Yoshiki Iwamoto

Cell Growth Arrest and Induction of Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1 Mediated by STAT1

Signal transducers and activators of transcription (STAT) proteins can be conditionally activated in response to epidermal growth factor (EGF) and interferon (IFN)-γ. STAT activation was correlated with cell growth inhibition in response to EGF and IFN-γ. Activated STAT proteins specifically recognized the conserved STAT-responsive elements in the promoter of the gene encoding the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1 and regulated the induction of p21 messenger RNA. IFN-γ did not inhibit the growth of U3A cells, which are deficient in STAT1, but did inhibit the growth of U3A cells into which STAT1α was reintroduced. Thus, STAT1 protein is essential for cell growth suppression in response to IFN-γ. The STAT signaling pathway appears to negatively regulate the cell cycle by inducing CDK inhibitors in response to cytokines.

STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality : a critical role of STAT3 in innate immunity

Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional mediator for many cytokines and is essential for normal embryonic development. We have generated a unique strain of mice with tissue-specific disruption of STAT3 in bone marrow cells during hematopoiesis. This specific STAT3 deletion causes death of these mice within 4–6 weeks after birth with Crohns disease-like pathogenesis in both the small and large intestine, including segmental inflammatory cell infiltration, ulceration, bowel wall thickening, and granuloma formation. Deletion of STAT3 causes significantly increased cell autonomous proliferation of cells of the myeloid lineage, both in vivo and in vitro. Most importantly, Stat3 deletion during hematopoiesis causes overly pseudoactivated innate immune responses. Although inflammatory cytokines, including tumor necrosis factor α and IFN-γ, are overly produced in these mice, the NAPDH oxidase activity, which is involved in antimicrobial and innate immune responses, is inhibited. The signaling responses to lipopolysaccharide are changed in the absence of STAT3, leading to enhanced NF-κB activation. Our results suggest a model in which STAT3 has critical roles in the development and regulation of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in myeloid cells and causes Crohns disease-like pathogenesis.

Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age

Cytokines and inflammation have been implicated in the pathogenesis of heart failure. For example, IL-6 family cytokines and the gp130 receptor play important roles in cardiac myocyte survival and hypertrophy. Signal transducer and activator of transcription 3 (STAT3) is a major signaling protein that is activated through gp130. We have created mice with a cardiomyocyte-restricted deletion of STAT3. As measured by serial echocardiograms, mice with cardiac specific deletion of STAT3 are significantly more susceptible to cardiac injury after doxorubicin treatment than age-matched controls. Intriguingly, STAT3 appears to have a critical role in protection of inflammation-induced heart damage. STAT3-deficient mice treated with lipopolysaccharide demonstrated significantly more apoptosis than their WT counterparts. At the cellular level, cardiomyocytes with STAT3 deleted secrete significantly more tumor necrosis factor α in response to lipopolysaccharide than those with WT STAT3. Furthermore, histologic examination of the cardiomyocyte-restricted STAT3-deficient mice reveals a dramatic increase in cardiac fibrosis in aged mice. Although no overt signs of heart failure are present in young STAT3-deficient mice, they spontaneously develop heart dysfunction with advancing age. These results indicate the crucial functions of STAT3 in cardiomyocyte resistance to inflammation and other acute injury and in pathogenesis of age-related heart failure.

Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inflammation

Endothelial cells (ECs) are believed to be an important component in the protection from lipopolysaccharide (LPS)-induced endotoxic shock. However, the cellular and molecular mechanism is not well defined. Here, we report that signal transducer and activator of transcription (STAT) 3 is an essential regulator of the antiinflammatory function of ECs in systemic immunity. Because STAT3 deficiency results in early embryonic lethality, we have generated mice with a conditional STAT3 deletion in endothelium (STAT3E−/−). STAT3E−/− mice are healthy and fertile, and isolated ECs initiate normal tube formation in vitro. Conditional endothelial but not organ-specific (i.e., hepatocyte or cardiomyocyte) STAT3 knockout mice show an increased susceptibility to lethality after LPS challenge. The LPS response in STAT3E−/− mice shows exaggerated inflammation and leukocyte infiltration in multiple organs combined with elevated activity of serum alanine aminotransferase and aspartate aminotransferase, indicating organ damage. Concomitantly, proinflammatory cytokines are produced at an exaggerated level and for a prolonged period. This defect cannot be explained by lack of antiinflammatory cytokines, such as interleukin 10 and transforming growth factor β. Instead, we have shown that a soluble activity derived from endothelia and dependent on STAT3 is critical for suppression of interferon γ. These data define STAT3 signaling within endothelia as a critical antiinflammatory mediator and provide new insight to the protective function of ECs in inflammation.

2-Arachidonoylglycerol: A Novel Inhibitor of Androgen-Independent Prostate Cancer Cell Invasion

Endocannabinoids have been implicated in cancer. Increasing endogenous 2-arachidonoylglycerol (2-AG) by blocking its metabolism inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. Noladin ether (a stable 2-AG analog) and exogenous CB1 receptor agonists possess similar effects. Conversely, reducing endogenous 2-AG by inhibiting its synthesis or blocking its binding to CB1 receptors with antagonists increases the cell invasion. 2-AG and noladin ether decrease protein kinase A activity in these cells, indicating coupling of the CB1 receptor to downstream effectors. The results suggest that cellular 2-AG, acting through the CB1 receptor, is an endogenous inhibitor of invasive prostate cancer cells.

Role of STAT3 in liver regeneration: survival, DNA synthesis, inflammatory reaction and liver mass recovery.

The hepatoprotective effect of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) has been well documented. However, reports on the role of IL-6/STAT3 in liver regeneration are conflicting probably due to the fact that the model of Stat3 knockout mice were complicated with obesity and fatty liver, which may cause some secondary effects on liver regeneration. To study the direct role of STAT3 and to circumvent the problems of obesity and fatty liver in liver regeneration, we generated conditional STAT3 knockout in the liver (L-Stat3−/−) using a transthyretin-driven Cre-lox method. The L-Stat3−/− mice were born with the expected Mendelian frequency and showed no obesity or other obvious phenotype. After partial hepatectomy, mortality in the L-Stat3−/− mice was significantly higher than the littermate Stat3f/+ controls in the early time points (<24 h). Hepatocyte DNA synthesis in the survived L-Stat3−/− mice slightly decreased as compared with Stat3f/+ mice at 40 h after partial hepatectomy, whereas similar hepatocyte DNA synthesis was found at other time points and liver mass could be completely recovered in the L-Stat3−/− mice. In another model of liver regeneration induced by subcutaneous injection of carbon tetrachloride (CCl4), hepatocyte DNA synthesis in the CCl4-treated L-Stat3−/− mice also decreased as compared with Stat3f/+ mice at 40 h after injection but not at other time points. In addition, infiltration of neutrophils and monocyte increased in the liver of CCl4-treated L-Stat3−/− mice compared to wild-type mice. In conclusion, STAT3 is required for survival in the acute stage after 70% hepatectomy and plays a role in inflammatory reaction after hepatocyte necrosis. However, the hepatocytic STAT3 may have limited role in liver mass recovery although DNA synthesis may be impaired.

