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BBA - Molecular Basis of Disease (v.1782, #4)

Editorial Board (pp. ii).

Role of transforming growth factor-β superfamily signaling pathways in human disease by Kelly J. Gordon; Gerard C. Blobe (pp. 197-228).

Transforming growth factor β (TGF-β) superfamily signaling pathways are ubiquitous and essential regulators of cellular processes including proliferation, differentiation, migration, and survival, as well as physiological processes, including embryonic development, angiogenesis, and wound healing. Alterations in these pathways, including either germ-line or somatic mutations or alterations in the expression of members of these signaling pathways often result in human disease. Appropriate regulation of these pathways is required at all levels, particularly at the ligand level, with either a deficiency or an excess of specific TGF-β superfamily ligands resulting in human disease. TGF-β superfamily ligands and members of these TGF-β superfamily signaling pathways also have emerging roles as diagnostic, prognostic or predictive markers for human disease. Ongoing studies will enable targeting of TGF-β superfamily signaling pathways for the chemoprevention and treatment of human disease.

Keywords: TGF-β superfamily; Hereditary disease; Sporadic disease

Novel functions of the α-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease by Qingli Shi; Hui Xu; Wayne A. Kleinman; Gary E. Gibson (pp. 229-238).

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H₂O₂ and to probe the mechanism underlying these changes. Since the response to H₂O₂ is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H₂O₂ decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H₂O₂ inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H₂O₂ converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H₂O₂ diminished glutathione to a similar level after 24 h. However, H₂O₂, but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H₂O₂ together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.

Keywords: α-ketoglutarate dehydrogenase complex; Alzheimer's disease; Glutathione; Malate dehydrogenase; Reactive oxygen species

Transcripts of ceruloplasmin but not hepcidin, both major iron metabolism genes, exhibit a decreasing pattern along the portocentral axis of mouse liver by Marie-Bérengère Troadec; Alain Fautrel; Bernard Drénou; Patricia Leroyer; Emilie Camberlein; Bruno Turlin; André Guillouzo; Pierre Brissot; Olivier Loréal (pp. 239-249).

During iron overload of dietary origin, iron accumulates predominantly in periportal hepatocytes. A gradient in the basal and normal transcriptional control of genes involved in iron metabolism along the portocentral axis of liver lobules could explain this feature. Therefore, we aimed at characterizing, by quantitative RT-PCR, the expression of iron metabolism genes in adult C57BL/6 mouse hepatocytes regarding lobular localisation, with special emphasis to cell ploidy, considering its possible relationship with lobular zonation.We used two methods to analyse separately periportal and perivenous liver cells: 1) a selective liver zonal destruction by digitonin prior to a classical collagenase dissociation, and 2) laser capture microdissection. We also developed a method to separate viable 4N and 8N polyploid hepatocytes by flow cytometer.Transcripts of ceruloplasmin, involved in iron efflux, were overexpressed in periportal areas and the result was confirmed by in situ hybridization study. By contrast, hepcidin 1, hemojuvelin, ferroportin, transferrin receptor 2, hfe andl-ferritin mRNAs were not differentially expressed according to either lobular zonation or polyploidisation level.At variance with glutamine or urea metabolism, iron metabolism is not featured by a metabolic zonation lying only on a basal transcriptional control. The preferential periportal expression of ceruloplasmin raises the issue of its special role in iron overload disorders involving a defect in cellular iron export.

Keywords: Abbreviations; LDH; lactate dehydrogenase; SSC; Side Scatter Channel; FSC; Forward Scatter Channel; PI; Propidium Iodide; 2N; diploid cells; 4N; tetraploid cells; 8N; octoploid cells; LCM; Laser Capture MicrodissectionIron metabolism; Liver zonation; Ploidy; Ceruloplasmin; Hepcidin; Gene expression; Laser microdissection; Cytometry; Mouse

Gene expression profiling of mucolipidosis type IV fibroblasts reveals deregulation of genes with relevant functions in lysosome physiology by Andrea Bozzato; Sergio Barlati; Giuseppe Borsani (pp. 250-258).

Mucolipidosis type IV (MLIV,MIM 252650) is an autosomal recessive lysosomal storage disorder that causes mental and motor retardation as well as visual impairment. The lysosomal storage defect in MLIV is consistent with abnormalities of membrane traffic and organelle dynamics in the late endocytic pathway. MLIV is caused by mutations in the MCOLN1 gene, which codes for mucolipin-1 (MLN1), a member of the large family of transient receptor potential (TRP) cation channels. Although a number of studies have been performed on mucolipin-1, the pathological mechanisms underlying MLIV are not fully understood. To identify genes that characterize pathogenic changes in mucolipidosis type IV, we compared the expression profiles of three MLIV and three normal skin fibroblasts cell lines using oligonucleotide microarrays. Genes that were differentially expressed in patients' cells were identified. 231 genes were up-regulated, and 116 down-regulated. Real-Time RT-PCR performed on selected genes in six independent MLIV fibroblasts cell lines was generally consistent with the microarray findings. This study allowed to evidence the modulation at the transcriptional level of a discrete number of genes relevant in biological processes which are altered in the disease such as endosome/lysosome trafficking, lysosome biogenesis, organelle acidification and lipid metabolism.

