br Experimental br Results br

Experimental
Results
Discussion In the last two decades, several studies have shown an important role for progestogens, and in particular ALLO, in the modulation of Schwann cell physiology, regulating several biochemical and functional parameters. The classical PR has been found both in rat Schwann cell primary cultures and in the sciatic nerve [14], [38], [39], [40], [41]. Schwann cells also express the GABA-A receptor [16], which is allosterically modulated by ALLO and mediates most of the progestogen-mediated actions in Schwann cells. Progestogens control Schwann cell proliferation, myelin protein expression, GABA synthesis, GABA-B receptor modulation and glutamate trafficking [13], [14], [15], [16], [42], [43]. However, the exact mechanism underlying progestogen modulation of Schwann cell physiology is not completely clear and other mechanisms, involving classic and non-classic steroid receptors, may be involved. After their initial identification in seatrout ovaries [24], mPRs have been identified in several tissues, including the central nervous system. They are expressed both in the ARRY-614 and the spinal cord [25], [26], [28], [29] and mPRα expression in glial cells was up-regulated following traumatic brain injury [29]. Despite these findings suggesting that mPRs may have a role in the nervous system, their presence or activation in the peripheral nervous system and in Schwann cells had not been investigated. In the present study, the S42 Schwann cell line model, derived from primary rat Schwann cell cultures, was used to study the potential role of mPRs in Schwann cell functions. These cells resemble Schwann cells at an early pre-myelinating stage and express some typical Schwann cell markers, such as MAG, which is expressed at high levels, and P0, that is instead expressed at lower levels than in sciatic nerves [44]. RT-PCR identification of mPR mRNAs provided the first evidence that all five mPR subtypes are present in peripheral glial cells. The subsequent qRT-PCR analysis defined the basal gene expression of the five mPRs and PGRMC1. The results showing that transcript levels in the Schwann cells are greatest for mPRβ followed by mPRδ are in line with previous findings; indeed, mPRβ was previously reported to be the main isoform in rat brain [28], while mPRδ was described as a brain specific isoform [25]. The immunofluorescence analysis revealed that all the mPR isoforms and PGRMC1 are present on the cell membrane, but they also appear to be present in the cytoplasm, with strong immunoreactivity in the perinuclear area and weaker staining in the cellular processes. Furthermore, the localization of the five mPR receptors and PGRMC1 as well as functional mPRs on the cell membrane were confirmed by Western blot analysis and receptor binding studies, respectively. All the receptors, except for mPRδ, appear to have a higher molecular weight than predicted, roughly double. This phenomenon has been reported before in rat brain and human umbilical vein endothelial cells for mPRs [28], [45], and in zebrafish ovarian and oocytes membranes in zebrafish for PGRMC1 [46], and could be linked to dimer formation. The results of the two-point binding assay demonstrating the presence of receptors with the characteristics of mPRs, because they are able to bind P4 and the specific mPR agonist 02, but not the specific PR agonist R5020, provide clear evidence that the mPRs are the major progestogen membrane receptors on S42 Schwann cells [34]. After nerve injury, myelinating Schwan cells change their differentiation status, assuming a phenotype that promotes nerve regeneration [4], becoming proliferative and migrating [47]. Recent studies have clarified some of the pathways involved in Schwann cell myelination (see [1] for review), whereas the mechanisms involved in Schwann cell migration are less clear. Several mediators and receptors have been proposed to be involved in the regulation of cell migration, including retinoic acid [48], fibroblast-derived factors [49], Egfr [50], Netrin-1 [51], the NMDA receptor [47], and proBDNF [52]. Our data suggest that the mPR-mediated progestogen action on Schwann cells may be important in the early stages of this process, because Schwann cell migration was significantly increased after 2 and 4h of 02 and P4 treatment. An involvement of the PR in this progesterone action is also possible, because cell migration appeared to be also stimulated by R5020, although it was not significantly different from the vehicle group. Progestogens have previously been proposed to stimulate Schwann cell proliferation [13], [53], [54]. However, to our knowledge, this is the first study to demonstrate that progestogens can regulate Schwann cell migration.