Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha 1.

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16 to 20 kDa) including two well-characterized isoforms (NDPK-A and -B). NDPK catalyzes the conversion of nucleoside diphosphates to nucleoside triphosphates, regulates a diverse array of cellular events, and can act as a protein histidine kinase. AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that responds to the cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of acetyl coenzyme A carboxylase 1 (ACC1), a regulator of cellular fatty acid synthesis. We recently reported that NDPK-A (but not NDPK-B) selectively regulates the alpha1 isoform of AMPK independently of the AMP concentration such that the manipulation of NDPK-A nucleotide trans-phosphorylation activity to generate ATP enhanced the activity of AMPK. This regulation occurred irrespective of the surrounding ATP concentration, suggesting that "substrate channeling" was occurring with the shielding of NDPK-generated ATP from the surrounding medium. We speculated that AMPK alpha1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK alpha1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK alpha1 and that the phosphorylation status of S122, but not S144, determines whether substrate channeling can occur. We report the cellular effects of the S122 mutation on ACC1 phosphorylation and demonstrate that the presence of E124 (absent in NDPK-B) is necessary and sufficient to permit both AMPK alpha1 binding and substrate channeling.

Nm23-H1 suppresses tumor cell motility by down-regulating the lysophosphatidic acid receptor EDG2.

Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1-suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through down-regulation of EDG2 expression.

FGFR3 contributes to intestinal crypt cell growth arrest.

Fibroblast growth factors (FGFs) are important regulators of the dynamic development and turnover of tissues. Among FGF receptors, FGFR3 expression is confined in the intestinal crypts. We examined FGFR3-deficient mice and saw increased intestinal crypt depth but no change in villae length or in the distribution of differentiated intestinal cells, suggesting that the impact of lack of FGFR3 was limited to the progenitor cell compartment. Accordingly, enhancement of intestinal crypt proliferation was observed in FGFR3 mutant mice and interestingly, upon anti-FGFR3 antibody administration in wild type mice. Moreover, injection of FGF18, a ligand of FGFR3, in wild type mice resulted in decreased cell proliferation within the intestinal crypts. In addition, we found that ERK level of activation was increased in FGFR3-deficient intestinal epithelium. In vitro studies showed that ERK, AKT and activation was regulated by FGFs and that ERK level of activation was inversely correlated to FGFR3 level of expression in the intestinal crypt cells. Furthermore, effects of FGF18 on ERK and AKT activation paralleled FGFR3 effects on these intracellular targets. Our data indicate that FGF18 and FGFR3 are involved, possibly as partners, in the control of intestinal precursor cell proliferation.

FGFR3 is a negative regulator of the expansion of pancreatic epithelial cells.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are key signaling molecules for pancreas development. Although FGFR3 is a crucial developmental gene, acting as a negative regulator of bone formation, its participation remains unexplored in pancreatic organogenesis. We found that FGFR3 was expressed in the epithelia in both mouse embryonic and adult regenerating pancreata but was absent in normal adult islets. In FGFR3 knockout mice, we observed an increase in the proliferation of epithelial cells in neonates, leading to a marked increase in islet areas in adults. In vitro studies showed that FGF9 is a very potent ligand for FGFR3 and activates extracellular signal-related kinases (ERKs) in pancreatic cell lines. Moreover, FGFR3 blockade or FGFR3 deficiency led to increased proliferation of pancreatic epithelial cells in vivo. This was accompanied by an increase in the proportion of potential islet progenitor cells. Thus, our results show that FGFR3 signaling inhibits the expansion of the immature pancreatic epithelium. Consequently, this study suggests that FGFR3 participates in regulating pancreatic growth during the emergence of mature islet cells.

Protein kinase CK2 acts as a signal molecule switching between the NDPK-A/AMPK alpha1 complex and NDPK-B.

