Essential role of Cdc42 in Ras-induced transformation revealed by gene targeting

The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies.     

Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting

Knockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in alpha-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency.     

STAP-2/BKS,an adaptor/docking protein,modulates STAT3 activation in acute-phase response through its YXXQ motif

As a c-fms-interacting protein, we cloned a novel adaptor molecule, signal-transducing adaptor protein-2 (STAP-2), which contains pleckstrin homology- and Src homology 2-like (PH and SRC) domains and a proline-rich region. STAP-2 is structurally related to STAP-1/BRDG1 (BCR downstream signaling-1), which we had cloned previously from hematopoietic stem cells. STAP-2 is a murine homologue of a recently identified adaptor molecule, BKS, a substrate of BRK tyrosine kinase. STAP-2 was tyrosine-phosphorylated and translocated to the plasma membrane in response to epidermal growth factor when overexpressed in fibroblastic cells. To define the function of STAP-2, we generated mice lacking the STAP-2 gene. STAP-2 mRNA was strongly induced in the liver in response to lipopolysaccharide and in isolated hepatocytes in response to interleukin-6. In the STAP-2(-/-) hepatocytes, the interleukin-6-induced expression of acute-phase (AP) genes and the tyrosine-phosphorylation level of STAT3 were reduced specifically at the late phase (6-24 h) of the response. These data indicate that STAP-2 plays a regulatory role in the AP response in systemic inflammation. STAP-2 contains a YXXQ motif in the C-terminal region that is a potential STAT3-binding site. Overexpression of wild-type STAP-2, but not of mutants lacking this motif, enhanced the AP response element reporter activity and an AP protein production. These data suggest that STAP-2 is a new class of adaptor molecule that modulates STAT3 activity through its YXXQ motif.     

Fasting in alpha1-antitrypsin deficient liver: constitutive [correction of consultative] activation of autophagy

Alpha1-antitrypsin (alpha1-AT) deficiency causes severe liver injury in a subgroup of patients. Liver injury is thought to be caused by retention of a polymerized mutant alpha1-ATZ molecule in the endoplasmic reticulum (ER) of hepatocytes and is associated with an intense autophagic response. However, there is limited information about what physiologic stressors might influence liver injury. In this study, we examined the effect of fasting in the PiZ mouse model of alpha1-AT deficiency, because fasting is a well-characterized physiological stressor and a known stimulus for autophagy. Results show that there is a marked increase in fat accumulation and in alpha1-AT-containing globules in the liver of the PiZ mouse induced by fasting. Although fasting induced a marked autophagic response in wild-type mice, the autophagic response was already activated in PiZ mice and did not further increase with fasting. PiZ mice also had a significantly decreased tolerance for prolonged fasting compared with wild-type mice (PiZ mice 0% survival of 72-h fast; wild-type 100% survivial). These results demonstrate an altered response to stress in the alpha1-AT-deficient liver, including inability to further increase an activated autophagic response, a developmental state-specific increase in alpha1-AT-containing globules, and increased mortality.     

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.     

SKAnking with Ska3: Essential role of Ska3 in cell division revealed by combined phenotypic profiling

Comment on: Theis M, et al. EMBO J 2009; 28:1453-65.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.     

STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration

STAT3 is rapidly induced during liver regeneration in an interleukin 6 (IL-6)-dependent fashion, and IL-6 is required for normal liver regeneration. We wanted to know whether STAT3 was also required for liver regeneration but disruption of the STAT3 gene during embryonic stages causes lethality. Therefore, an albumin promoter-driven Cre-loxP recombination system was used to create a STAT3 deletion in the adult mouse liver to study the role of STAT3 in liver regeneration. After partial hepatectomy, there was virtually no STAT3 RNA or protein induction in Alb(+) STAT3(fl/fl) livers. STAT3 DNA binding activity was also absent in Alb(+) STAT3(fl/fl) livers. Unlike in control livers, STAT1 was activated in STAT3 conditional-mutant livers posthepatectomy. Hepatocyte DNA synthesis at 40 h posthepatectomy in Alb(+) STAT3(fl/fl) livers was reduced to approximately one-third of the control. Alb(+) STAT3(fl/fl) livers had abnormalities in immediate-early gene activation that largely correlated with but were not identical to those seen in IL-6-/- livers. G(1) phase cyclins including cyclins D1 and E had lower expression levels in Alb(+) STAT3(fl/fl) livers, indicating an abnormal G(1) to S phase transition. Therefore, STAT3 accounts for part of the DNA synthetic response of the hepatocytes during liver regeneration, which cannot be compensated for by induction of STAT1. Normal activation of the MAPK pathway in Alb(+) STAT3(fl/fl) livers reinforces the fact that at least part of the effect of IL-6 on hepatocyte proliferation is not mediated by STAT3. This study provides the first in vivo evidence that STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.