Rf-2 gene modulates proteinuria and albuminuria independently of changes in glomerular permeability in the fawn-hooded hypertensive rat

We report that Rab38 , a gene within the Rf-2 locus appears to influence the development of proteinuria (UPV) and albuminuria (UAV) in fawn-hooded hypertensive rats (FHH). Using congenic animals, we narrowed the region to eight genes; however, only one gene had a sequence variant. Rab38 has a

BCG directly induces cell cycle arrest in human transitional carcinoma cell lines as a consequence of integrin cross-linking

BackgroundCurrent models of the mechanism by which intravesical BCG induces an anti-tumor effect in urothelial carcinoma propose a secondary cellular immune response as principally responsible. Our group has demonstrated that BCG mediated cross-linking of α51 integrin receptors present on the tumor surface elicits a complex biologic response involving AP1 and NF-κB signaling as well as the transactivation of immediate early genes. This study evaluated the direct biologic effect of cross-linking α5β1 integrin on cell cycle progression and apoptosis in two human urothelial carcinoma cell lines.MethodsTwo independent assays (MTT and Colony forming ability) were employed to measure the effect of α5β1 cross-linking (antibody mediated or BCG) on cellular proliferation. Flow cytometry was employed to measure effect of BCG and α5β1 antibody mediated cross-linking on cell cycle progression. Apoptosis was measured using assays for both DNA laddering and Caspase 3 activation.ResultsResults demonstrate that integrin cross-linking by BCG, or antibody mediated crosslinking of α5β1 resulted in a decrease in proliferating cell number. BCG treatment or α5β1 cross-linking increased the percentage of cells in G0/G1, in both 253J and T24 cell lines. Peptide mediated blockade of integrin binding site using RGDS reversed the effect BCG on both proliferation and cell cycle arrest. Apoptosis in response to BCG was not identified by either DNA laddering or Caspase 3 activation.ConclusionThese findings show that BCG exerts a direct cytostatic effect on human urothelial carcinoma cell lines. Cell cycle arrest at the G1/S interface is a mechanism by which BCG inhibits cellular proliferation. This effect is duplicated by antibody mediated cross-linking of α5β1 and likely occurs as a consequence of crosslink-initiated signal transduction to cell cycle regulatory genes.

c-Jun NH2-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt

Obesity is associated with advanced prostate cancer. Here we demonstrate that in mouse prostate cancer TRAMP-C1 cells epididymal fat extracts from high-fat diet-fed obese mice stimulate androgen-independent cell growth more significantly than those from low-fat diet-fed lean mice or genetically obese leptin-deficient ob/ob mice in correlation with leptin concentrations. This result suggests that obesity promotes androgen-independent prostate cancer cell growth via adipose leptin. We have reported that added leptin stimulates androgen-independent prostate cancer cell proliferation through c-Jun NH(2)-terminal kinase (JNK). As with JNK, signal transducer and activator of transcription 3 (STAT3) and Akt are implicated in androgen-independent prostate cancer. In this study, we identify novel interaction of these three molecules in leptin-stimulated androgen-independent cell proliferation. Leptin activates JNK, STAT3 and Akt in a biphasic manner with a similar time-course. Pharmacological JNK inhibition suppresses leptin-stimulated DNA binding activity, as well as Ser-727 phosphorylation, of STAT3. Since JNK upregulates STAT3 activity via Ser-727 phosphorylation, JNK mediates leptin-stimulated STAT3 activation through Ser-727 phosphorylation. Moreover, JNK inhibition impairs leptin-stimulated Ser-473 phosphorylation of Akt that is required for its activation. Thus, JNK is involved in leptin-stimulated Akt activation. These findings together indicate that JNK mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via STAT3 and Akt.

Crystallization of cytochrome P-450scc from bovine adrenocortical mitochondria

Cytochrome P‐450scc (P‐450scc), a cholesterol side‐chain cleavage enzyme from bovine adrenocortical mitochondria, has been crystallized for the first time. Upon removal of glycerol from the solution of the native enzyme complexed with pyridoxal 5′‐phosphate (PLP) by microdialysis against distilled water, reddish and planar crystals appeared. The crystals of native P‐450scc were also obtained by the same procedure. We identified the crystals as the P‐450scc‐PLP complex or native P‐450scc by absorption spectroscopy and SDS‐polyacrylamide gel electrophoresis, and characterized them under a polarization microscope.