Previously we elucidated the molecular interaction between the nucleoside diphosphate kinase A (NDPK-A)/AMP-activated protein kinase (AMPK) alpha1 complex, discovering a process we termed "substrate channeling." Here, we investigate the protein-protein interaction of the substrate channeling complex with the pleiotropic protein kinase, CK2 (formerly casein kinase 2). We show that CK2 is part of the NDPK-A/AMPK alpha1 complex under basal (background AMPK activity) conditions, binding directly to each of the complex components independently. We report that when S122 on NDPK-A is phosphorylated by AMPK alpha1 in vivo, (i.e., stimulation of AMPK using either metformin or phenformin) initiating the substrate channeling mechanism, the catalytic subunit of CK2 (CK2alpha) is expelled from the complex and translocates to bind NDPK-B, a closely related but independent isoform of NDPK. Thus, we find that the AMPK-dependent phospho-status of S122 on NDPK-A determines whether CK2alpha swaps partners between NDPK-A and NDPK-B. This is the first reported linkage between NDPK-A and NDPK-B via a phosphorylation pathway and could explain the complex biology of NDPK. This study also offers an explanation as to how CK2alpha exclusion mutations (S120A or S122D of NDPK-A) on NDPK-A might have implications in cancer biology and general cellular energy metabolism.

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes.

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.

Acute oxidative stress is associated with cell proliferation in the mouse liver.

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.

Epithelial progenitor 1, a novel factor associated with epithelial cell growth and differentiation.

The growth and renewal of epithelial tissue is a highly orchestrated and tightly regulated process occurring in different tissue types under a variety of circumstances. We have been studying the process of pancreatic regeneration in mice. We have identified a cell surface protein, named EP1, which is expressed on the duct epithelium during pancreatic regeneration. Whereas it is not detected in the pancreas of normal mice, it is found in the intestinal epithelium of normal adult mice, as well as during pancreatic repair following cerulein-induced destruction of the acinar tissue. The distinctive situations in which EP1 is expressed, all of which share in common epithelial cell growth in the gastrointestinal tract, suggest that EP1 is involved in the growth and renewal of epithelial tissues in both the intestine and the pancreas.

Infrared spectroscopy: a reagent-free method to distinguish Alzheimer's disease patients from normal-aging subjects.

The physiopathogenesis of Alzheimer's disease (AD) is related to various biochemical mechanisms that may be reflected by changes in plasma components. In the current study, Fourier transform-infrared (FT-IR) spectroscopy was used to identify these biochemical variations by monitoring spectral differences in the plasma of 40 AD patients compared with those of 112 control subjects. A hierarchical classification in the whole mid-infrared region allowed a clear separation between AD and controls (C) that was optimized by using a restricted spectral range (1480-1428 cm(-1)). Spectral changes confirmed vibration differences between AD and C mostly related to modified lipid and nucleic acid structures involved in oxidative stress-dependent processes of AD. Moreover, the analysis of samples in the 1480-910-cm(-1) region allowed the distinction between C and AD with an accuracy of 98.4% and showed 2 subgroups C(1) and C(2) within the C group. Interestingly, the C(1) subgroup was located closer to the AD group than the C(2) subgroup, which suggests biochemical differences within the nondemented subjects. Biochemical studies revealed a significant increase in a specific marker of oxidative stress, F8-isoprostanes (8-epi-PGF2alpha) levels, in the plasma of AD patients as compared with total controls and subgroup C(2) but not subgroup C(1). Thus, these results suggest that use of FT-IR spectroscopy could be valuable to distinguish AD patients from normal-aging subjects.

Tyrosine kinase receptors are crucial for normal ß-cell development and function.

Signaling pathways play critical roles in most physiological and pathological processes and convert an extracellular stimulus into a change of function in the recipient cell. Intracellular messages originate from the activation of membrane receptors by a variety of ligands, such as hormones, nutrients or growth factors. The receptors subsequently interact with specific intracellular cascades, triggering the phosphorylation of cell effectors. In the pancreas, these processes control the organogenesis, maintenance and function of endocrine cells within the islets. Growth factors acting through tyrosine kinase receptors play a prominent role among the multitude of signaling pathways active in pancreatic ß cells. Deregulation of these processes leads to the development of disorders such as hypoglycemia or diabetes. This review will describe recent advances made on the understanding of the roles of major tyrosine kinase receptors in pancreatic ß-cell physiology.

