HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells

Background

Heparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2). Human endosulfatase 1 (HSULF-1) is an enzyme that selectively removes 6-O sulfate groups from HS side chains and alter their level and pattern of sulfation and thus biological activity. It is known that HSULF-1 is expressed at low levels in some cancer cell lines and its enhanced expression can inhibit cancer cell growth or induce apoptosis, but the mechanism(s) involved has not been identified.

Methods

HSULF-1 mRNA expression was assessed in five normal cells (primary human lung alveolar type 2 (hAT2) cells, adult lung fibroblasts (16Lu), fetal lung fibroblasts (HFL), human bronchial epithelial cells (HBE), and primary human lung fibroblasts (HLF)) and five lung cancer cell lines (A549, H292, H1975, H661, and H1703) using quantitative real time polymerase chain reaction (qRT-PCR). H292 and hAT2 cells over-expressing HSULF-1 were analyzed for cell viability, apoptosis, and ERK/Akt signaling, by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and Western Blot, respectively. Apoptosis pathway activation was confirmed by PCR array in hAT2, H292, and A549 cells.

Results

HSULF-1 was expressed at a significantly lower level in epithelial cancer cell lines compared to normal cells. Infection with recombinant adenovirus for HSULF-1 over-expression resulted in decreased cell viability in H292 cells, but not in normal hAT2 cells. HSULF-1 over-expression induced apoptosis in H292 cells, but not in hAT2 cells. In addition, apoptosis pathways were activated in both H292 and A549 cells, but not in hAT2 cells. HSULF-1 over-expression reduced ERK and Akt signaling activation in H292 cells, which further demonstrated its inhibitory effects on signaling related to proliferation.

Conclusions

These results indicate that HSULF-1 is expressed at lower levels in H292 lung cancer cells than in normal human alveolar cells and that its over-expression reduced cell viability in H292 cells by inducing apoptotic pathways, at least in part by inhibiting ERK/Akt signaling. We hypothesize that HSULF-1 plays important roles in cancer cells and functions to modify cell signaling, inhibit cancer proliferation, and promote cancer cell death.     

Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen

Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone.     

Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone

Thickening of the basement membrane zone (BMZ) is a characteristic of several airway diseases; however, very little is known about how this process occurs. The purpose of this study was to define development of the BMZ in the trachea of growing rhesus monkeys at 1, 2, 3, and 6 mo of age. We measured immunoreactivity of collagen types I, III, and V to detect structural changes in the developing BMZ. To detect more dynamic, functional components of the epithelial-mesenchymal trophic unit, we evaluated the distribution of perlecan, fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor-1 (FGFR-1). One-month-old monkeys had a mean collagen BMZ width of 1.5 +/- 0.7 microm that increased to 4.4 +/- 0.4 microm in 6-mo-old monkeys. Perlecan was localized in the BMZ of the epithelium at all ages. FGF-2 was strongly expressed in basal cells at 1-3 mo. At 6 mo, FGF-2 was expressed throughout the BMZ and weakly in basal cells. FGFR-1 immunoreactivity was expressed by basal cells and cilia and weakly in the nuclei of columnar cells at all time points. These data indicate that development of the BMZ is a postnatal event in the rhesus monkey that involves FGF-2.     

Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1-10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.     

Geographical distribution of antimicrobial resistance among Escherichia coli causing acute uncomplicated pyelonephritis in the United States

The susceptibility to 12 antimicrobial agents of 165 Escherichia coli isolates from women with acute uncomplicated pyelonephritis of mild to moderate severity was analyzed by geographic region in the US. Ampicillin, trimethoprim, and trimethoprim/sulfamethoxazole resistance exhibited a descending prevalence gradient from west to east. Composite antimicrobial resistance phenotypes also exhibited significant regional differences, with a greater prevalence of most combined resistance profiles seen in the Pacific region of the US, but with significant north-south variation for combined ampicillin/sulfisoxazole resistance. These findings suggest geographical segregation of resistant clones and/or resistance elements among uropathogenic E. coli within the US, which is relevant both to clinical practice and to understanding the basis for the current epidemic of antimicrobial resistance in E. coli.     

Candidate SNP of CACNA2D1 Gene Associated with Clinical Mastitis and Production Traits in Sahiwal (Bos taurus indicus) and Karan Fries (Bos taurus taurus × Bos taurus indicus)

The present study was conducted to identify polymorphisms in CACNA2D1 gene and their association with clinical mastitis and production traits. Exon 18 and its flanking regions were screened for the presence of SNPs. Statistical analysis was performed to identify association of period of birth, breed, and genotype with mastitis incidence on randomly selected 103 Sahiwal and 102 Karan Fries cattle. PCR-RFLP analysis revealed that g.38819398G?>?A mutation in exon 18 (269?bp amplicon) of CACNA2D1 gene resolved into AA, AG, and GG genotypes in Sahiwal and Karan Fries cattle. Wald chi-square analysis revealed that the period of birth, breed, and genotype were significantly associated with mastitis incidence. GG genotyped cattle were found to be less susceptible to mastitis. Least square analysis revealed that GG and AG genotype animals of G38819398A SNP of CACNA2D1 gene in Sahiwal as well as in Karan Fries cattle were associated with higher average milk yields during 1st, 2nd, and 3rd lactations (P?<?0.01). These observations and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits; however, validation of results on large number of animals is warranted.     

Fibroblast growth factor-binding protein and N-deacetylase/N-sulfotransferase-1 expression in type II cells is modulated by heparin and extracellular matrix

Fibroblast growth factors (FGFs) play critical roles in development, maintenance, and repair following injury or disease in the lung. Their activity is modulated by a variety of factors, including FGF-binding protein (FGF-BP; HBp-17) and N-deacetylase/N-sulfotransferase-1 (NDST-1). Functionally, FGF-BP shuttles FGFs from binding sites in ECMs to cell surfaces and enhances FGF binding and signaling, whereas NDST-1 adds sulfate groups to FGF coreceptor proteoglycans and modulates alveolar type II (ATII) cell maturation and differentiation. Since the sulfated nature of ECMs is a critical determinant of their relationship with FGFs, we predicted that ECMs and their sulfation would modulate the expression of FGF-BP and NDST-1. To examine this question, selected culture conditions of rat ATII cells were manipulated [with and without coculture with rat lung fibroblasts (RLFs)] by treatment with heparin or sodium chlorate (inhibitor of sulfation) for 24-96 h. In addition, ECMs biosynthesized by RLFs for up to 10 days before coculture were used as model intervening barriers to communication between alveolar cells and fibroblasts. FGF-BP expression was enhanced in ATII cells by coculture with RLF cells and least suppressed by desulfated heparin. NDST-1 expression in ATII cells was most sensitive to the amount of sulfation in medium and ECM and enhanced by fully sulfated heparin. Preformed ECM appears to supply factors that modify subsequent treatment effects. These results demonstrate a potentially important modulatory influence of sulfated ECMs and fibroblasts on FGF-BP and NDST-1 at the gene expression level.