Axon guidance proteins are critical for the correct wiring of the nervous system during development. Several axon guidance cues and their family members have been well characterized. More unidentified axon guidance cues are assumed to participate in the formation of the extremely complex nervous system. We identified a secreted protein, draxin, that shares no homology with known guidance cues. Draxin inhibited or repelled neurite outgrowth from dorsal spinal cord and cortical explants in vitro. Ectopically expressed draxin inhibited growth or caused misrouting of chick spinal cord commissural axons in vivo. draxin knockout mice showed defasciculation of spinal cord commissural axons and absence of all forebrain commissures. Thus, draxin is a previously unknown chemorepulsive axon guidance molecule required for the development of spinal cord and forebrain commissures.

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.

The objective of this study was to investigate the role of miR-148a-3p in lupus nephritis (LN) based on data from previous studies and a microRNA assay. We evaluated the miR-148a-3p expression level in LN renal tissues and blood serum to determine its clinicopathological significance and effect on glomerular cell proliferation. Then, we collected renal glomeruli from LN mice and determined the miR-148a-3p, proliferating cell nuclear antigen (PCNA), and PCNA/Thy1 expression. We performed functional analyses of miR-148a-3p in vitro and in vivo. We also investigated the target gene of miR-148a-3p in LN. The results showed that miR-148a-3p expression levels were significantly higher not only in glomeruli but also in the blood serum during LN and increased in the glomeruli of LN mice and that at the same time there was positive correlation between miR-148a-3p and PCNA expression of glomruli. Overexpression of miR-148a-3p accelerated cell proliferation and PCNA expression, while a miR-148a-3p inhibitor inhibited cell proliferation via the Akt/cyclin D1 pathway. Furthermore, miR-148a-3p overexpression reduced the phosphatase and tensin homology deleted on chromosome ten (PTEN) expression level, while miR-148a-3p silencing increased its expression in high-mobility group box 1 (HMGB1)-induced mouse mesangial cells (MMCs). Luciferase assays demonstrated that miR-148a-3p could directly bind to the PTEN 3'-UTR. PTEN overexpression inhibited MMC proliferation considerably, resembling the results observed during miR-148a-3p inhibition. Reducing miR-148a-3p expression upregulated PTEN in the glomeruli and improved renal function in LN mice. Thus miR-148a-3p may promote proliferation and contribute to LN progression by targeting PTEN.

Our previous experiment confirmed that high-mobility group box chromosomal protein 1 (HMGB1) was involved in the pathogenesis of Lupus nephritis (LN) by upregulating the proliferation of the mouse mesangial cell line (MMC) through the cyclin D1/CDK4/p16 system, but the precise mechanism is still unknown. Therefore, in the present study, we demonstrated that HMGB1 induced the proliferation of MMC cells in a time- and concentration-dependent manner, downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, increased the level of Akt serine 473 phosphorylation, and induced p65 subunit nuclear translocation. The overexpression of PTEN prevented the upregulation of HMGB1-induced proliferation by blocking the activation of Akt. The knockdown of Akt by siRNA technology and blocking the nuclear factor-κB (NF-κB) pathway using pyrrolidine dithiocarbamate (PDTC) and SN50, inhibitors of NF-κB, both attenuated the HMGB1-induced proliferation by counteracting the activation of the cyclin D1. In addition, while sh-Akt partly blocked the nuclear translocation of the p65 subunit, PDTC did not affect the activation of the Akt induced by HMGB1 in MMC cells. These findings indicate that HMGB1 induced the proliferation of MMC cells by activating the PTEN/phosphoinositide-3-kinase (PI3K)/Akt/NF-κB signaling pathway.

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