OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Animals ; Cell Cycle Proteins/genetics* ; Cell Line ; Cell Survival ; Chondroitin Sulfate Proteoglycans/genetics* ; Chromosomal Proteins, Non-Histone/genetics* ; Gene Expression Regulation ; Gene Knockdown Techniques ; Infertility, Male ; Male ; Meiosis/genetics* ; Mice ; Mice, Knockout ; Mice, Transgenic ; Phosphate-Binding Proteins/genetics* ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Rad51 Recombinase/genetics* ; Real-Time Polymerase Chain Reaction ; Spermatocytes/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism*

OBJECTIVETo explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism.

METHODSK562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2.

RESULTSAfter exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect.

CONCLUSIONSHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

Cell Proliferation ; Drug Resistance, Neoplasm ; Genetic Vectors ; Humans ; Imatinib Mesylate ; pharmacology ; K562 Cells ; drug effects ; Phosphorylation ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; Signal Transduction ; Transfection

BACKGROUNDSrc homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase. Studies have revealed its roles in various disease, however, whether SHP-2 involves in renal fibrosis remains unclear. The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.

METHODSMyeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system, and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO). The total collagen deposition in the renal interstitium was assessed using picrosirius red stain. F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium. Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney. Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.

RESULTSSrc homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO. Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice. Meanwhile, the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice. However, no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.

CONCLUSIONSOur observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis, and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibrosis ; enzymology ; pathology ; Immunohistochemistry ; Kidney Diseases ; enzymology ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myeloid Cells ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; genetics ; metabolism ; Ureteral Obstruction ; enzymology ; pathology

PTPL1 is a protein with a predicted MW of 270 kD, and plays a major role in many cellular functions, including cell survival, proliferation, differentiation and motility. Evidence has demonstrated that PTPL1 is associated with tumor. Although many conflicting results suggested that PTPL1 has two contradictory effects (supressing or promoting ) on tumor, the real effect depends on the involved substrate and the cellular context. Expression of PTPL1 is low in lymphoma, while it is high in myeloid leukemia. PTPL1 has been regarded as a tumor suppressor in lymphoma, the methylation of PTPL1 promoter leads to gene expression reduced or disappeared, playing a lymphoma tumor suppressor role. This review focuses on PTPL1 domain and its interacting proteins, the relationship between PTPL1 and hematological malignancies.
Cell Movement ; Cell Survival ; Genes, Tumor Suppressor ; Hematologic Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Protein Tyrosine Phosphatase, Non-Receptor Type 13 ; genetics ; metabolism

This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of 5-aza-CdR demethylation on expression of SHP-1 and C-kit genes. RT-PCR was used to detect the mRNA expression level of SHP-1 and C-kit mRNA in HL-60 cells of the drug-treated group and control group.The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 and C-kit genes in HL-60 cells.The results showed that after being treated with 5-aza-CdR, the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally was not expressed. Meanwhile, the high expression level of C-kit mRNA in HL-60 cells was decreased. When HL-60 cells were treated with 0, 0.5, 1.0, 2.0 µmol/L 5-aza-CdR, the demethylation effect was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and the expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05) . It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with 5-aza-CdR suggest that the hypermethylation of SHP-1 gene relates with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of 5-aza-CdR on expression of SHP-1 and C-kit shows dose-dependency, the higher the 5-aza-CdR concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of 5-aza-CdR shows time-dependency in specific concentration.The SHP-1 mRNA expression negatively correlates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.
Azacitidine ; pharmacology ; DNA Methylation ; drug effects ; HL-60 Cells ; Humans ; Leukemia ; genetics ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; RNA, Messenger

This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of inhibitor As2O3 demethylation on SHP-1 and C-kit genes expression. RT-PCR was used to detect the expression level of SHP-1 and C-kit mRNA in drug-treated cell group and control group. The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 gene in HL-60 cells. The results showed that after being treated with As2O3 the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally did not expressed, meanwhile the expression level of C-kit mRNA in HL-60 cells with high expression decreased. When HL-60 cells were treated with As2O3 of 1.0, 2.5, 5.0 µmol/L, the demethylation effects was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P < 0.05). It is concluded that the absence of SHP-1 mRNA expression in HL-60 cells and recovery of expression after treatment with As2O3 suggest the hypermethylation of SHP-1 gene related with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of As2O3 on expression of SHP-1 and C-kit shows dose-dependency, the higher the As2O3 concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of As2O3 shows time-dependency in specific concentration. The SHP-1 mRNA expression negatively relates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.
Arsenicals ; metabolism ; pharmacology ; DNA Methylation ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Oxides ; metabolism ; pharmacology ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; Proto-Oncogene Proteins c-kit ; genetics

