BBA - Molecular Basis of Disease (v.1762, #9)

The expression of the Na+,K+-ATPase α and β subunit isoforms in rat skeletal muscle and its age-associated changes have been shown to be muscle-type dependent. The cellular basis underlying these findings is not completely understood. In this study, we examined the expression of Na+,K+-ATPase isoforms in individual fiber types and tested the hypothesis that, with age, the changes in the expression of the isoforms differ among individual fibers. We utilized immunohistochemical techniques to examine the expression of the subunit isoforms at the individual fiber levels. Immunofluorescence staining of the subunit isoforms in both white gastrocnemius (GW) and red gastrocnemius (GR) revealed a predominance of staining on the sarcolemmal membrane. Compared to the skeletal muscle of 6-month-old rats, there were substantial increases in the levels of α1, β1, and β3 subunit isoforms, and decreases in the levels of α2 and β2 in 30-month-old rats. In addition, we found distinct patterns of staining for the α1, α2, β1, and β2 isoforms in tissue sections from young and aged rats. Muscle fiber-typing was performed to correlate the pattern of staining with specific fiber types. Staining for α1 and α2 isoforms in the skeletal muscle of young rats was generally evenly distributed among the fibers of GW and GR, with the exception of higher α1 levels in slow-twitch oxidative Type I fibers of GR. By contrast, staining for the β1 and β2 isoforms in the mostly oxidative fibers and the mostly glycolytic fibers, respectively, was almost mutually exclusive. With age, there was a fiber-type selective qualitative decrease of α2 and β2 in Type IIB fibers, and increase of β1 in Type IIB fibers and β2 in Type IID fibers of white gastrocnemius. These results provide, at the individual fiber level, a cellular basis for the differential expression of the Na+,K+-ATPase subunit isoforms in the muscle groups. The data further indicate that the aged-associated changes in expression of the subunit isoforms occur in both a fiber-type specific as well as an across fiber-type manner. Because of the differing biochemical properties of the subunit isoforms, these changes add another layer of complexity in our understanding of the adaptation of the Na-pump in skeletal muscle with advancing age.
Keywords: Aging; Na+–K+ pump; Subunit isoforms;

Sarcoidosis and NOD1 variation with impaired recognition of intracellular Propionibacterium acnes by Tsuyoshi Tanabe; Ikuo Ishige; Yoshimi Suzuki; Yukie Aita; Asuka Furukawa; Yuki Ishige; Keisuke Uchida; Takashige Suzuki; Tamiko Takemura; Soichiro Ikushima; Masaru Oritsu; Tetsuji Yokoyama; Yukari Fujimoto; Koichi Fukase; Naohiro Inohara; Gabriel Nunez; Yoshinobu Eishi (794-801).
Sarcoidosis is a systemic granulomatous disease of unknown etiology. NOD2 mutations have been shown to predispose to granulomatous diseases, including Crohn's disease, Blau syndrome, and early-onset sarcoidosis, but not to adult sarcoidosis. We found that intracellular Propionibacterium acnes, a possible causative agent of sarcoidosis, activated NF-κB in both NOD1- and NOD2-dependent manners. Systematic search for NOD1 gene polymorphisms in Japanese sarcoidosis patients identified two alleles, 796G-haplotype (156C, 483C, 796G, 1722G) and 796A-haplotype (156G, 483T, 796A, 1722A). Allelic discrimination of 73 sarcoidosis patients and 215 healthy individuals showed that the frequency of 796A-type allele was significantly higher in sarcoidosis patients and the ORs were significantly elevated in NOD1-796G/A and 796A/A genotypes (OR [95% CI] = 2.250 [1.084, 4.670] and 3.243 [1.402, 7.502], respectively) as compared to G/G genotype, showing an increasing trend across the 3 genotypes (P  = 0.006 for trend). A similar association was found when 52 interstitial pneumonia patients were used as disease controls. Functional studies showed that the NOD1 796A-allele was associated with reduced expression leading to diminished NF-κB activation in response to intracellular P. acnes. The results indicate that impaired recognition of intracellular P. acnes through NOD1 affects the susceptibility to sarcoidosis in the Japanese population.
Keywords: NOD2; TLR2; Granulomatous disease; NF-κB;

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53 by Vanessa A. Morais; Catarina Brito; Donald S. Pijak; Adam S. Crystal; Ryan R. Fortna; Tong Li; Phil C. Wong; Robert W. Doms; Júlia Costa (802-810).
The γ-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. γ-Secretase cleavage of APP releases the Aβ peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active γ-secretase complex.
Keywords: Alzheimer's disease; Nicastrin; γ-secretase complex; N-glycosylation; Calnexin; ERGIC-53;

