Objective: To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods: PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(fl/fl) AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31(+)c-Kit(high) cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results: In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31(+)c-Kit(high) in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion: PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.
3-Phosphoinositide-Dependent Protein Kinases ; Aorta ; Endothelial Cells ; Gonads ; Hematopoiesis ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Mesonephros ; Proto-Oncogene Proteins c-kit

OBJECTIVETo study the effect of homcysteine on the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the adipose tissue and explore whether PDK1 inhibits p-Akt(Thr-308) expression and affect PI3K/Akt signal pathway to decrease glucose uptake and utilization.

METHODSForty mice were randomly divided into 4 groups (n=10), namely the fasting control group, feeding control group, fasting hyperhomocysteinemia group, and feeding hyperhomocysteinemia group. In the two hyperhomocysteinemia groups, the mice were given water containing 1.5% methionine to induce hyperhomocysteinemia. Blood glucose and insulin levels in each group were determined, and the expressions of PDK1 and Akt mRNA in the adipose tissue were detected using RT-PCR; the expressions of PDK1, p-Akt(Thr-308) and Akt proteins were detected using Western blotting.

RESULTSIn the fasting and feeding hyperhomocysteinemia groups, blood glucose and insulin levels were significantly higher than those in the two control groups. The expressions of PDK1 mRNA and PDK1 and p-Akt(Thr-308) proteins were reduced in the two hyperhomocysteinemia groups, but Akt mRNA and protein expressions were comparable with those in the control groups.

CONCLUSIONHomocysteine lowers the uptake and utilization of glucose by down-regulating PDK1 expression and affecting PI3K/Akt signal pathway to cause disturbance of glucose metabolism.

3-Phosphoinositide-Dependent Protein Kinases ; metabolism ; Adipose Tissue ; metabolism ; Animals ; Blood Glucose ; metabolism ; Hyperhomocysteinemia ; metabolism ; Insulin ; blood ; Male ; Mice ; Mice, Inbred Strains ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

BACKGROUNDPDK1 is an essential protein kinase that plays a critical role in mammalian development. Mouse lacking PDK1 leads to multiple abnormalities and embryonic lethality at E9.5. To elucidate the role of PDK1 in the heart, we investigated the cardiac phenotype of mice that lack PDK1 in the heart in different growth periods and the alteration of PDK1 signaling in human failing heart.

METHODSWe employed Cre/loxP system to generate PDK1(flox/flox): α-MHC-Cre mice, which specifically deleted PDK1 in cardiac muscle at birth, and tamoxifen-inducible heart-specific PDK1 knockout mice (PDK1(flox/flox):MerCreMer mice), in which PDK1 was deleted in myocardium in response to the treatment with tamoxifen. Transmural myocardial tissues from human failing hearts and normal hearts were sampled from the left ventricular apex to analyze the activity of PDK1/Akt signaling pathways by Western blotting.

RESULTSPDK1(flox/flox): α-MHC-Cre mice died of heart failure at 5 and 10 weeks old. PDK1(flox/flox) -MerCreMer mice died of heart failure from 5 to 21 weeks after the initiation of tamoxifen treatment at 8 weeks old. We found that expression levels of PDK1 in human failing heart tissues were significantly decreased compared with control hearts.

CONCLUSIONOur results suggest that PDK1 signaling network takes part in regulating cardiac viability and function in mice, and may be also involved in human heart failure disease.

3-Phosphoinositide-Dependent Protein Kinases ; Adult ; Animals ; Female ; Glycogen Synthase Kinase 3 ; physiology ; Heart ; physiology ; Heart Failure ; enzymology ; etiology ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Middle Aged ; Myosin Heavy Chains ; physiology ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; physiology ; Signal Transduction ; Tamoxifen ; pharmacology

OBJECTIVETo explore the silencing effects of RNA interference on the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) gene, and the effects on malignant phenotypes of esophageal carcinoma EC9706 cells.

METHODSPDK1 siRNAs was transfected into the EC9706 cells. The expression of PDK1 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). At the same time, expressions of PDK1, Akt and phosphorylated Akt proteins were detected by Western blot. Methyl thiazolyl tetrazolium assay (MTT) was used to examine the cell proliferation after transfection. Flow cytometry was used to determine the percentage of apoptosis cells, and Transwell chambers were used to detect the invasion ability of the cells. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo.

RESULTSCompared with the non-transfected group, PDK1 siRNA effectively inhibited the expression of PDK1 mRNA in EC9706 cells, with an inhibition rate of (28.5 ± 4.2)% at 24 h, (51.1 ± 5.7)% at 48 h and (60.6 ± 4.1)% at 72 h after transfection. The expressions of PDK1 and phosphorylated Akt protein were also knocked down by PDK1 siRNA (P < 0.05). PDK1 siRNA significantly inhibited the cell proliferation and invasion, promoted the cell apoptosis, and inhibited the EC9706 cells proliferation in vivo and the expression of PDK1 protein in the transplanted tumors (P < 0.05).

CONCLUSIONPDK1 may play an important role in esophageal cancer cell proliferation, invasion and apoptosis, and may serve as an effective target for cancer gene therapy.

3-Phosphoinositide-Dependent Protein Kinases ; Animals ; Apoptosis ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Burden