Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.
Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment.


BACKGROUND
The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined.


METHODS
Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities.


RESULTS
ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3.


CONCLUSIONS
Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc.
Uchiyama
Yoshiyasu
Y
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Cheng
Chin-Chang
CC
Danielson
Keith G
KG
Mochida
Joji
J
Albert
Todd J
TJ
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D062565:Acid Sensing Ion Channels; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D020932:Nerve Growth Factor / Q000096:biosynthesis; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009995:Osmosis; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D015222:Sodium Channels / Q000096:biosynthesis* / Q000235:genetics; D013314:Stress, Mechanical
C568269:ASIC3 protein, rat; D062565:Acid Sensing Ion Channels; D000906:Antibodies; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D015222:Sodium Channels; C068313:Ngfr protein, rat; D020932:Nerve Growth Factor; D011494:Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C041311:tropomyosin kinase; D048369:MAP Kinase Kinase 1; C505410:Casp3 protein, rat; D053148:Caspase 3