Cytokines are secreted proteins and interact with their specific cell surface receptors, triggering intracellular signal transduction pathways that activate a number of genes crucial for the biological functions of cytokines. These cytokine signal transduction pathways are tightly regulated processes. The negative regulations of cytokine signaling are achieved by receptor internalization and degradation, dephosphorylation of signaling intermediates, expression of protein inhibitors such as suppressor of cytokine signaling (SOCS) and protein inhibitors of activated STAT (PIAS). The observation that cytokines are central to the inflammatory and destructive process in several autoimmune diseases suggests that interventions targeting the cytokine intracellular signaling will be a new therapeutic strategy in autoimmune diseases. We review the current knowledge about negative regulation of cytokine signal transduction.
Autoimmune Diseases ; Cytokines ; Protein Inhibitors of Activated STAT ; Receptors, Cell Surface ; Signal Transduction* ; Social Control, Formal

OBJECTIVETo construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

METHODSPIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.

RESULTSWe successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

CONCLUSIONThe construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.

Animals ; Cell Line ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Mice ; Plasmids ; Protein Inhibitors of Activated STAT ; genetics ; Spermatocytes ; cytology ; Transfection

OBJECTIVETo screen and identify PIAS2-interacting proteins from the mouse spermatogonial cDNA library using the yeast two-hybrid system, and to investigate the action mechanism of PIAS2 in spermatogenesis.

METHODSWith pGBKT7-PIAS2 as a bait plasmid, the positive clones interacting with pGBKT7-PIAS2 were screened from the mouse spermatogonial cDNA library, the inserted fragments were sequenced and underwent bioinformatic analysis, and their interaction was verified using the yeast two-hybrid system.

RESULTSThrough screening, sequencing, homology analysis and yeast two-hybrid verification, we obtained 8 different candidate proteins interacting with PIAS2, including Cyfip2, Psmb3, Nmel, nischarin, Ints10, Nsun5, Gnb211 and Ndufaf3.

CONCLUSIONEight different genes were successfully obtained using the yeast two-hybrid system, and their encoding proteins interacted with PIAS2, which might be related with male fertility regulation. Our findings have offered some new clues to the action mechanism of PIAS2 in spermatogenesis.

Animals ; Base Sequence ; Gene Library ; Male ; Mice ; Protein Binding ; Protein Inhibitors of Activated STAT ; genetics ; Protein Interaction Domains and Motifs ; Spermatogenesis ; genetics ; Spermatogonia ; metabolism ; Two-Hybrid System Techniques

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.
Cell Line ; DNA-Binding Proteins/*metabolism ; Histone Deacetylases/antagonists & inhibitors/metabolism ; Humans ; Protein Binding ; Protein Inhibitors of Activated STAT/*metabolism ; Protein Structure, Tertiary ; Repressor Proteins/*metabolism ; Small Ubiquitin-Related Modifier Proteins/*metabolism ; Transcription, Genetic ; Two-Hybrid System Techniques ; Zinc Fingers

OBJECTIVETo investigate the expression of STAT3 and PIAS3 in the renal tissues of rats with lupus nephritis.

METHODSThe kidneys were harvested from 18-week-old female MRL/lpr mice (lupus nephritis model group) and age-matched female BALB/C mice (normal control group). The expressions of STAT3 and PIAS3 mRNA in the renal tissues were measured by real-time quantitative RT-PCR, and the protein levels of STAT3 and p-STAT3 were examined using Western blotting and immunohistochemistry. The laboratory indices and renal histopathology of the mice were also investigated.

RESULTSThe urinary protein, blood urea nitrogen (BUN) and creatinine levels were significantly higher in MRL/lpr mice than in the control mice (P<0.05). The renal histopathology of MRL/lpr mice was characterized by increased mesangiocapillary proliferation and prominent inflammatory infiltration in the tubulo-interstitium, which were absent in the kidneys of normal control mice. Compared with the control mice, MRL/lpr mice showed significantly increased STAT3 mRNA and p-STAT3 protein levels in the renal tissues (P<0.05) with significantly lowered levels of PIAS3 mRNA (P<0.01). No significant difference was noted in the total STAT3 protein levels between MRL/lpr and control mice.

CONCLUSIONMRL/lpr mice have significantly increased expressions of STAT3 mRNA and p-STAT3 protein and decreased expression of mRNA PIAS3 in the kidneys compared with BALB/C mice.

Animals ; Female ; Kidney ; metabolism ; pathology ; Lupus Nephritis ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred MRL lpr ; Protein Inhibitors of Activated STAT ; metabolism ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

OBJECTIVESTo investigate the function and possible mechanisms of PIAS3 expression on the invasion of TJ905 cells.

METHODSPIAS3 overexpression vectors were constructed and PIAS3 siRNA were chemically synthesized, which were separately transfected into TJ905 cells for upregulation or downregulation of PIAS3 expression levels in TJ905 cells. After that, the invasive effects of TJ905 cells were measured by Transwell assay, and the expression of PIAS3, tissue inhibitor of metalloproteinases (TIMP)3, matrix metalloprotease (MMP)-2, and MMP-9 were identified by Western blot.

RESULTSIn vitro transfection efficiency of plasmids and oligonucleotides were separately 85.3% ± 3.1% and 95.1% ± 2.9%. PIAS3 overexpression plasmid transfection in vitro could effectively improve the expression of PIAS3 protein in TJ905 cells and inhibit the invasion of TJ905 cells (P < 0.05), and cell penetration ratio reduced from 87.9% ± 9.3% to 37.3% ± 7.9% compared with control group, while it upregulated TIMP3 and downregulated MMP-2, MMP-9 protein expression (P < 0.05); PIAS3 siRNA transfection could inhibit the PIAS3 protein expression of TJ905 cells and promote the invasion of TJ905 cells (P < 0.05), and cell penetration ratio increased from 83.9% ± 7.1% to 93.2% ± 3.1% compared with control group, while it downregulated TIMP3 and upregulated MMP-2, MMP-9 protein expression (P < 0.05).

CONCLUSIONPIAS3 expression is closely related to the invasion properties of glioma TJ905 cells.

Cell Line, Tumor ; Genetic Vectors ; Glioma ; metabolism ; pathology ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Molecular Chaperones ; genetics ; metabolism ; Neoplasm Invasiveness ; Protein Inhibitors of Activated STAT ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Tissue Inhibitor of Metalloproteinase-3 ; metabolism ; Transfection