Limits...
Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Show MeSH

Related in: MedlinePlus

NHE5 knockdown affects NGF signaling. (A–D) Control PC12, shNHE5, and shNHE1 cells were serum starved overnight and then treated with 50 ng/ml NGF for the indicated times. The amounts of phosphorylated and total Akt and Erk1/2 were detected by Western blot (A and B, respectively). Representative Western blots are shown. The experiments were repeated three times, and the intensities of the bands were determined by densitometry. The relative levels of phospho-Akt (C) and phospho-Erk1/2 (D) are expressed as means ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814139&req=5

Figure 6: NHE5 knockdown affects NGF signaling. (A–D) Control PC12, shNHE5, and shNHE1 cells were serum starved overnight and then treated with 50 ng/ml NGF for the indicated times. The amounts of phosphorylated and total Akt and Erk1/2 were detected by Western blot (A and B, respectively). Representative Western blots are shown. The experiments were repeated three times, and the intensities of the bands were determined by densitometry. The relative levels of phospho-Akt (C) and phospho-Erk1/2 (D) are expressed as means ± SEM.

Mentions: Because NHE5 knockdown affects TrkA trafficking and its cellular distribution, we reasoned that NHE5 might modulate TrkA downstream signaling. Control PC12 or shNHE5 cells were treated with 50 ng/ml NGF for 0–60 min, and the phosphorylation status of Akt (also known as PKB) and ERK1/2, two major downstream kinases, were evaluated by Western blot using antibodies that specifically recognize phosphorylated Akt and Erk1/2, respectively. NGF treatment of serum-starved PC12 cells caused a clear increase in the phosphorylation levels of Akt and Erk1/2 within 5 min. In contrast, phosphorylated Akt in response to NGF was significantly suppressed in shNHE5 cells under the same experimental conditions (Figure 6, A and C). Erk1/2 phosphorylation in shNHE5 cells was reduced at the 5-min time point, reaching ∼30% of that observed in control cells, and then formed a slightly delayed peak at the 10-min time point (Figure 6, B and D). Of interest, although shNHE1 cells showed weaker and delayed Akt phosphorylation in response to NGF treatment, Erk1/2 phosphorylation was not affected. To examine the role of endosomal alkalinization in NGF-TrkA signaling, we treated PC12 cells with bafilomycin for 6 h, a condition that significantly reduced cell-surface TrkA levels (Figure 5, A and B), and assessed its effect on NGF-induced phosphorylation of Akt and Erk1/2. Bafilomycin treatment markedly decreased NGF-induced phosphorylation of Akt (∼20% of control) and Erk1/2 to a lesser extent (∼60% of control; Figure 7, A–C). Thus NHE5 knockdown and bafilomycin treatment commonly have a greater effect on the phosphorylation of Akt than the phosphorylation of Erk1/2 in response to NGF stimulation. Of interest, 1-h bafilomycin treatment, a condition that did not affect TrkA cell-surface abundance, reduced Akt phosphorylation but not Erk1/2 phosphorylation (Figure 7, D–F).


Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

NHE5 knockdown affects NGF signaling. (A–D) Control PC12, shNHE5, and shNHE1 cells were serum starved overnight and then treated with 50 ng/ml NGF for the indicated times. The amounts of phosphorylated and total Akt and Erk1/2 were detected by Western blot (A and B, respectively). Representative Western blots are shown. The experiments were repeated three times, and the intensities of the bands were determined by densitometry. The relative levels of phospho-Akt (C) and phospho-Erk1/2 (D) are expressed as means ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814139&req=5

Figure 6: NHE5 knockdown affects NGF signaling. (A–D) Control PC12, shNHE5, and shNHE1 cells were serum starved overnight and then treated with 50 ng/ml NGF for the indicated times. The amounts of phosphorylated and total Akt and Erk1/2 were detected by Western blot (A and B, respectively). Representative Western blots are shown. The experiments were repeated three times, and the intensities of the bands were determined by densitometry. The relative levels of phospho-Akt (C) and phospho-Erk1/2 (D) are expressed as means ± SEM.
Mentions: Because NHE5 knockdown affects TrkA trafficking and its cellular distribution, we reasoned that NHE5 might modulate TrkA downstream signaling. Control PC12 or shNHE5 cells were treated with 50 ng/ml NGF for 0–60 min, and the phosphorylation status of Akt (also known as PKB) and ERK1/2, two major downstream kinases, were evaluated by Western blot using antibodies that specifically recognize phosphorylated Akt and Erk1/2, respectively. NGF treatment of serum-starved PC12 cells caused a clear increase in the phosphorylation levels of Akt and Erk1/2 within 5 min. In contrast, phosphorylated Akt in response to NGF was significantly suppressed in shNHE5 cells under the same experimental conditions (Figure 6, A and C). Erk1/2 phosphorylation in shNHE5 cells was reduced at the 5-min time point, reaching ∼30% of that observed in control cells, and then formed a slightly delayed peak at the 10-min time point (Figure 6, B and D). Of interest, although shNHE1 cells showed weaker and delayed Akt phosphorylation in response to NGF treatment, Erk1/2 phosphorylation was not affected. To examine the role of endosomal alkalinization in NGF-TrkA signaling, we treated PC12 cells with bafilomycin for 6 h, a condition that significantly reduced cell-surface TrkA levels (Figure 5, A and B), and assessed its effect on NGF-induced phosphorylation of Akt and Erk1/2. Bafilomycin treatment markedly decreased NGF-induced phosphorylation of Akt (∼20% of control) and Erk1/2 to a lesser extent (∼60% of control; Figure 7, A–C). Thus NHE5 knockdown and bafilomycin treatment commonly have a greater effect on the phosphorylation of Akt than the phosphorylation of Erk1/2 in response to NGF stimulation. Of interest, 1-h bafilomycin treatment, a condition that did not affect TrkA cell-surface abundance, reduced Akt phosphorylation but not Erk1/2 phosphorylation (Figure 7, D–F).

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Show MeSH
Related in: MedlinePlus