Sympathetic neurons express and secrete MMP-2 and MT1-MMP to control nerve sprouting via pro-NGF conversion

Abstract

Recently, we have shown that high frequency electrical field stimulation (HFES) of sympathetic neurons (SN) induces nerve sprouting by up-regulation of nerve growth factor (NGF) which targets the tyrosine kinase A receptor (TrkA) in an autocrine/paracrine manner. There is increasing evidence that matrix metalloproteinase-2 (MMP-2) is not only involved in extracellular matrix (ECM) turnover but may also exert beneficial effects during neuronal growth. Therefore, this study aimed to investigate the regulation and function of MMP-2 and its major activator membrane type 1-matrix metalloproteinase (MT1-MMP) as well its inhibitor TIMP-1 in SN under conditions of HFES. Moreover, we analyzed molecular mechanisms of the beneficial effect of losartan, an angiotensin II type I receptor (AT-1)blocker on HFES-induced nerve sprouting. Cell cultures of SN from the superior cervical ganglia (SCG) of neonatal rats were electrically stimulated for 48 h with a frequency of 5 or 50 Hz. HFES increased MMP-2 and MT1-MMP mRNA and protein expression, whereas TIMP-1 expression remained unchanged. Under conditions of HFES, we observed a shift from pro- to active-MMP-2 indicating an increase in MMP-2 enzyme activity. Specific pharmacological MMP-2 inhibition contributed to an increase in pro-NGF amount in the cell culture supernatant and significantly reduced HFES-induced neurite outgrowth. Losartan abolished HFES-induced nerve sprouting in a significant manner by preventing HFES-induced NGF, MMP-2, and MT1-MMP up-regulation. In summary, specific MMP-2 blockade prevents sympathetic nerve sprouting (SNS) by inhibition of pro-NGF conversion while losartan abolishes HFES-induced SNS by reducing total NGF, MMP-2 and MT1-MMP expression.

Fig. 1

High frequency electrical field stimulation of cultured sympathetic neurons increases the mRNA and protein expression of MMP-2 and MT1-MMP. a, b, c On mRNA level we observed an increase in MMP-2 and MT1-MMP expression after 48 h of HFES, whereas TIMP-1 mRNA expression remained on control level (n = 5). Losartan (1 μmol/l) was able to prevent HFES-induced changes in MMP-2 and MT1-MMP mRNA expression, whereas specific MMP-2 (1 μmol/l) inhibition could not show any effect (n = 3). Regarding TIMP-1, neither losartan nor MMP-2 inhibition showed any difference in mRNA expression (n = 3). d, e On protein level measured with western blotting, we could confirm our mRNA data. In the presence of losartan, HFES induced MMP-2 and MT1-MMP up-regulation reversed to control levels (n = 3). On the other hand, specific MMP-2 inhibition could not prevent HFES-induced MMP-2 and MT1-MMP protein up-regulation (n = 3). f To further characterize the activation of MMP-2, we performed high sensitive ELISA experiments, to investigate the amount of active- and pro-MMP-2 as well the amount of MMP-2 in complex with TIMP-1. HFES induced an increase in unbound active-MMP-2 and a decrease in pro-MMP-2. MMP2- TIMP-1 complex also decreased, although the mRNA expression of TIMP-1 remained unchanged as shown before (n = 3). * P < 0.05 versus 5 Hz; ** P < 0.05 versus 50 Hz

Fig. 2

MMP-2 inhibition contributes to an increase in pro-NGF amount in the cell culture supernatant. a Cell cultures of sympathetic neurons were stimulated for 48 h as described in the M&M section and MMP-2 inhibition was performed with a specific pharmacological inhibitor (1 μmol/l) as described elsewhere (Ould-yahoui et al. 2009). Measured with the ELISA technique, MMP-2 inhibition resulted in an increase in pro-NGF amount in the cell-culture supernatant (n = 3). These data indicate that pro-NGF in predominantly converted by unbound active-MMP-2. * P < 0.05 versus 50 Hz

Fig. 3

Losartan prevents HFES-induced sympathetic nerve outgrowth by reducing NGF mRNA and protein expression. a Light microscopic images of un-stimulated and stimulated SCG cell-cultures. SNS indicates sympathetic nerve sprouting. b In the presence of losartan (1 μM) HFES-induced GAP-43 protein expression as a marker for neuronal outgrowth completely returned to control levels (n = 4). c, d To analyze whether losartan prevents HFES-induced nerve outgrowth by preventing NGF up-regulation in stimulated cell-cultures of sympathetic neurons, we further analyzed the mRNA and protein expression of NGF with the real-time PCR method and western blotting. * P < 0.05 versus 5 Hz; ** P < 0,05 versus 50 Hz

Fig. 4

MMP-2 activation by HFES does not depend on NGF signaling. a To investigate whether HFES induced MMP-2 secretion and activation depends on NGF signaling, we performed loss of function experiments with NGF-neutralizing antibodies. NGF neutralizing did not reduce HFES-induced MMP-2 secretion and activation in the cell-culture supernatant (n = 3). b A proposed model for pro-NGF conversion under conditions of HFES. The cartoon shows a single SN exposed to electrical stimulation. In summary, our results demonstrate that SN respond to HFES with an increase in NGF, MMP-2 and MT1-MMP expression. In the extracellular matrix, MT1-MMP activates MMP-2 which further cleaves pro-NGF to mature-NGF to promote neurite growth via TrkA signaling. These findings strengthen the hypothesis that neuronal remodeling in terms of sympathetic hyperactivity is coupled to parallel expression and secretion of NGF and MMP-2. * P < 0.05 versus 5 Hz