5) neurons were transfected with control or Myc-tagged Spry4 construct together with a GFP expression vector. In agreement with the results obtained in PC12 cells, neurite outgrowth stimulated by NGF was significantly reduced in cells transfected with Myc-Spry4 (neurite length ± SEM in m, control: 119.4±21.0; Myc-tagged Spry4: 59.6±4.9, n = 6; *p<0.05) (Fig. 5A, 5B, left histogram and 5C). No differences were observed in NGF-promoted neuron survival between the two groups (Fig. 5B, right histogram). Data expressed as average values ± SEM are as follow: control: 94.1±4.2; Spry4 vector: 92.0±3.4, n = 3; p>0.05). These results indicate that inhibition of TrkA signaling by Spry4 specifically restrict neuronal differentiation but not survival.


Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF.

Alsina FC, Irala D, Fontanet PA, Hita FJ, Ledda F, Paratcha G - PLoS ONE (2012)

Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285629&req=5

pone-0032087-g005: Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.
Mentions: Next, we also examined the role of Spry4 on neurite outgrowth induced by NGF in primary DRG neurons. To this end, dissociated DRG (E14.5) neurons were transfected with control or Myc-tagged Spry4 construct together with a GFP expression vector. In agreement with the results obtained in PC12 cells, neurite outgrowth stimulated by NGF was significantly reduced in cells transfected with Myc-Spry4 (neurite length ± SEM in m, control: 119.4±21.0; Myc-tagged Spry4: 59.6±4.9, n = 6; *p<0.05) (Fig. 5A, 5B, left histogram and 5C). No differences were observed in NGF-promoted neuron survival between the two groups (Fig. 5B, right histogram). Data expressed as average values ± SEM are as follow: control: 94.1±4.2; Spry4 vector: 92.0±3.4, n = 3; p>0.05). These results indicate that inhibition of TrkA signaling by Spry4 specifically restrict neuronal differentiation but not survival.

Bottom Line: Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA.In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF.Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Neuroscience, Institute of Cellular Biology and Neuroscience Prof Dr E De Robertis-CONICET, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.

Show MeSH
Related in: MedlinePlus