Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice

<p>Abstract</p> <p>Background</p> <p>Tobacco smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD), though the mechanisms of its toxicity are still unclear. The ABC transporters multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp/MDR1) extrude a wide variety of toxic substances across cellular membranes and are highly expressed in bronchial epithelium. Their impaired function may contribute to COPD development by diminished detoxification of noxious compounds in cigarette smoke.</p> <p>Methods</p> <p>We examined whether triple knock-out (TKO) mice lacking the genes for <it>Mrp1 </it>and <it>Mdr1a/1b </it>are more susceptible to develop COPD features than their wild-type (WT) littermates. TKO and WT mice (six per group) were exposed to 2 cigarettes twice daily by nose-only exposure or room air for 6 months. Inflammatory infiltrates were analyzed in lung sections, cytokines and chemokines in whole lung homogenates, emphysema by mean linear intercept. Multiple linear regression analysis with an interaction term was used to establish the statistical significances of differences.</p> <p>Results</p> <p>TKO mice had lower levels of interleukin (IL)-7, KC (mouse IL-8), IL-12p70, IL-17, TNF-alpha, G-CSF, GM-CSF and MIP-1-alpha than WT mice independent of smoke exposure (<it>P </it>< 0.05). IL-1-alpha, IL-6, IL-8, IL-13, IL-17, TNF-alpha, G-CSF, GM-CSF and MCP-1 increased after smoke exposure in both groups, but the increase in IL-8 was lower in TKO than WT mice (<it>P </it>< 0.05) with a same trend for G-CSF (<it>P </it>< 0.10). Smoke-induced increase in pulmonary inflammatory cells in WT mice was almost absent in TKO mice. The mean linear intercept was not different between groups.</p> <p>Conclusion</p> <p><it>Mrp1/Mdr1a/1b </it>knock-out mice have a reduced inflammatory response to cigarette smoke. In addition, the expression levels of several cytokines and chemokines were also lower in lungs of <it>Mrp1/Mdr1a/1b </it>knock-out mice independent of smoke exposure. Further studies are required to determine whether dysfunction of MRP1 and/or P-gp contribute to the pathogenesis of COPD.</p

Effect of COPD treatments on MRP1-mediated transport in bronchial epithelial cells

Margaretha van der Deen1, Sandra Homan1, Hetty Timmer-Bosscha1, Rik J Scheper2, Wim Timens3, Dirkje S Postma4, Elisabeth G de Vries1Departments of 1Medical Oncology, 3Pathology, 4Pulmonary Diseases, University Medical Center Groningen and University of Groningen, The Netherlands; 2Department of Pathology, VU University Medical Center, Amsterdam, The NetherlandsBackground: Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD.Methods: The immortalized human bronchial epithelial cell line 16HBE14o- was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry.Results: Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner.Conclusions: Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD.Keywords: bronchus epithelium, COPD, drugs, MRP1, multidrug resistance, oxidative stres

Role of multidrug resistance-associated protein 1 in airway epithelium

In this thesis we investigated whether multidrug resistance-associated protein 1 (MRP1) levels in bronchi and parenchymal lung tissue are related to chronic obstructive pulmonary disease (COPD) development. MRP1 is a protective protein against toxic substances and oxidative stress in which glutathione (GSH) plays a central role [1, 2]. MRP1 is highly expressed in lung airway epithelium and might be important with respect to protection against inhaled toxic substances. Development of COPD is highly related to smoking and is, amongst others, characterized by an imbalance between oxidants and antioxidants [3]. Diminished MRP1 function may therefore predispose smokers to a higher susceptibility to COPD [4].

The HiNF-P/p220NPAT cell cycle signaling pathway controls nonhistone target genes

HiNF-P and its cofactor p220(NPAT) are principal factors regulating histone gene expression at the G(1)-S phase cell cycle transition. Here, we have investigated whether HiNF-P controls other cell cycle- and cancer-related genes. We used cDNA microarrays to monitor responsiveness of gene expression to small interfering RNA-mediated depletion of HiNF-P. Candidate HiNF-P target genes were examined for the presence of HiNF-P recognition motifs, in vitro HiNF-P binding to DNA, and in vivo association by chromatin immunoprecipitations and functional reporter gene assays. Of 177 proliferation-related genes we tested, 20 are modulated in HiNF-P-depleted cells and contain putative HiNF-P binding motifs. We validated that at least three genes (i.e., ATM, PRKDC, and CKS2) are HiNF-P dependent and provide data indicating that the DNA damage response is altered in HiNF-P-depleted cells. We conclude that, in addition to histone genes, HiNF-P also regulates expression of nonhistone targets that influence competency for cell cycle progression

Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (Delta Psi(m)) and the production of reactive oxygen species. Fluorescent probes were used to assess Delta Psi(m) (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P <0.05) and a two-fold increase in Fluo-3 fluorescence (P <0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither Delta Psi(m), ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis

Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells

Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o(-), MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRPI downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% 0.22%) with MK571 further decreased cell survival 31% (p = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p <0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p=0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r=0.99, p=0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development. (c) 2007 Wiley Periodicals, Inc

The cancer-related Runx2 protein enhances cell growth and responses to androgen and TGFbeta in prostate cancer cells

Prostate cancer cells often metastasize to bone where osteolytic lesions are formed. Runx2 is an essential transcription factor for bone formation and suppresses cell growth in normal osteoblasts but may function as an oncogenic factor in solid tumors (e.g., breast, prostate). Here, we addressed whether Runx2 is linked to steroid hormone and growth factor signaling, which controls prostate cancer cell growth. Protein expression profiling of prostate cell lines (i.e., PC3, LNCaP, RWPE) treated with 5alpha-dihydrotestosterone (DHT) or tumor growth factor beta (TGFbeta) revealed modulations in selected cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors that are generally consistent with mitogenic responses. Endogenous elevation of Runx2 and diminished p57 protein levels in PC3 cells are associated with faster proliferation in vitro and development of larger tumors upon xenografting these cells in bone in vivo. To examine whether TGFbeta or DHT signaling modulates the transcriptional activity of Runx2 and vice versa, we performed luciferase reporter assays. In PC3 cells that express TGFbetaRII, TGFbeta and Runx2 synergize to increase transcription of synthetic promoters. In LNCaP cells that are DHT responsive, Runx2 stimulates the androgen receptor (AR) responsive expression of the prostate-specific marker PSA, perhaps facilitated by formation of a complex with AR. Our data suggest that Runx2 is mechanistically linked to TGFbeta and androgen responsive pathways that support prostate cancer cell growth