TI - Alteration of signaling pathways after MMP-28 inhibition . AB - To begin to understand the mechanism responsible for increased myelination seen in DRG cultures with anti-MMP-28 antibodies , we evaluated the ability of MMP-28 to alter signaling pathways that are known to be important in the development of myelin . DRG co cultures were induced to myelinate for 14 days . At this time point endogenous MMP-28 is reduced and a substantial percentage of cells are undergoing myelination and would be expected to have activated myelination related pathways . Cells were then treated with fresh myelination media containing NGF with or with out 10 nM MMP-28 . Myelination media contains NGF and is expected to activate signaling pathways including MAPK and PI3K downstream of cognate neurotrophin ( Trk or p75 ) receptors [21] but is not expected to result in PHOSphorylation of ErbB receptors . The addition of fresh media resulted in a minor increase in PHOSphorylated MAPK within 10 minutes which was enhanced in the presence of MMP-28 (Fig 6A) . In contrast , the PHOSphorylation of the p55 subunit of PI3K was inhibited in the presence of additional MMP-28 suggesting a resultant decrease in signaling responsible for the development of myelin . Interestingly , both ErbB2 and ErbB3 were rapidly PHOSphorylated following MMP-28 treatment while the addition of fresh myelination media alone did not result in activation of these receptors . The receptors ErbB2 and ErbB3 are known to be involved in both myelination and proliferation [6 , 22] however , the presence of MAPK PHOSphorylation suggests a proliferative rather than myelination related response [23] . As the DRG co culture system contains a mixed population of cell types , we were interested in determining if the altered signaling occurred in cells involved in the ongoing process of myelination . To identify the cell type with altered signaling following MMP-28 treatment , immunofluorescence was performed on d14 myelinating DRG co cultures treated with 30 nM IgG alone , or 10 nM MMP-28 with 30 nM IgG , pAb180 or pAb183 . PHOSphorylated ErbB3 was not detected in IgG treated control cultures or in cultures treated with MMP-28 in the presence of pAb180 or pAb183 but was detectable along axons in cells of IgG+MMP-28 treated cultures (Fig 6B) . While it remains to be shown if the response localized to this area is within the neurons or glial cells , it is clear that the signaling changes do occur in the microenvironment of myelinating cells . The loss of ErbB3 PHOSphorylation in the presence of inhibitory MMP-28 antibodies suggests that the proteolytic activity of MMP-28 is needed for the observed changes in signaling following treatment . These data indicate that MMP-28 alters the signaling in myelinating DRG cultures , resulting specifically in increased PHOSphorylation of pathways associated with proliferation rather than myelination in undifferentiated Schwann cells , and that pAb180 and pAb183 inhibit this activity . PHOSphorylation of these signaling pathways in myelinating cells within the context of these DRG co cultures may not be sufficient to elicit a proliferative response , however , to address this possibility DRG co cultures grown under myelinating conditions for 14 days were treated with 10 nM MMP-28 and analyzed for incorporation of a fluorescently labeled DNA binding agent to detect any increases in DNA content (Fig 6C) . No increase in cell number as measured by DNA content was detected 24 hours after MMP-28 treatment . Alternatively , DRG co cultures prior to myelination induction ( 2 days in culture ) were treated with 10 nM MMP-28 for 24 hours and analyzed for the presence of the proliferation marker proliferating cell nuclear antigen ( PCNA ) (Fig 6D) . No increase in proliferation was detected under these conditions , suggesting that the alteration in signaling due to MMP-28 treatment may not induce proliferation in this system under these conditions .