Biological Role Of β-1,4-GalT Ⅰ And Ⅴ In Two Types Of Astrocytes And Schwann Cells

Objective: To observe the expressions ofβ-1,4-GalT I and V in two types of astrocytes and Schwann cells during physiological and pathological conditions, and to explore biological role ofβ-1,4-GalT I and V in these cells.Methods: Two types of astrocytes and Schwann cells were cultured in vitro, in view of TNFα, different concentration and different time of LPS were used to treat astrocytes and Schwann cells, ELISA was used to determine the expression of TNFαin supernatant. The expression levels of TNFα, TNFR1 and TNFR2 mRNAs were examined by using RT-PCR. Immunocytochemistry stainning were used to investigate the cellular localization ofβ-1,4-GalT I,V. The expression ofβ-1,4-GalT I,V mRNA were detected by real-time PCR. Preparing the sciatic nerve injury models, sciatic functional index of rat was investigated by feet trace test. Inflammatory model was made by intraperitoneal injection of LPS. The expression ofβ-1,4-GalT I,V mRNAs in rat injury and inflammatory sciatic nerves were detected by real-time PCR. The products of RT-PCR were used to label probes for in situ hybridization,and the expressions and changes ofβ-1,4-GalT I,V in normal, injured and inflammtory rat sciatic nerves were analyzed by in situ hybridization and image analysis. Combined in situ hybridization and immunohistochemistry were used to determine the cellular localization ofβ-1,4-GalT I,V. RNAi and cell transfection technique were used to interfere with the expression ofβ-1,4-GalT V in Schwann cells. Galβ1-4GlcNAc synthesis in physiological and pathological Schwann cells were detected by using Lectin Blot. MAPK special inhibitors: U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor) were used to pretreat Schwann cell to investigate the effect of MAPK signal pathways on the expression ofβ-1,4-GalT I,V.Results: (1) Real-time PCR showed that TNFαor LPS affectedβ-1, 4-GalT I mRNA expression in a time- and dose- dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type 2 astrocytes, and that TNFα, TNFR1 and TNFR2 increased in type 2 astrocytes after exposure to TNFαor LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type 2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type 2 astrocytes, and distributed mainly in the processes of type 2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressedβ-1,4-GalT I mRNA expression induced by TNF-αor LPS.(2)β-1, 4-GalT I and V mRNAs are present in normal control type 1 astrocytes and affected by TNFαand LPS stimuli. Type 1 astrocytes express TNFαreceptor 1 (TNFR1), and increased slightly after exposure to LPS. TNFαand TNFR2 are not detected in control astrocytes, and upregulated significantly with LPS stimulation. And that activation of these receptors by TNFαaffects expressions ofβ-1, 4-GalT I and V mRNAs. In addition, we observed that not only exogenous TNF-αbut also TNFαproduced by astrocytes affectedβ-1, 4-GalT I and V mRNAs production in astrocytes.(3) Real-time PCR revealed that theβ-1, 4-GalT I andβ-1, 4-GalT V mRNAs reached peaks at 2w after sciatic nerve crush and 1w after sciatic nerve transection. Combined in situ hybridization forβ-1, 4-GalT I orβ-1, 4-GalT V mRNA and immunohistochemistry for S100 showed thatβ-1, 4-GalT I andβ-1, 4-GalT V mRNAs were mainly located in Schwann cells after sciatic nerve injury. In other pathology, such as inflammation, we found that LPS administration affectsβ-1, 4-GalT I andβ-1, 4-GalT V mRNAs expressions in sciatic nerve in a time- and dose-dependent manner, andβ-1, 4-GalT I andβ-1, 4-GalT V mRNA expressed mainly in Schwann cells. Similarly, we found that andβ-1, 4-GalT V in Schwann cells and the binding with RCA-I on the Schwann cell surface in vitro were affected in a time- and concentration dependent manner in response to LPS stimulation, and the trend of the binding with RCA-I on the cell surface was similar to the trend ofβ-1, 4-GalT V. In addition,β-1, 4-GalT V production and overall lectin binding were drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor).Conclusions: (1) TNFαsignaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induceβ-1, 4-GalT I mRNA. Not only exogenous TNFαbut also TNFαproduced by type 2 astrocytes affectedβ-1, 4-GalT I mRNA production in type 2 astrocytes. These results suggest that an autocrine loop involving TNFαcontributes to the production ofβ-1, 4-GalT I mRNA in response to inflammation. (2) An autocrine loop involving TNFαcontributes to the production ofβ-1, 4-GalT I and V mRNAs in response to inflammation.(3)β-1, 4-GalT V and Galβ1-4GlcNAc containing glycan structure plays an important role in inflammation. Schwann cells regulated expression ofβ-1, 4-GalT V and galactosylation of membrane glycoproteins after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.