Comparison of protein tyrosine phosphorylation and morphological changes induced by IL-2 and IL-3.

We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R. Comparison of protein tyrosine phosphorylation profiles induced by IL-2 and IL-3 among a variety of cell lines revealed that the pp90 band is the common tyrosine phosphorylation substrate in the cell lines examined, although the general tyrosine phosphorylation pattern differed in each cell line. Mutant p75 molecules incapable of inducing tyrosine phosphorylation could bind and internalize IL-2, but could not support cell growth. We also found that swift changes of cytoskeletal protein organization are one of the early events caused by signal transduction through either IL-2R and IL-3R. Reorganization of cytoskeletal proteins seems to be associated with protein phosphorylation, as a significant portion of pp90 was found in a detergent-soluble fraction in IL-2 or IL-3 treated cells.     

Increased expression levels of integrin alphavbeta5 on scleroderma fibroblasts

Integrin alphavbeta5 is a receptor for vitronectin, a plasma glycoprotein that is also distributed in extracellular matrix of various tissues. Matrix-bound vitronectin has the potential to stabilize the active form of plasminogen activator inhibitor-1, resulting in the inhibition of the plasmin-mediated pericellular proteolytic cascade. In this study, we compared the levels of alphavbeta5 and matrix-bound vitronectin between normal and scleroderma fibroblasts and investigated the association with fibrosis. We demonstrated that alphavbeta5 was up-regulated on scleroderma fibroblasts. The up-regulated alphavbeta5 contributed to the increase in vitronectin-binding ability in scleroderma fibroblasts, which led to the vitronectin-dependent activation of plasminogen activator inhibitor-1. In immunohistochemistry, the alphav and beta5 subunits were stained strongly on scleroderma fibroblasts and the amount of vitronectin was increased in the pericellular matrix of those cells. The transient overexpression of alphavbeta5 on normal fibroblasts enhanced the human alpha2(I) collagen promoter activity through Sp-1 and Smad3 as well as the vitronectin-dependent plasminogen activator inhibitor-1 activity. This effect on the promoter activity was also observed in the absence of vitronectin and completely disappeared in the presence of anti-alphavbeta5 antibody. These results indicate that the up-regulated alphavbeta5 may contribute to the phenotypical alteration of scleroderma fibroblasts, while at the same time suppressing the plasmin-mediated pericellular proteolytic cascade.     

C-reactive protein levels in the serum of asthmatic patients.

BACKGROUND: Asthma is a chronic airway inflammatory disease caused by immune cells such as T lymphocytes and eosinophils. Recently, highly sensitive C-reactive protein (hs-CRP) assays have become available for detecting small changes in CRP levels within the reference range, allowing for the evaluation of clinical inflammation. OBJECTIVE: To investigate the relationship between hs-CRP levels and bronchial asthma. METHODS: We collected blood samples from 109 patients with bronchial asthma, with or without attacks, and measured serum eosinophil cationic protein levels, pulmonary function, and serum CRP levels using an hs-CRP assay. RESULTS: Mean serum hs-CRP levels were significantly higher in patients without attacks (0.473 mg/L) and with attacks (0.908 mg/L) (P < .001 for both) than in controls (0.262 mg/L). Serum hs-CRP levels were inversely correlated with forced expiratory volume in 1 second/forced vital capacity in asthmatic patients (r = -0.4915; P < .01). CONCLUSION: Serum hs-CRP levels may be related to the state of asthma exacerbation and allergic inflammation.     

Overexpression of monocyte chemotactic protein-1/CCL2 in beta-amyloid precursor protein transgenic mice show accelerated diffuse beta-amyloid deposition

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.     

Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, PI-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the PI3-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEK1-MAPK and PI3K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymorphisms and the differential antiviral activity of the chicken Mx gene

The nucleotide sequence of chicken Mx cDNA was reported earlier using the White Leghorn breed in Germany, but it showed no enhanced resistance to viruses. In this study, the nucleotide sequences of chicken Mx cDNA were determined in many breeds. A total of 25 nucleotide substitutions, of which 14 were deduced to cause amino acid exchanges, were detected, suggesting that the chicken Mx gene is very polymorphic. Transfected cell clones expressing chicken Mx mRNA were established after the Mx cDNA was constructed with an expression vector and introduced into mouse 3T3 cells, and the Mx genes from some breeds were demonstrated to confer positive antiviral responses to influenza virus and vesicular stomatitis virus. On the basis of the comparison among the antiviral activities associated with many Mx variations, a specific amino acid substitution at position 631 (Ser to Asn) was considered to determine the antivirally positive or negative Mx gene. Thus, a single amino acid substitution influences the antiviral activity of Mx in domesticated chickens.     

Functional analysis of the effect of forced activation of STAT3 on M1 mouse leukemia cells

M1 mouse myeloid leukemia cells exhibit growth arrest and differentiation to monocytes/macrophages in response to leukemia inhibitory factor (LIF) stimulation. Although recent studies have demonstrated that STAT3 plays a central role in this process, it is unknown whether STAT3 activation alone is sufficient. To address this issue, we have established M1/STAT3ER cells, where STAT3 is selectively activated by 4-hydroxytamoxifen (4HT). 4HT stimulation did not have any effect on growth and morphology of M1/ STAT3ER cells, and did not induce the down-regulation of mRNA of c-myc and c-myb, which is necessary for M1 cell differentiation. On the other hand, mRNA of jun-B, IRF1 and p19 was increased by 4HT. DNA precipitation assay indicated that both stimulation of LIF and 4HT similarly activated STAT3ER. Introduction of a constitutive active MAP kinase kinase (MEK1) into M1/STAT3ER cells did not induce differentiation either. Together, our present data suggest that signaling other than the activation of STAT3 and MEK1 may be necessary for M1 cell-growth arrest and differentiation, while a set of early genes of LIF are induced by only STAT3 activation.     

The effect of peroxisome proliferator-activated receptor-gamma ligand on urological cancer cells

Peroxisome proliferator activator-receptor (PPAR)-gamma ligand induces growth arrest of cancer cells through apoptosis. In this study, we examined the effects of PPAR-gamma inhibitors on cell proliferation in renal cell carcinoma (RCC), bladder tumor (BT), and prostatic carcinoma (PC) cell lines. We investigated the inhibitory effect of PPAR-gamma ligands, troglitazone and 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) on RCC, BT and PC-derived cell lines using MTT assay and Hoechst staining. PPAR-gamma ligands (troglitazone and 15dPGJ2) induced the reduction of cell viability with the half-maximal concentration of growth inhibition of RCC, BT, and PC cell lines. Furthermore, counting cells at days 1, 2 and 3, clearly showed marked inhibition of cell proliferation using troglitazone and 15dPGJ2. All PPAR-gamma inhibitors stopped the growth of all RCC, BT and PC cells. Cells treated with PPAR-gamma inhibitors showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These cellular changes were typically redundant characteristics of apoptosis. PPAR-gamma ligands may mediate potent antiproliferative effects against RCC, BT and PC cells through differentiation. Thus, PPAR-gamma may become a new target in treatment of urological tumors.