Chronic or recurrent inflammation plays a role in the development of many types of cancer including prostate cancer. CXCL10 (interferon-γ inducible protein-10, IP-10) is a small secretory protein of 8.7 kDa. Recently, it has been shown that normal prostate epithelial (PZ-HPV-7) cells produce lower amounts of angiogenic CXC chemokines (GRO-α, IL-8) and higher amounts of angiostatic chemokines (CXCL10, CXCL11) as compared to prostate cancer cells (CA-HPV-10 and PC-3). Accordingly, we studied the effects of overexpression of CXCL10 in human prostate cancer LNCaP cells. LNCaP cells were transiently transfected with CXCL10 cDNA in pIRES2-EGFP vector. CXCL10, CXCR3, PSA and G3PDH mRNA levels were determined by semi-quantitative conventional and quantitative real-time RT-PCR and fluorescence-activated cell sorting (FACS). The expression of CXCL10 was markedly enhanced in the transfected cells at mRNA and protein levels in the cells. Overexpression of CXCL10 inhibited cell proliferation of the transfected cells by 30%–40% in serum-limited medium (1% FCS in RPMI1640 medium) and decreased PSA production. CXCR3 expression was significantly induced by the overexpression of CXCL10 as determined by RT-PCR and FACS. These results indicated that CXCL10 inhibited LNCaP cell proliferation and decreased PSA production by up-regulation of CXCR3 receptor. CXCL10 may be potentially useful in the treatment of prostate cancer.
Keywords: CXCL10; Prostate cancer; CXCR3; Cell proliferation; PSA;

Proteasomal degradation regulates expression of porphobilinogen deaminase (PBGD) mutants of acute intermittent porphyria by Nurit Grünberg-Etkovitz; Lior Greenbaum; Borislava Grinblat; Zvi Malik (819-827).
Acute intermittent porphyria (AIP) is a neuropathic disease caused by a dominant inherited deficiency in porphobilinogen deaminase (PBGD). We investigated the expression and the degradation of the human PBGD-mutations G748A, G748C and 887insA following transfection into human SH-SY5Y neuroblastoma cells. Mutant proteins exhibited reduced protein expression compared to transfected wild-type (wt) PBGD as revealed by Western blotting. The transcription levels assessed by real-time PCR of these mutant species were identical to those of the wild type. Immuno-fluorescence microscopy revealed reduced cellular distribution of the mutated PBGDs in the cytosol and the nucleus in comparison to the wild-type PBGD. Enhanced cellular accumulation of the mutated and wild-type PBGDs was detected following inhibition of the proteasome by the inhibitors CLBL and hemin. Elevated expression of wt and mutated PBGD protein levels was either achieved by hemin or heme-arginate treatment. On the other hand, enhanced PBGD degradation was achieved by lead poisoning of ALAD in the SH-SY5Y cells concomitant with acceleration of proteasomal activity, most probably by ALAD participation in proteasomal regulation [G.G. Guo, M. Gu, J.D. Etlinger, 240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. J Biol Chem 1994; 269:12399–402.] Our results suggest that the difference in expression between the wild-type and mutant proteins appears to be regulated on the level of protein degradation. In conclusion, we demonstrate that the PBGD cellular pool is controlled by the proteasome activity, which in turn is down regulated by hemin or up-regulated by Pb-ALAD.
Keywords: AIP; PBGD; Proteasome; Hemin; ALAD;

ANKHD1, ankyrin repeat and KH domain containing 1, is overexpressed in acute leukemias and is associated with SHP2 in K562 cells by Fabíola Traina; Patricia M.B. Favaro; Samuel de Souza Medina; Adriana da Silva Santos Duarte; Sheila Maria Brochado Winnischofer; Fernando F. Costa; Sara T.O. Saad (828-834).
In the present study, increased levels of ANKHD1 mRNA and protein expression in leukemia cell lines are reported, as compared with normal hematopoietic cells. Furthermore, a higher expression of ANKHD1 mRNA was detected in primary acute leukemia samples than in normal hematopoietic cells (P  = 0.002). ANKHD1 was detected in the cytosolic and membrane fraction of cells and was co-immunoprecipitated with SHP2 in protein extracts of K562 and LNCaP cell lines. These findings suggest a role for ANKHD1 as a scaffolding protein that may be associated with the abnormal phenotype of leukemia cells.
Keywords: ANKHD1; MASK; Ankyrin repeat; SHP2 phosphatase; Acute leukemia;

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation–inflammation–thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IκB kinase β (IKKβ) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-κB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKβ and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKβ and ERK2, activations of NF-κB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKβ/proteasome/NF-κB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation–inflammation–thrombosis circuit during infections by VT-producing E. coli.
Keywords: Tissue factor; Verotoxin-1; Shiga toxin; Blood coagulation; c-Yes; NF-κB; Macrophage-like cell;

Complex regulation of mucosal pentraxin (Mptx) revealed by discrete micro-anatomical locations in colon by Janice E. Drew; Andrew J. Farquharson; Jaap Keijer; Lawrence N. Barrera B. (844-848).
Recently a mucosal pentraxin, Mptx, regulated by heme and calcium was reported in rat gut mucosal scrapings using microarray strategies. Considering the heterogeneity of gut mucosa scrapings and the widespread use of the rat as a model to study colon pathologies this study was undertaken to generate detailed mapping of micro-anatomical locations of Mptx and gain further insight into potential functions of this mucosal pentraxin in rat colon. Differential regulation was also examined in colon from different rat strains and rat models of oxidative stress and in pre-cancerous colon tissue. Different regional patterns of expression and discrete localisation in epithelial cells within transverse and distal colon crypts and an absence of expression in proximal colon were confirmed by regional PCR analysis and in situ hybridisation studies of colon. This study demonstrates that consideration of regional differences in Mptx gene expression and micro-anatomical location is necessary in the interpretation and deciphering of its regulation in colon.
Keywords: Colon crypt; Gene regulation; Epithelium; Oxidative stress; Pre-cancerous;