Lentivirus-Mediated Silencing of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase 2 Inhibits Release of Inflammatory Cytokines and Apoptosis in Renal Tubular Epithelial Cells Via Inhibition of the TLR4/NF-kB Pathway in Renal Ischemia-Reperfusion Injury.

BACKGROUND/AIMS: Renal reperfusion injury occurs after the blood flow to the ischemic kidney is re-established under various clinical conditions, such as organ transplantation, renal artery stenosis, embolic disease, and the repair of descending aortic. The current study aims to explore the effects of src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) on the release of inflammatory cytokines and the apoptosis of renal tubular epithelial cells by regulating the TLR4/NF-κB signaling pathway in rats with renal ischemia-reperfusion (I/R) injury. METHODS: A total of 60 normal clean Sprague Dawley (SD) (WT) rats were used in this study. The levels of creatinine (Cr) and blood urea nitrogen (BUN) were determined using an automatic biochemical analyzer. The apoptosis in renal tissue was detected by TUNEL assay. The renal tubular epithelial cells of rats were cultured, infected and treated with different lentivirus vectors. The serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1ß and SHP27 were measured. Reverse transcription quantitative polymerases chain reaction and Western blot analysis were performed to measure the expression of relevant genes and proteins. Furthermore, the effect of SHP-2 on the proliferation, cell cycle and apoptosis of renal tubular epithelial cells was also investigated. RESULTS: In the serum of rats with renal I/R injury and prolonged reperfusion time, the contents of Cr and BUN were increased, the positive expression of SHP-2 was higher, the level of apoptosis was promoted, IL-6, TNF-α, IL-1ß and SHP27 expression in the serum was increased, the expression of SHP2, TLR4, NF-κB, IL-6, TNF-α and Bax was up-regulated, and the expression of Bcl-2 was down-regulated. Lentivirus-mediated silencing of SHP-2 promoted the proliferation of renal tubular epithelial cells, inhibited their apoptosis, and reduced the expression of inflammatory factors in these cells by functionally suppressing the TLR4/NF-κB signaling pathway. CONCLUSION: The results indicated that lentivirus-mediated silencing of SHP-2 inhibited the release of inflammatory cytokines and the apoptosis of renal tubular epithelial cells, and promoted the proliferation of these cells by inhibiting the TLR4/NF-κB signaling pathway in rats with renal I/R injury.

SHP2 tyrosine phosphatase stimulates CEBPA gene expression to mediate cytokine-dependent granulopoiesis.

G-CSF signals contribute to granulocyte lineage specification. We previously found that G-CSF induces SHP2 tyrosine phosphorylation and that chemical inhibition of SHP1/SHP2 reduces CFU-G and prevents G-CSF but not M-CSF activation of ERK. We now find that SHP2 shRNA knockdown in the 32Dcl3 granulocytic line reduces ERK activation, diminishes CEBPA protein and RNA expression and promoter histone acetylation, and inhibits granulopoiesis. Exogenous, shRNA-resistant SHP2 rescues these effects of SHP2 knockdown, exogenous C/EBPα rescues granulocytic markers, and exogenous RUNX1 rescues C/EBPα. 32Dcl3 lines with knockdown of ERK1 and ERK2 retain normal levels of C/EBPα and differentiate normally in G-CSF despite also having reduced proliferation. SHP2 knockdown reduces CEBPA levels in lineage-negative murine marrow cells cultured in TPO, Flt3 ligand, and SCF, without affecting the rate of cell expansion. On transfer to IL-3, IL-6, and SCF to induce myelopoiesis, levels of granulocytic RNAs are reduced and monocyte-specific RNAs are increased by SHP2 knockdown, and there is a reduction in the percentage of CFU-G that form in methylcellulose and of granulocytes that develop in liquid culture. In summary, SHP2 is required for induction of C/EBPα expression and granulopoiesis in response to G-CSF or other cytokines independent of SHP2-mediated ERK activation.