Precursor/stem cell substitutive therapy to promote remyelination is a well-established effective strategy for the treatment of central nervous system injuries such as SCI. However, lesions are not conducive to the survival, differentiation, and function of transplanted cells. Identifying potential factors that impair cell survival in lesions will be important for the treatment of central nervous system diseases such as SCI.
After SCI, increased levels of proteins such as BMP4 in lesions might inhibit remyelination and repair [12]. After antagonizing BMPs with Noggin, the function of injured spinal cord was partially recovered [18, 19]. However, some experiments also showed that blocking BMP signaling was not conducive for repair [20, 21], whereas the activation of BMP signaling promoted nerve regeneration in the injured spinal cord [22]. These results suggest that the roles of BMPs in injured spinal cord are complex and contradictory. These different consequences may be due to the activation of different downstream target genes of the BMP signaling pathway. However, the above studies investigated the regulation of BMPs (ligand proteins), BMP receptors, and Smad levels in the cytoplasm and no interventions in BMP signaling were performed in the nucleus. Sip1 is a transcription factor that functions as a transcript target DNA in the nucleus and participates in regulating TGF-β signaling including BMP. The overexpression of Sip1 promoted oligodendrocyte differentiation [14]. However, the expression of Sip1 in SCI and the mechanism of promoting the maturation of oligodendrocytes is still unclear.
Our in vivo results revealed that the expression of Sip1 was increased in the early stages of SCI and peaked at 1 day. The increased expression of Sip1 was predominantly located in oligodendrocytes of the white matter, and then gradually decreased close to normal levels at 7 days post-injury. Previous studies suggested that mRNA levels of BMPs were up-regulated several hours after central nervous system injury [23]. After SCI, various molecules related to BMP signaling were significantly up-regulated and lasted until 1 month after injury. Oligodendrocytes also responded quickly. Newly-proliferating OPCs were detected in the remaining white matter of the spinal cord at 1 day post-injury, reached a peak at 3 days post-injury, and then gradually decreased [24]. Our results suggest that Sip1 may be involved in the response of oligodendrocytes to BMPs after SCI. With reference to a previous study [14], we speculated that the up-regulation of Sip1 may be involved in the differentiation of recruited/generated OPCs after SCI.
Next, the specific timepoint where Sip1 participates in oligodendrocyte differentiation was investigated in vitro. The development of oligodendrocytes can be divided into three phases, OPCs, immature oligodendrocytes, and mature oligodendrocytes. MBP is a marker of mature oligodendrocytes [25, 26]. Our results showed that Sip1 was weakly expressed in OPCs and predominantly located in the cytoplasm. During the process of differentiation, the Sip1 expression gradually increased, and peaked approximately 2 days post-differentiation. The location of Sip1 transferred from the cytoplasm to the nucleus. At this timepoint, most cells were immature oligodendrocytes and only a small amount of MBP was detected. Then, the expression of Sip1 decreased close to the level of undifferentiated OPCs at 7 days post-differentiation, although Sip1 was in the nucleus. At this timepoint, most cells had differentiated into mature oligodendrocytes.
The above results suggest that Sip1 may play important roles in the early stage of OPC differentiation to oligodendrocytes or when oligodendrocytes respond to injury, but it has limited functions in matured or stable oligodendrocytes. At the same time, Sip1 was increased transiently in the early stage of SCI, which is not completely consistent with the trend of increased BMPs and OPC proliferation, especially the former, which suggests that Sip1 may become exhausted. This might explain new OPC differentiation disorders and remyelination difficulties.
Finally, we interfered with the expression of Sip1 in OPCs to observe the effect on the differentiation and maturation of oligodendrocytes. The results showed MBP levels in the OPCs were significantly decreased after intracellular interference with SIP1 siRNA. This indicated that the differentiation of oligodendrocytes was significantly restricted after the down-regulation of Sip1 levels.
Although Sip1 is indispensable for the differentiation of oligodendrocytes, the targets of Sip1 in the process of oligodendrocyte differentiation are still unclear. The MAPK/ERK signaling pathway is a recognized and classical pathway that regulates cell differentiation [27]. ERK1/2 is the ultimate factor of this pathway [27, 28]. In addition, previous articles reported a strong correlation between ERK activity and OPC differentiation [29, 30]. The number of OPCs differentiated into mature oligodendrocytes was reduced with MAPK/ERK signaling inhibitors. This indicated that the MAPK/ERK pathway plays an important role in OPC differentiation [31–33, 27, 34, 17, 35]. Our experimental results showed that when the expression of Sip1 was decreased, the expression of P-ERK1/2, the final executor of the MAPK/ERK signaling pathway, was also significantly decreased. We speculate that Sip1 may affect OPC differentiation by regulating the expression or activation of ERK, and this specific regulatory relationship and locations need further study.