Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit

MicroRNAs (miRNAs) have attracted attention because of their key regulatory functions in many biological events, including differentiation and tumorigenesis. Recent studies have reported the existence of a reciprocal regulatory loop between the family of let-7 miRNAs and an RNA-binding protein, Lin28, both of which have been documented for their important roles during cell differentiation. Hence, using bipotent K562 human leukemia cells and human CD34+ hematopoietic progenitor cells as research models, we demonstrate that let-7 and Lin28 have contrary roles in megakaryocytic (MK) differentiation with a dynamic balance; expression of miR-181 is capable of effectively repressing Lin28 expression, disrupting the Lin28-let-7 reciprocal regulatory loop, upregulating let-7, and eventually promoting MK differentiation. However, miR-181 lacks a significant effect on hemin-induced erythrocyte differentiation. These results demonstrate that miR-181 can function as a 'molecular switch' during hematopoietic lineage progression specific to MK differentiation, thus providing insight into future development of miRNA-oriented therapeutics.     

Variable agronomic practices, cultivar, strain source and initial contamination dose differentially affect survival of Escherichia coli on spinach

Aims: Greenhouse and field trials were conducted under different agronomic practices and inoculum doses of environmental Escherichia coli and attenuated E. coli O157:H7, to comparatively determine whether these factors influence their survival on leaves and within the rhizosphere. Methods and Results: Hydroponic conditions: E. coli spray‐inoculated at log 4 CFU ml^?1 was recovered from leaf surfaces at a mean population of 1·6 log CFU g^?1 at 15 days. E. coli O157:H7 sprayed at log 2 or 4 CFU ml^?1 levelled off on spinach leaf surfaces at a mean average population of 1·4 log CFU g^?1 after 14 days, regardless of initial dose. Quantitative recovery was inconsistent across leaf developmental age. Field conditions: Average populations of E. coli O157:H7 spray‐inoculated at log 1·45 or 3·4 CFU m^?2 levelled off at log 1·2 CFU g^?1 over a 14‐day period. Pathogen recovery from leaves was inconsistent when compared to regularly positive detection on basal shoot tissue. Pathogen recovery from soil was inconsistent among sampling locations. Moisture content varied up to 40% DW and was associated with 50% (P < 0·05) decrease in positive locations for E. coli O157:H7 but not for E. coli. Conclusions: Overall, similar populations of environmental E. coli and E. coli O157:H7 were recovered from plants despite differences in inoculum dose and agronomic conditions. Strain source had a significant impact on the quantitative level and duration of survival on leaves and in soil. Water availability appeared to be the determinant factor in survival of E. coli and E. coli O157:H7; however, E. coli showed greater environmental fitness. Significance and Impact of the Study: Persistence of surrogate, indicator E. coli and E. coli O157:H7, irrespective of variable growing conditions in spinach is predominantly limited by water availability, strain source and localization within the plant. These findings are anticipated to ultimately be adopted into routine and investigative pathogen testing protocols and mechanical harvest practices of spinach.     

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.     

Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: Report of a new case and review of the literature

Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA). The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient’s bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.     

Expression profiling after induction of demethylation in MCF-7 breast cancer cells identifies involvement of TNF-α mediated cancer pathways

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethylation by 5-Aza-2′-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confirmed by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change ≥ 2), respectively, in common in cells treated with 5 and 10 μM of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-α in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.