Catalytic Domain ; genetics ; Dual Specificity Phosphatase 3 ; chemistry ; genetics ; metabolism ; Genetic Code ; Humans ; Mutation ; Phosphorylation ; Protein Conformation ; Signal Transduction ; genetics ; Structure-Activity Relationship ; Tyrosine ; analogs & derivatives ; chemistry ; genetics

OBJECTIVETo explore the role of SHP-1 promoter methylation on the pathogenesis and progression in myelodysplastic syndromes (MDS) and its related mechanism.

METHODS63 MDS patients were divided into low-grade (LG) group and high-grade (HG) group according to IPSS score system. Bone marrow samples were collected. Methylation specific-PCR (MSP) were used to detect the status of SHP-1 promoter methylation in bone marrow (BM) samples from different risk MDS patients and MDS cell line, SKK-1. Western blot was used to detect signal transduction and activator of transcription (STAT3) activation in SKK-1 cell line and MDS patients.

RESULTSNo SHP-1 promoter methylation could be detected in healthy controls BM. Partially methylation was found in SKK-1 cell line. Methylation rate of SHP-1 gene promoter was found in BM of 24.2% of low-grade MDS patients and 63.3% of high-grade MDS patients, the difference between these two groups was statistically significant (P < 0.05); Patients were divided into different groups according to WHO subtype, chromosomal karyotype and blast cells in bone marrow, methylation rates of SHP-1 were significantly higher in RAEB-II, poor karyotype group and samples with 0.11-0.19 blast cells (P < 0.05); The phosphorylation protein of STAT3 was detected in SKK-1 cell line. The expression of phosphorylation STAT3 was significantly higher in HG group than in LG group (66.7% vs 18.2%) (P < 0.05). There was a significant correlation between SHP-1 promoter methylation and STAT3 phosphorylation.

CONCLUSIONAbnormal methylation of SHP-1 gene promoter might have tentative role in the pathogenesis and progression of MDS, which may be involved in STAT3 activation. Detection of SHP-1 promoter methylation may be helpful to evaluate the prognosis of MDS.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; STAT3 Transcription Factor ; metabolism ; Young Adult

OBJECTIVETo explore the effect of DNAX-associated protein 12 (DAP12) pathway on the transformation from mouse monocytes RAW264.7 to osteoclasts induced by tensile strain.

METHODSDAP12shRNA plasmid was constructed and introduced to RAW264.7 cells. Then we supplied tensile strain to RAW264.7 cells by four-point bending system. The mRNA or protein expression of DAP12, tartrate-resistant acid phosphatase (TRAP), tyrosine kinases Btk and Tec and nuclear facior of activated T cells 1 (NFATc1) was measured by reverse transcription PCR (RT-PCR) and Western blotting respectively.

RESULTSThe expression of DAP12 mRNA (0.112 ± 0.025) and protein (0.193 ± 0.015) both declined sharply after plasmid being introduced into monocytes RAW264.7 (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, tensile strain-induced TRAP mRNA expression of RAW264.7 cells increased at 6 h (0.671 ± 0.031) and 12 h (0.800 ± 0.043) (P < 0.05), but it was weaker than non-RNA-interference-groups at each time point (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, the expressions of Btk, Tec, NFATc1 increased as time passed (6, 12 h) (P < 0.05), but the expressions on corresponding time decreased sharply compared with those in control groups (P < 0.05).

CONCLUSIONSDAP12 pathway play an important role in regulating osteoclast differentiation induced by tensile strain.

Acid Phosphatase ; genetics ; metabolism ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Differentiation ; Cell Line ; Gene Expression Regulation ; Gene Silencing ; Isoenzymes ; genetics ; metabolism ; Mice ; Monocytes ; cytology ; metabolism ; NFATC Transcription Factors ; metabolism ; Osteoclasts ; cytology ; Plasmids ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Tensile Strength