TI - Discussion . AB - We have shown previously that MMP-28 is expressed in the developing nervous system in a temporally regulated manner [18] . This expression is inversely correlated with the expression of MAG in two models of Xenopus laevis nerve regeneration and in myelinating rat DRG cultures . These data suggested the possibility that MMP-28 may play a role in the development of myelin during normal development and in neural regeneration . Here we show that exogenously added MMP-28 reduces myelination , that activity in vitro can be inhibited by antibodies to MMP-28 , and that such inhibition results in enhanced formation of myelin . Specific inhibitors of MMP-28 are not known ; therefore , we generated antibodies to two distinct regions of MMP-28 with the goal of developing inhibitory antibodies . The peptides used were unique to MMP-28 and not expected to cross react with other MMPs . In addition , the locations of the epitopes were expected to be near the active site based on the known sequence of MMP-28 . Both antibodies were shown to bind and inhibit MMP-28 activity but did not bind or inhibit MMP-2 or MMP-10 . MMP-28 is expressed by DRG neurons within the first 14 days after initiation of myelination by ascorbic/ acid [18] . As MMP-28 expression is downregulated during the period of myelination in vitro as well as in vivo during nerve regeneration , it is possible that MMP-28 activity is antagonistic to myelination . If so , blocking its proteolytic activity may result in enhancement of the myelination program . Addition of either of the two neutralizing anti-MMP-28 antibodies ( pAb 180 or pAb 183 ) to myelinating DRG cultures resulted in an increase in axon associated MAG staining . This strengthens the hypothesis that neuronal MMP-28 expression after the onset of myelination acts as an inhibitor of the development of myelin . It is not known if MMP-28 acts on the matrix of the neural micro-environment or cleaves cell surface molecules involved in signaling . Illman et al. [20] have shown that MMP-28 is membrane localized , can induce an epithelial-to-mesenchymal transition in lung adenocarcinoma cells and alters signaling mediated through TGF beta . We were therefore interested in the signaling changes that may be mediated by MMP-28 in our cell culture system and chose to evaluate changes in the ErbB activated pathways as signaling downstream of ErbB receptors regulates myelination . Our initial observations suggest that addition of MMP-28 results in rapid phosphorylaTION of ErbB2 and ErbB3 and enhanced MAPK PHOSphorylation . Activation of ErbB2 and ErbB3 on glial cells can result in proliferative signals or myelinating signals [22,23] and does not on its own suggest which pathway might be affected . However , ErbB receptor activity in myelinating glial cells is characterized by reduced phosphorylaTION of MAPK and enhanced PI3K PHOSphorylation . The enhanced MAPK PHOSphorylation following MMP-28 treatment in DRG co cultures coupled with decreased activation of the p55 subunit of PI3K , the active signaling pathway during myelination , are consistent with MMP-28 activity enhancing the non-myelination pathway downstream of the ErbB receptors . It is not yet clear if MMP-28 activity is directly involved in the generation of these intracellular PHOSphorylation events but there is evidence for MMP -mediated signaling within the nervous system . Of particular interest , both Neuregulin-1 and the ErbB receptors are known to be processed by MMP proteolysis [9,13,25] . As MMP processing of NRG-1 leads to soluble NRG and the myelinating signal is dependent on juxtacrine signaling , it is possible that MMP-28 cleaves NRG-1 in this system and that inhibiting MMP-28 activity results in accumulation of membrane bound NRG-1 . In previous studies , we identified increased proteolysis of NCAM and Nogo-A following MMP-28 treated of embryonic rat brains [18] . Alterations in NCAM expression may be involved in the development of myelin [26 , 27] and Nogo-A , a component of myelin , regulates multiple aspects of glial and neural cell biology [28] . Cleavage of either protein could potentially result in soluble biologically active fragments or loss of function through degradation . Alternatively , the degradation of MAG by MMP-28 is a possibility . Previously , we showed that in vitro , at much higher doses than used in these experiments , MAG may be a SUBstrate for MMP-28 [18] and it could be that MMP-28 is degrading MAG expressed in the tissues as it develops until the down regulation of MMP-28 . The inverse expression of MMP-28 and MAG suggests that MMP-28 regulation is atleast temporally coordinated with the myelination program even if it relates only to the post-transcriptional control of MAG . However , the correlation of MAG staining within myelinated and demyelinated regions of both mouse and human nervous tissue with Luxol Blue staining , which stains the lipid component of myelin , suggests that MAG can be used as marker of myelination even in the presence of varying levels of MMP-28 . When taken together with data suggesting that signaling related to the myelination program is altered when exogenous MMP-28 is added , these data support the use of MAG reduction to represent a delay or inhibition of myelination . Further work will be required to determine the essential target of MMP-28 action in vivo . While the experiments performed in vitro demonstrate a role for MMP-28 in regulating PNS myelination , we were curious if a similar role might be involved in the development or maintenance of CNS myelin . We report in this study for the first time that increased MMP-28 expression can be detected within demyelinated lesions of mouse EAE and human MS nervous system tissue . The increase in MMP-28 detected in both cases lends further support to the hypothesis that MMP-28 activity is involved in the regulation of myelin . It can not be determined from these experiments if MMP-28 activity in these tissues is responsible for the demyelination or if it expressed prior to or during any remyelination , however , the timing of the EAE progression in the tissue ( 21 days after induction ) and the increased nuclei ( presumably infiltrating immune cells ) in both tissues , suggest that these lesions may be actively demyelinating rather than undergoing the process of remyelination . While these experiments represent a small sample size and need to be followed up to determine the specific lesion types that demonstrate this altered expression , the results suggest that MMP-28 may be a relevant target for therapeutic intervention in MS . It is important to note that while there is consistency in MMP-28 expression during development in the CNS and PNS , and between dysmyelination states ( PNS renervation , mouse EAE and MS ) the functional implications of altered MMP-28 expression in the CNS may not be the same as in the PNS . Also remaining to be characterized is the role of activation of MMP-28 . MMP function is carefully controlled by cleavage of the pro - form to generate the active form of the protein . The sequence of proMMP-28 contains a putative furin recognition sequence but the details of activation for this enzyme in vivo are unknown . Validation of the functional role of MMP-28 in the CNS remains to be carried out . Although the specific mechanism is unknown , MMP-28 appears to play a role in the development of myelin . We suggest that in the nervous system , signaling pathways such as activation of the ErbB-MAPK cascade can be altered in response to MMP-28 -induced proteolysis and that continued expression of this protease results in inhibition of robust myelination . If MMP-28 activity plays a similar role in modulating myelination in vivo , inhibition of this protease may represent a therapeutic mechanism for enhancing remyelination in demyelinating diseases .