Brain-derived neurotrophic factor (BDNF) acts primarily via the JAK/STAT pathway to promote neurite growth in the major pelvic ganglion of the rat: part 2.
BACKGROUND
Surgical and radiation therapies of bladder and prostate cancers may damage cavernous nerves and cause erectile dysfunction (ED). We previously showed that brain-derived neurotrophic factor (BDNF) could restore erectile function in a neurogenic ED rat model. We now investigated the signaling mechanism of BDNF in major pelvic ganglia (MPG) explants.


OBJECTIVE
To identify the signaling mechanism that mediates the neurotrophic effect of BDNF in cultured MPG.


METHODS
Major pelvic ganglia was isolated from male rats for immunohistochemistry and immunofluorescence staining to locate BDNF receptors, pan-neurotrophin 75 (p75), tropomyosin-related kinase B (TrkB), and tropomyosin-related kinase C (TrkC). The dorso-caudal region of MPG was treated with BDNF to determine the optimal dosage for promoting neurite growth. Specific kinase inhibitors AG490, KT5720, LY294002, and U0126 were then used to treat MPG either alone or prior to BDNF treatment. The treated MPG was examined for neurite growth and for expression and phosphorylation of JAK2, STAT1, and STAT3 by Western blot analysis.


METHODS
Lengths of neurite growth from MPG were measured to quantify the effects of BDNF and to identify specific signaling pathways. Ratios of phosphorylated vs. unphosphoryated proteins of JAK2, STAT1, and STAT2 in control and treated MPG were determined to confirm JAK/STAT as the principal signaling pathway.


RESULTS
Tropomyosin-related kinase B and TrkC were localized to neurons whereas p75 to perineuronal satellite glial cells (SGC). The optimal dosage of BDNF for promoting MPG neurite growth was between 25 and 50 ng/mL. Among the four specific kinase inhibitors, AG490 was the strongest in suppressing MPG neurite growth as well as BDNF-induced phosphorylation of JAK2, STAT1, and STAT3.


CONCLUSIONS
In rat MPG, TrkB and TrkC were expressed in neurons, whereas p75 in SGC. Optimal BDNF dosage for promoting MPG neurite growth was between 25 and 50 ng/mL. BDNF promotes MPG neurite growth primarily by activating the JAK/STAT pathway.
Lin
Guiting
G
Knuppe Molecular Urology, Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA. Electronic address: cslin@urology.ucsf.edu.
Bella
Anthony J
AJ
Knuppe Molecular Urology, Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA.
Lue
Tom F
TF
Knuppe Molecular Urology, Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA.
Lin
Ching-Shwun
CS
Knuppe Molecular Urology, Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA.
D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005726:Ganglia, Parasympathetic / Q000378:metabolism*; D066298:In Vitro Techniques; D053613:Janus Kinase 1 / Q000378:metabolism*; D008297:Male; D016501:Neurites / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000378:metabolism*; D015398:Signal Transduction
D019208:Brain-Derived Neurotrophic Factor; D050791:STAT Transcription Factors; C507906:Jak1 protein, rat; D053613:Janus Kinase 1