PYY in the expanding pancreatic epithelium.

Gut peptide YY (PYY) plays an important role in regulating metabolism and is expressed during the ontogeny of the pancreas. However, its biological role during endocrine cell formation is not fully understood, and its role, if any, during pancreatic regeneration in the adult has not yet been explored. The knowledge of factors involved in beta cell renewal in adult animals is clearly relevant for the design of treatment strategies for type 1 diabetes. We therefore sought to determine if observations during fetal pancreas formation also apply to pancreatic growth in adult animals. Indeed, we have found marked expansion of the PYY-expressing population during pancreatic regeneration. In addition, we demonstrate the presence of cells co-expressing PYY and the critical pancreatic transcription factor pancreatic duodenal homeobox1 (PDX-1). Interestingly, these cells also co-expressed specific islet hormones during pancreatic development and re-growth, suggesting a developmental relationship. Furthermore, we have found that PYY can act in concert with IGF-1 to stimulate cellular responsiveness in pancreatic epithelial cells in vitro. Our data suggest that PYY may be a mediator of islet cell development, as well as a cofactor for growth factor responses, not only during fetal pancreas formation but also during regeneration in adult animals.

Increased lung metastasis in transgenic NM23-Null/SV40 mice with hepatocellular carcinoma.

BACKGROUND: The metastasis-suppressing role of the NM23 gene in the metastatic spread of solid tumors is still debated. We examined the role of NM23 in tumor development and metastatic dissemination by using transgenic mice that lack mouse NM23 (NM23-M1) in two mouse models of hepatocellular carcinoma (HCC) that recapitulate all steps of tumor progression. METHODS: We induced HCC in mice that contained (NM23-M1(+/+)) or lacked (NM23-M1(-/-)) NM23-M1 by diethylnitrosamine injection or by a crossing scheme that transferred a transgene that leads to liver expression of simian virus 40 large T antigen (ASV mice). We used microscopic examination and immunohistochemistry to analyze tumor progression. Expression of Nm23 protein isoforms (Nm23-M1 and Nm23-M2) and several tumor markers was analyzed in the primary tumor and in metastases by Western blotting. The statistical significance of differences in the incidence of Nm23-M2 overexpression in null mice relative to that in wild-type mice was tested by a one-sided Fisher's exact test. The statistical significance of differences in the incidence of metastases was examined using one-sided chi-square tests. All other statistical tests were two-sided. RESULTS: In both models, Nm23-M1 and/or Nm23-M2 were overexpressed in the primary liver tumors compared with nontumor liver tissue; however, the lack of the NM23-M1 gene had no effect on primary tumor formation in either model. ASV mice developed pulmonary metastases that were positive for the Hep-Par 1 antibody, which recognizes a specific hepatocyte antigen, whereas the few pulmonary nodules that developed in diethylnitrosamine-injected mice were negative for this antigen. Statistically significantly more ASV/NM23-M1(-/-) mice than ASV/NM23-M1(+/+) mice developed lung metastases (69.2% versus 37.5%; difference = 31.7%, 95% confidence interval = 13.1% to 50.3%; P<.001). In ASV/NM23-M1(+/+) mice, immunohistochemical staining for Nm23-M1 was highly heterogeneous among the primary liver tumors, but weak or negative among lung metastases. CONCLUSIONS: The lack of NM23-M1 expression promotes metastasis in the SV40 animal model of liver carcinogenesis.

Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death.

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c-myc promoter and transactivates the c-myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c-myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c-myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c-myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c-myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c-myc overexpression or H(2)O(2) exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response.