Keywords: Mucolipidosis type IV; Lysosomal diseases; Microarray; Gene expression profiling; Gene ontology

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer by Teresa Golden; Ileana V. Aragon; Beth Rutland; J. Allan Tucker; Lalita A. Shevde; Rajeev S. Samant; Guofei Zhou; Lauren Amable; Danalea Skarra; Richard E. Honkanen (pp. 259-270).

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis ( P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Keywords: Abbreviations; DCIS; ductal carcinoma in situ; IDC; invasive ductal carcinoma; FACS; fluorescence activated cell sorting; ASK1; apoptosis signal regulating kinase 1; HIF-1; hypoxia inducible factor 1; MKK4; Map-kinase kinase-4; DNA-PKcs; DNA-dependent ser/thr protein kinase; ATM; ataxia-telangiectasia mutated-kinase; ATR; ATM/rad 3 related kinase; GR; glucocorticoid receptor; ER; estrogen receptor; PR; progesterone receptor; JNK; c; -Jun N-terminal kinasePhosphatase; PP5; UVC; Adriamycin; Vinblastine; Oxidative stress; MCF-7 cells; Cancer

Changes in the expression and dynamics of SHP-1 and SHP-2 during cerulein-induced acute pancreatitis in rats by Nancy Sarmiento; Carmen Sánchez-Bernal; Manuel Ayra; Nieves Pérez; Angel Hernández-Hernández; José J. Calvo; Jesús Sánchez-Yagüe (pp. 271-279).

Protein tyrosine phosphatases (PTPs) are important regulators of cell functions but data on different PTP expression and dynamics in acute pancreatitis (AP) are very scarce. Additionally, both c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK1/2), together with intracellular cAMP levels in inflammatory cells, play an essential role in AP. In this study we have detected an increase in PTP SHP-1 and SHP-2 in the pancreas at the level of both protein and mRNA as an early event during the development of Cerulein (Cer)-induced AP in rats. Nevertheless, while SHP-2 protein returned to baseline levels in the intermediate or later phases of AP, SHP-1 protein expression remained increased throughout the development of the disease. The increase in SHP-2 protein expression was associated with changes in its subcellular distribution, with higher percentages located in the fractions enriched in lysosomes+mitochondria or microsomes. Furthermore, while the increase in SHP-2 protein was also observed in sodium-taurocholate duct infusion or bile-pancreatic duct obstruction AP, that of SHP-1 was specific to the Cer-induced model. Neutrophil infiltration did not affect the increase in SHP-1 protein, but favoured the return of SHP-2 protein to control levels, as indicated when rats were rendered neutropenic by the administration of vinblastine sulfate. Inhibition of JNK and ERK1/2 with SP600125 pre-treatment further increased the expression of both SHP-1 and SHP-2 proteins in the early phase of Cer-induced AP, while the inhibition of type IV phosphodiesterase with rolipram only suppressed the increase in SHP-2 protein expression during the same phase. Our results show that AP is associated with increases in the expression of SHP-1 and SHP-2 and changes in the dynamics of SHP-2 subcellular distribution in the early phase of Cer-induced AP. Finally, both JNK and ERK1/2 and intracellular cAMP levels are able to modulate the expression of these PTPs.

Keywords: SHP-1; SHP-2; Acute pancreatitis; Cerulein; SP600125; Rolipram

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria by Christina Piskernik; Susanne Haindl; Tricia Behling; Zanoni Gerald; Ingeborg Kehrer; Heinz Redl; Andrey V. Kozlov (pp. 280-285).

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O₂⁻) from rat heart mitochondria (RHM), while O₂⁻ generated in intact RHM do not escape from mitochondria. This was shown by a set of O₂⁻-sensitive spin probes with varying hydrophobicity. The levels of O₂⁻ detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O₂⁻ levels. Elevated O₂⁻ levels became sensitive to SOD but in a different manner. The determination of O₂⁻ with water-soluble PPH was fully sensitive to SOD, while the determination of O₂⁻ with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O₂⁻ into the surrounding medium.

Keywords: Mitochondria; Superoxide radical; Electron spin resonance; Spin probe; Endotoxic shock; Apoptosis

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BBA - Molecular Basis of Disease (v.1782, #4)